Sequence-structure based phylogeny of GPCR Class A Rhodopsin receptors

Molecular Phylogenetics and Evolution 74 (2014) 66–96

Contents lists available at ScienceDirect

Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev

Sequence-structure based phylogeny of GPCR Class A Rhodopsin receptors Kavita Kumari Kakarala ⇑, Kaiser Jamil Centre for Biotechnology and Bioinformatics (CBB), School of Life Sciences, Jawaharlal Nehru Institute of Advanced Studies (JNIAS), 6th Floor, Buddha Bhawan, M.G. Road, Secunderabad 500003, Andhra Pradesh, India

a r t i c l e

i n f o

Article history: Received 6 November 2013 Revised 17 January 2014 Accepted 24 January 2014 Available online 3 February 2014 Keywords: GPCR Alignment Phylogeny Maximum Likelihood Orphan receptors Ligands

a b s t r a c t Current methods of G protein coupled receptors (GPCRs) phylogenetic classification are sequence based and therefore inappropriate for highly divergent sequences, sharing low sequence identity. In this study, sequence structure profile based alignment generated by PROMALS3D was used to understand the GPCR Class A Rhodopsin superfamily evolution using the MEGA 5 software. Phylogenetic analysis included a combination of Neighbor-Joining method and Maximum Likelihood method, with 1000 bootstrap replicates. Our study was able to identify potential ligand association for Class A Orphans and putative/unclassified Class A receptors with no cognate ligand information: GPR21 and GPR52 with fatty acids; GPR75 with Neuropeptide Y; GPR82, GPR18, GPR141 with N-arachidonylglycine; GPR176 with Free fatty acids, GPR10 with Tachykinin & Neuropeptide Y; GPR85 with ATP, ADP & UDP glucose; GPR151 with Galanin; GPR153 and GPR162 with Adrenalin, Noradrenalin; GPR146, GPR139, GPR142 with Neuromedin, Ghrelin, Neuromedin U-25 & Thyrotropin-releasing hormone; GPR171 with ATP, ADP & UDP Glucose; GPR88, GPR135, GPR161, GPR101with 11-cis-retinal; GPR83 with Tackykinin; GPR148 with Prostanoids, GPR109b, GPR81, GPR31with ATP & UTP and GPR150 with GnRH I & GnRHII. Furthermore, we suggest that this study would prove useful in re-classification of receptors, selecting templates for homology modeling and identifying ligands which may show cross reactivity with other GPCRs as signaling via multiple ligands play a significant role in disease modulation. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction GPCRs are key regulators for many physiological processes and hence are the target for multiple diseases including cancer. The Rhodopsin family is the largest of the five families of GPCRs (Fredriksson et al., 2003) with 3000 members. In addition to these receptors there are 155 Class A Orphan and 127 putative/unclassified Class A Orphan receptors present (as per GLIDA database) (Okuno et al., 2008). Thus, due to the vast diversity within the family, the mechanism of molecular recognition and signaling process is yet to be realized completely. Adding to this complexity, the signaling molecules of these GPCRs include a wide array of ligands such as peptides, proteins, amines, sugars, lipids, nucleotides, and photons (Gether, 2000). Research based on sequence based phylogenetic classification (Attwood and Findlay, 1994; Kolakowski, 1994; Josefsson, 1999; Fredriksson et al., 2003; Fredriksson and Schioth, 2005), multidimensional scaling analysis, binding site analysis and ligand based classification (Pelé et al., 2011; Surgand ⇑ Corresponding author. Fax: +91 04027541551. E-mail address: [email protected] (K.K. Kakarala). http://dx.doi.org/10.1016/j.ympev.2014.01.022 1055-7903/Ó 2014 Elsevier Inc. All rights reserved.

et al., 2006; Gloriam et al., 2009; van der Horst et al., 2010) gave new insights to GPCR superfamily evolution. A step further in this process was achieved by phylogenetic clustering methods; this method was successful in the identification of natural ligands for many Orphan GPCRs (An et al., 1998; Narumiya et al., 1999; Joost and Methner, 2002; Im, 2010; Szekeres et al., 2000; Metpally and Sowdhamini, 2005a,b). Understanding class A GPCR phylogenetic relationship is challenging and needs to be improved to characterize the ligands for orphan receptors/putative unclassified Class A GPCRs accurately, and to separate some of the GPCRs without ambiguity. Characterizing superfamily evolution solely on sequence based methods may give partial information when applied to divergent, dataset like GPCRs. Although it is well known that proteins similar in sequence often exhibit comparable functions, but if the sequence identity is less than 30% (twilight zone), predictions based on sequence search methods alone may not be enough. Furthermore, they may not classify receptors with similar structure and function in spite of having a low sequence identity. The aim of the present study was to reanalyze the Class A Rhodopsin superfamily including Orphan and putative/unclassified

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

Class A receptors with a unique sequence–structure – profile based multiple sequence alignment program Promals3D (Pei et al., 2008; Pei and Grishin, 2007) combined with robust evolutionary analysis software MEGA 5 (Tamura et al., 2011). Our study is based on the reports, that proved the utility of structural alignments in guiding and evaluating sequence alignments (Hubbard and Blundell, 1987; Russell and Barton, 1994). Moreover, previous studies have also suggested that although evolution works at the sequence level, it always proceeds under robust structural and functional constraints (Fitch, 1971; Studer and Robinson-Rechavi, 2010; Tuffley and Steel, 1998).

67

3. Results and discussion There are more than 3257 entries of Human, Rat and Mouse species of which there are more than 3000 entries of Class A Rhodopsin alone. So, Class A Rhodopsin family represents an evolutionarily active set of GPCRs. In humans, this family includes about 900 GPCRs (534 of which are olfactory receptors) (based on the information given in a GLIDA database at the time of preparation of the manuscript) (Okuno et al., 2008). Analysis of sequence alignment generated by PROMALS3D showed that the conserved residues characteristic for each helix were aligned for most of the receptors in the dataset i.e., TM1-N, TM2-D, TM3-R TM4-W, TM5-F/P, TM6-P, TM7-P (Fig. 1).

2. Methods 2.1. Preparation of the sequence set

3.1. Phylogenetic analysis

Sequences of Class A Rhodopsin family, Class A Orphan receptors and Class A putative/unclassified receptors excluding olfactory receptors from GLIDA database (1083) (Okuno et al., 2008) were selected for the present study (Table S1).

The analysis of Class A receptors resulted in the formation of 36 clusters. This classification resulted in a scattered distribution of subfamilies of GPCRs receptors among different clusters. Sometimes even subtypes were distributed in different clusters. In this study some of the Class A Orphan and putative/unclassified Class A receptors remained unassociated with any of annotated receptors. In addition Neuropeptide Y subtype 6 of rat and human also were unassociated with any group Table 1. Cluster 1 is composed of Class A Orphan receptors of GPR type. These receptors are Q6NWM5 HUMAN, GPR21 HUMAN, Q8BX79 MOUSE, GPR52 HUMAN out of which Q6NWM5 HUMAN, Q8BX79 MOUSE are placed closer to GPR21 HUMAN showing possible similarity in structure and function. However, GPR52 forms a separate clade indicating distant relationship. There are no known endogenous ligands known for GPR21 and GPR52, but studies have indicated that these receptors may play a significant role in diet induced obesity, inflammation and glucose metabolism (Gardner et al., 2012). Thus, free fatty acids and/or leukotrienes may activate these receptors. (Fig. 2). However, the receptors of this cluster are not associated with any annotated Class A Rhodopsin receptors. Cluster 2 represents putative/unclassified Class A receptors which are known as putative neurotransmitters, belonging to type Super Conserved Receptor Expressed in Brain (SERB). Our classification suggests the possibility of these receptors as ancestral in origin as the members of this family share high sequence identity with rat and zebrafish orthologues, thus indicating important functions of SERB family and their endogenous ligand(s) (Matsumoto et al., 2000) (Fig. 3). The receptors of this cluster are not associated with any annotated Class A Rhodopsin receptors. Cluster 3 represents peptide family representatives of type vasopressin-like. Arginine vasopressins (AVP) and oxytocin (OT) are both neurohypophysial hormones with high sequence and structure homology (IUPHAR database, Mouillac et al., 2012). Arginine vasopressin receptors are of two types based on the locationV1 (hepatic AVP) and V2 (Renal AVP). V1 receptors are further divided to V1A (the hepatic receptor) and V1B (adenohyposical receptor) (Jard et al., 1986). Our analysis shows that oxytocin receptors are closer to vasopressin type 1 (77% bootstrap replicates) in comparison to vasopressin type 2 (V2R). This classification is in agreement with the experimental finding that oxytocin activates V1A but with low affinity. Furthermore, our analysis also have clearly distinguished V2 receptors from V1A, V1B and OT receptors, confirming earlier studies where V2 receptor was reported to be different. V2 receptors are different as their activation is brought about by coupling to Gs followed by activation of adenylate cyclase and protein kinase A (IUPHAR database, Mouillac et al., 2012). Our results also showed a close relationship between peptide hormone receptors, gonadotrophin releasing hormone type (GNRHR HUMAN, GNRHR RAT, Q6P8H4 MOUSE, GNRHR

2.2. Multiple sequence alignments The sequences used for the present study were aligned with PROMALS3D, which is best fitted for divergent clusters. The advantage of using this method is that it generates alignment combining sequence structure and profile matching (Pei and Grishin, 2007). The PDB IDs of the GPCR crystal structures that were used to impose structural constraints to the alignment were as follows: 3OE6, 4EA3, 4DJH, 3PBL, 2R4R, 3SN6, 3RZE, 3EML.

2.3. Phylogenetic analysis using MEGA 5 2.3.1. Neighbor-Joining method The multiple sequence alignment obtained from PROMALS3D analysis was used as the input for further investigation. The evolutionary history was inferred initially using the Neighbor-Joining method (Saitou and Nei, 1987) using 1000 bootstrap replications (Felsenstein, 1985). The Branches corresponding to less than 50% bootstrap replicates were collapsed. The distances were calculated using JTT matrix-based method (Jones et al., 1992). Our study involved 1083 amino acid sequences. All ambiguous positions were removed for each sequence pair. There were a total of 5450 positions in the final dataset. Evolutionary analyses were conducted using MEGA 5 (Tamura et al., 2011). The receptors which formed clusters were again subjected to analysis using much more accurate Maximum Likelihood method.

2.3.2. Maximum Likelihood method The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model (Jones et al., 1992). The bootstrap consensus tree inferred from 1000 replicates (Felsenstein, 1985) was taken to represent the evolutionary history of the taxa analyzed Branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) were shown as branches (Felsenstein, 1985). Initial tree(s) for the heuristic search were obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using a JTT model. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011).

68

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

Fig. 1. Representative multiple sequence alignment of GPCR clusters. Analysis of sequence alignment of these sequences showed that the conserved residues characteristic for each helix were aligned for most of the receptors in the cluster i.e., TM1-N, TM2-D, TM3-R, TM4-W, TM5-F/P, TM6-P, TM7-P.

MOUSE GNRR2 HUMAN) and putative/unclassified Class A GPCRs: GP150_Mouse and GP150_Human (84% bootstrap replicates). Thus, GPR150 may share ligands of gonadotropin releasing hormone like GNRHI, GNRHII: triptorelin, leuprolide, triptorelin and antagonist like CMPD, antide, antarelix, NBI-42902, WAY-207024. The receptors belonging to Class A Orphan:GPR19_HUMAN, GPR19_MOUSE and putative/unclassified Class A receptors: Q8BYA1 MOUSE, Q8BZP8 MOUSE, Q6JSL8 HUMAN, Q6W5P3 HUMAN, Q6W5P4 HUMAN, Q6JSL4 HUMAN appeared to be distantly related to vasopressin-like receptors and peptide hormone receptors. These receptors may be ancestral proteins which later separated to form vasopressin, oxytocin and gonadotrophin Releasing Hormone type 1&2 receptors and hence may share same ligands (Fig. 4). Cluster 4 Chemokines are related to each other by sequence and structure homology. All of them have four cysteines and are divided into four classes CC, CXC, CX3C and C, based on the position of cysteines. CC and CXC are the most predominant classes of chemokines (IUPHAR database, Murphy et al., 2013). Our study grouped chemokines into separate subclusters. CCR7, CCR9, CCR10, CCR6, CXCR1, CX3C1, CXCR2, CXCR5, CXCR3, CXCR4, CCR8 in one group and subtypes CCR2, CCR5, CCR3, CCR1, CCRL1, CCRL2 to separate group (Fig. 5, Table 1) suggesting differences in the binding affinity and interacting ligands. Thus, our results are in agreement with the experimental finding that ten human chemokine receptors: CXCR1, CXCR4, CXCR5, CXCR6, CCR6, CCR8, CCR9, CCR10, XCR1 and CX3CR1 are highly selective for endogenous ligand chemokine (Kd  1 nM) in comparison with receptors CXCR1, CCR1 and CCR5 (IUPHAR database, Murphy et al., 2013). Chemokine receptors were reported to signal through a nonchemokine agonist, and interestingly chemokines were also reported to act as either agonist or antagonist to GPCRs other than chemokine receptors (IUPHAR database, Murphy et al., 2013) (Fig 5). Cluster 5 comprises GPCRs belonging to Class A Orphan/RDC1 (RDC1 HUMAN, RDC1 RAT, Q9JLZ0 RAT, RDC1 MOUSE), adrenomedulin (ADMR MOUSE, ADMR RAT, ADMR HUMAN, Q6LAJ3 HUMAN) and G protein-coupled estrogen receptor1 (GPER)/ chemokine receptor like type 2 (GPER HUMAN, Q63ZY2 HUMAN, GPER MOUSE, GPER RAT). Our results show that Class A Orphan/ RDC1, (RDC1 HUMAN, RDC1 RAT, Q9JLZ0 RAT, RDC1 MOUSE) and adrenomedulin (ADMR MOUSE, ADMR RAT, ADMR HUMAN, Q6LAJ3 HUMAN) may share similar ligands as reported by previous studies (Metpally and Sowdhamini, 2005a). Our results are in agreement with experimental studies, that reported adrenomedulin activating adrenomedulin receptors, RDC1 receptors and calcitonin receptor-like receptors (Kapas and Clark, 1995; Kapas et al., 1995; Njuki et al., 1993). G protein-coupled estrogen receptor (GPER), also known as the G protein-coupled receptor 30 (GPR30) encoded by the GPER gene (O’Dowd et al., 1998), also appears to be related to adrenomedulin and RDC receptors from our results. However, 17b-estradiol was suggested as its endogenous ligand (Southern et al., 2013). Further studies are required to evaluate the binding affinity of adrenomedulin with GPER receptors (Fig. 6). Cluster 6 represents peptide family of GPCRs. It is represented by APJ, somatostatin and angiotensin like peptide, angiotensin and bradykinin subfamilies. APELIN, a 36 amino acid peptide, was recognized as an endogenous ligand for APJ receptors, belonging to peptide family (Tatemoto et al., 1998). The APJ/Apelin receptor was reported to be involved in the regulation of cardiovascular function, fluid homeostasis, the adipoinsular axis, gastrointestinal and immunomodulatory functions, as reviewed by Pitkin et al. (2010). Our analysis shows the possibility that APJ subfamily and somatostatin and angiotensin like peptide, which has been renamed as relaxin, may share similar ligand. Relaxin and insulin like peptides are endogenous ligands of somatostatin- and angiotensin-

69

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

Table 1 List of GPCRs in each of the 36 clusters derived from phylogenetic analysis. These clusters were obtained after aligning sequences with PROMALS3D multiple alignment program followed by evolutionary analysis by Neighbor-Joining method using MEGA 5 software package. The dataset was derived from GLIDA database and is composed of Class A Rhodopsin, Class A Orphan, putative/unclassified Class A receptors excluding olfactory receptors. Cluster 1

Cluster 2

Cluster 3

Cluster 4

Cluster 5

Cluster 6

Q6NWM5 HUMAN GPR21 HUMAN Q8BX79 MOUSE GPR52 HUMAN

GPR85 RAT GPR85 MOUSE GPR85 HUMAN Q8NEN2 HUMAN GP173 HUMAN GP173 RAT GP173 MOUSE GPR27 HUMAN GPR27 RAT GPR27 MOUSE

V1BR MOUSE Q9JLP2 MOUSE V1BR RAT V1BR HUMAN OXYR HUMAN OXYR MOUSE Q8R561 RAT OXYR RAT V1AR HUMAN V1AR RAT V1AR MOUSE V2R RAT V2R MOUSE V2R HUMAN O43192 HUMAN Q8BYA1 MOUSE Q8BZP8 MOUSE Q6JSL8 HUMAN Q6W5P3 HUMAN Q6W5P4 HUMAN Q6JSL4 HUMAN GNRR2 HUMAN GNRHR HUMAN GNRHR RAT Q6P8H4 MOUSE GNRHR MOUSE GP150 MOUSE GP150 HUMAN GPR19 HUMAN GPR19 MOUSE Q8VDJ9 MOUSE Q810I4 MOUSE

Q8CH33 RAT CCR9 MOUSE Q9UQQ6 HUMAN CCR9 HUMAN CCR7 HUMAN Q8CAS2 MOUSE CCR7 MOUSE CCRL1 HUMAN Q8C0M1 MOUSE Q924I3 MOUSE Q8QZW9 MOUSE CCR6 HUMAN Q7Z7I1 HUMAN CCBP2 HUMAN Q5U1W0 RAT CCBP2 RAT Q8CEG1 MOUSE CCBP2 MOUSE Q8NH28 HUMAN 3OE6 CXCR4 HUMAN CXCR4 MOUSE Q8VD47 RAT Q62973 RAT CXCR4 RAT Q6T7X2 HUMAN CCR10 HUMAN CCR10 MOUSE Q6P3C2 MOUSE CXCR5 MOUSE CXCR5 RAT CXCR5 HUMAN Q7Z710 HUMAN CXCR3 HUMAN CXCR3 RAT Q9QWN6 MOUSE CXCR3 MOUSE Q810W6 MOUSE CXCR1 MOUSE CXCR1 RAT CXCR2 RAT CXCR2 MOUSE CXCR2 HUMAN CXCR1 HUMAN Q8N6T6 HUMAN Q6IN95 HUMAN XCR1 MOUSE Q8BN14 MOUSE XCR1 HUMAN CHEMOKINE Q8BR50 MOUSE CX3C1 MOUSE Q8CBJ0 MOUSE CX3C1 RAT CX3C1 HUMAN Q9BYX5 HUMAN CCR8 HUMAN CCR8 MOUSE Q96KP5 HUMAN O00421 HUMAN Q6IPX0 HUMAN Q9UPG0 HUMAN O75307 HUMAN O35457 MOUSE Q91YD7 MOUSE O70171 MOUSE Q91ZH4 RAT CCR4 MOUSE CCR4 HUMAN Q5QIP0 HUMAN Q5QIN9 HUMAN CCR5 HUMAN

GPER RAT GPER MOUSE Q63ZY2 HUMAN GPER HUMAN RDC1 MOUSE Q9JLZ0 RAT RDC1 RAT RDC1 HUMAN Q6LAJ3 HUMAN ADMR HUMAN ADMR RAT ADMR MOUSE

Q8BVF1 MOUSE APJ MOUSE APJ RAT APJ HUMAN GPR25 HUMAN GPR15 HUMAN BKRB2 RAT BKRB2 MOUSE BKRB2 HUMAN Q68DM8 HUMAN BKRB1 HUMAN BKRB1 RAT BKRB1 MOUSE AG22 RAT AG22 MOUSE AG22 HUMAN RL3R2 HUMAN RL3R2 MOUSE RL3R1 HUMAN RL3R1 MOUSE Q5Y987 RAT Q5Y986 RAT

(continued on next page)

70

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

Table 1 (continued) Cluster 1

Cluster 2

Cluster 3

Cluster 4

Cluster 5

Cluster 6

Q5QIP1 HUMAN CCR5 MOUSE Q68G28 RAT CCR5 RAT CCR2 RAT CCR2 MOUSE CCR2 HUMAN Q8K3M7 MOUSE CCR3 MOUSE Q8BHB8 MOUSE CCR3 RAT CCR3 HUMAN CC1L1 MOUSE CCR1 HUMAN Q9JLY8 RAT Q6ZWR7 MOUSE CCR1 MOUSE Q8BVW4 MOUSE Q8BMH9 MOUSE Cluster 7

Cluster 8

Cluster 9

Cluster 10

Cluster 11

Cluster 12

LT4R2 LT4R2 LT4R2 LT4R1 LT4R1 LT4R1

O88535 MOUSE FPRL1 MOUSE O88536 MOUSE FPRL1 HUMAN FPRL2 HUMAN Q71MR7 MOUSE Q71MR8 MOUSE O88538 MOUSE O88537 MOUSE FPR1 MOUSE FPR1 HUMAN GPR39 HUMAN Q6NWS5 HUMAN GPR32 HUMAN GPR44 MOUSE GPR44 HUMAN CML1 HUMAN CML1 RAT CML1 MOUSE GPR33 MOUSE Q8C6R2 MOUSE C3AR MOUSE C3AR RAT C3AR HUMAN C5AR RAT C5AR MOUSE C5AR HUMAN C5ARL RAT Q5XPY4 RAT C5ARL MOUSE Q6NWR0 HUMAN Q6NWQ9 HUMAN Q6NWQ8 HUMAN C5ARL HUMAN GPR1_MOUSE GPR1 RAT Q6NVX4 HUMAN GPR1 HUMAN

MRGRE RAT MRGRE MOUSE MAS1L HUMAN MRGRD HUMAN MRGRD RAT MRGRD MOUSE MRGRF RAT MRGRF MOUSE Q8N7J6 HUMAN Q8IXE2 HUMAN MRGRF HUMAN MRGRG RAT MRGRG MOUSE MAS1L HUMAN MRGRD HUMAN MRGRD RAT MRGRD MOUSE MRGRF RAT MRGRF MOUSE MRGA8 MOUSE MRGA4 MOUSE MRGA7 MOUSE MRGA3 MOUSE MRGA5 MOUSE MRGA1 MOUSE MRGA2 MOUSE MRGA6 MOUSE Q91YB7 RAT MRGRA RAT SNSR2 HUMAN MRGX3 HUMAN SNSR3 HUMAN MRGX1 HUMAN SNSR5 HUMAN MRGX4 HUMAN MRGX2 HUMAN Q8CDY4 MOUSE SNSR1 RAT MRGX1 RAT MRGB4 RAT MRGB5 RAT MRGB4 MOUSE Q91ZB9 MOUSE Q8CIP3 MOUSE Q91ZC3 MOUSE Q7TN48 RAT Q7TN47 RAT MRGB8 RAT Q91ZC2 MOUSE Q8BHI8 MOUSE

G109B HUMAN Q8NGE4 HUMAN Q8TDS4 HUMAN Q80Z39 RAT Q9EP66 MOUSE GPR81 HUMAN GPR81 MOUSE Q8TDS5 HUMAN Q86WP7 HUMAN Q8NGW4 HUMAN GPR31 HUMAN Q9JLS1 MOUSE P2RY1 MOUSE P2RY1 RAT P2RY1 HUMAN Q8BMJ5 MOUSE GPR80 HUMAN Q6IYF8 MOUSE Q6Y1R5 RAT GPR91 HUMAN SUCR1 RAT GPR91 MOUSE P2Y11 HUMAN P2RY6 MOUSE P2RY6 RAT P2RY6 HUMAN Q711G2 HUMAN P2RY4 HUMAN P2RY4 MOUSE P2RY4 RAT P2RY2 HUMAN P2RY2 RAT P2RY2 MOUSE GPR18 HUMAN Q8K1Z6 MOUSE Q9H2L2 HUMAN Q8BLT7 MOUSE Q7Z602 HUMAN Q7TQN5 RAT Q7TQP0 MOUSE DUFFY MOUSE Q7TSL2 MOUSE DUFFY HUMAN Q8BZR0 MOUSE GPR82 HUMAN Q76L88 HUMAN

Q8BZV8 MOUSE P2Y12 MOUSE P2Y12 RAT P2Y12 HUMAN GPR86 HUMAN Q6GUG4 RAT GPR86 MOUSE GPR87 MOUSE Q5XIX4 RAT P2Y14 RAT Q8IV06 HUMAN GP171 HUMAN Q8CIF3 MOUSE Q8BY85 MOUSE Q8BTN1 MOUSE Q8BG55 MOUSE GPR34 HUMAN Q6XCE7 RAT Q8BYI1 MOUSE GPR34 MOUSE PAFR RAT PAFR HUMAN Q8C017 MOUSE PAFR MOUSE Q8IV19 HUMAN CLTR1 HUMAN CLTR1 MOUSE CLTR1 RAT CLTR2 HUMAN CLTR2 RAT Q8R528 MOUSE CLTR2 MOUSE Q8N5S7 HUMAN GPR17 HUMAN Q6NS65 MOUSE GP174 HUMAN Q7TMV7 MOUSE EBI2 HUMAN SPR1 HUMAN Q6IX34 HUMAN SPR1 MOUSE Q8BUD0 MOUSE Q6NWM4 HUMAN GPR4 HUMAN PSYR MOUSE PSYR HUMAN G2A MOUSE G2A HUMAN GPR92 HUMAN GPR35 MOUSE Q9Y2T6 HUMAN Q8N580 HUMAN Q8BYC4 MOUSE O35797 RAT

UR2R RAT UR2R MOUSE UR2R HUMAN MCHR1 MOUSE MCHR1 HUMAN Q8K3M8 MOUSE Q6Q377 HUMAN MCHR2 HUMAN SOMATOSTATIN Q9JK40 MOUSE SSR5 MOUSE SSR5 RAT SSR5 HUMAN SSR3 HUMAN SSR3 RAT SSR3 MOUSE SSR2 HUMAN SSR2 RAT SSR2 MOUSE SSR4 MOUSE Q8BQ97 MOUSE SSR4 RAT SSR4 HUMAN Q7TT86 RAT SSR1 RAT SSR1 MOUSE SSR1 HUMAN Q6NWQ6 HUMAN Q6NWQ5 HUMAN GPR8 HUMAN Q6NTC7 HUMAN GPR7 HUMAN Q80WU7 MOUSE OPRX MOUSE OPRX RAT Tq8ixb0 HUMAN Q8CH83 RAT Q6FGM5 HUMAN 4EA3 OPRK RAT OPRK MOUSE Q8IWP3 HUMAN OPRK HUMAN 4DJH Q8BLP9 MOUSE OPRD MOUSE OPRD RAT OPRD HUMAN Q8VIP0 MOUSE Q86V80 HUMAN Q6UQ80 HUMAN Q9H573 HUMAN OPRM HUMAN Q5TBX0 HUMAN

RAT MOUSE HUMAN HUMAN RAT MOUSE

71

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96 Table 1 (continued) Cluster 7

Cluster 8

Cluster 9

Cluster 10

Cluster 11

Cluster 12

GPR20 HUMAN Q8VCK6 MOUSE GPR43 HUMAN GPR42 HUMAN GPR41 HUMAN GPR40 HUMAN Q8K3T4 RAT Q8K3T5 MOUSE Q76JU9 MOUSE Q8BZ77 MOUSE PAR4 MOUSE PAR4 RAT Q6DK42 HUMAN PAR4 HUMAN Q5U324 RAT PAR1 RAT PAR1 MOUSE PAR1 HUMAN Q86VZ1 HUMAN Q8R3I1 MOUSE PAR2 MOUSE PAR2 RAT PAR2 HUMAN PAR3 HUMAN Q7Z3W3 HUMAN PAR3 RAT Q8BJB7 MOUSE PAR3 MOUSE

Q8IWW4 HUMAN Q8IWW3 HUMAN Q6UPP1 HUMAN Q9R0D1 MOUSE Q6YC50 MOUSE Q8CAN5 MOUSE Q8CH73 MOUSE Q9R1M0 MOUSE Q8CGW2 MOUSE Q8VI71 MOUSE Q8CH75 MOUSE Q8VI69 MOUSE Q9JIY1 MOUSE OPRM MOUSE Q8VI70 MOUSE Q9R1L9 MOUSE

Cluster 13

Cluster 14

Cluster 15

Cluster 16

Cluster 17

Cluster 18

Q8BHU4 MOUSE Q8BHR6 MOUSE Q99LE2 MOUSE Q96CH1 HUMAN Q8TDU8 HUMAN Q6DWJ6 HUMAN Q7Z601 HUMAN Q7TQN9 MOUSE Q9R297 RAT Q9QWW3 RAT O88820 RAT Q9ERT2 MOUSE TRFR HUMAN TRFR MOUSE TRFR RAT Q9JIB2 RAT NMUR1 RAT NMUR1 MOUSE O55040 MOUSE Q7LDP6 HUMAN NMUR1 HUMAN Q8NE20 HUMAN Q7LC54 HUMAN NMUR2 HUMAN Q9JIB1 RAT Q8BZ39 MOUSE GHSR RAT GHSR MOUSE GHSR HUMAN MTLR HUMAN Q5U431 MOUSE GPR39 HUMAN NTR1 RAT NTR1 HUMAN NTR2 HUMAN NTR2 MOUSE NTR2 RAT Q920Q5 MOUSE Q8VIF5 MOUSE

Q8BHU4 MOUSE Q8BHR6 MOUSE Q99LE2 MOUSE Q96CH1 HUMAN Q8TDU8 HUMAN Q6DWJ6 HUMAN Q7Z601 HUMAN Q7TQN9 MOUSE

Q68CR4 HUMAN OPSR HUMAN OPSG HUMAN OPSG RAT OPSG MOUSE OPSB HUMAN OPSB MOUSE O70363 RAT OPSB RAT 4a4 OPSD HUMAN OPSD RAT Q8K0D8 MOUSE OPSD MOUSE OPN3 MOUSE OPN3 HUMAN RGR MOUSE RGR HUMAN OPSX MOUSE OPSX HUMAN Q7TQN6 RAT OPN5 MOUSE OPN5 HUMAN OPN4 RAT OPN4 MOUSE OPN4 HUMAN Q8NGQ9 HUMAN Q8NH39 HUMAN Q8IZ08 HUMAN Q7TQP2 MOUSE Q7TQN7 RAT Q9ESP4 RAT Q9EPB7 MOUSE Q9GZN0 HUMAN Q6VN48 HUMAN Q5TGK2 HUMAN Q5TGK0 HUMAN GP161 HUMAN GP101 HUMAN Q6NWS4 HUMAN GPR45 HUMAN Q6NXU6 HUMAN O43898 HUMAN Q8CA29 MOUSE

GPR26 RAT GPR26 MOUSE GPR26 HUMAN GPR78 HUMAN Q8TAM0 HUMAN GPR62 HUMAN Q8C010 MOUSE GPR61 HUMAN Q8TDV4 HUMAN Q6NWS0 HUMAN

TAR09 RAT TAR06 RAT TAR13 RAT TAR15 RAT Q5QD20 RAT Q5QD21 RAT Q5QD22 RAT TAR12 RAT Q5QD11 MOUSE Q5QD10 MOUSE Q5QD09 MOUSE Q5QD08 MOUSE TAR08 RAT Q5QD12 MOUSE TAR14 RAT Q5QD19 RAT TAR03 RAT Q5QD04 MOUSE TAR03 HUMAN TAR04 RAT Q5QD13 MOUSE TAR04 HUMAN TAR05 HUMAN Q5QD06 MOUSE Q5QD05 MOUSE Q5QD07 MOUSE TAR07 RAT TAR11 RAT TAR10 RAT Q5QD18 RAT Q5QD23 RAT Q5QD14 MOUSE O14804 HUMAN Q6NTA8 HUMAN Q5VUQ3 HUMAN TAR02 RAT Q5QD15 MOUSE TAR01 MOUSE TAR01 HUMAN Q5QD24 RAT Q5QD16_MOUSE Q5QD17 MOUSE Q5QD02 HUMAN GPR58_HUMAN

Q7Z5R9 HUMAN HRH2 HUMAN HRH2 RAT Q9QX37 MOUSE Q9D282 MOUSE HRH2 MOUSE

Q9R297 RAT Q9QWW3 RAT O88820 RAT Q9ERT2 MOUSE TRFR HUMAN TRFR MOUSE TRFR RAT Q9JIB2 RAT NMUR1 RAT(SPID) NMUR1 MOUSE (SPID) GP151 RAT GP151 MOUSE GP151 HUMAN KISSR RAT KISSR MOUSE KISSR HUMAN GALR1 RAT GALR1 MOUSE GALR1 HUMAN GALR3 RAT GALR3 MOUSE GALR3 HUMAN GALR2 HUMAN GALR2 RAT Q8BKB0 MOUSE GALR2 MOUSE

(continued on next page)

72

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

Table 1 (continued) Cluster 13

Cluster 14

Cluster 15

Cluster 16

GPR45 MOUSE Q8BZ93 MOUSE GPR63 MOUSE Q6NWR7 HUMAN Q6NWR9 HUMAN Q6NWR8 HUMAN GPR63 HUMAN

Cluster 17

Cluster 18

5HT4R MOUSE 5HT4R RAT Q712M9 HUMAN Q8IXH9 HUMAN 5HT4R HUMAN

Cluster 19

Cluster 20

Cluster 21

Cluster 22

Cluster 23

Cluster 24

DRD5 RAT DRD5 MOUSE DRD5 HUMAN Q8NGU3 HUMAN DRD1 HUMAN DRD1 RAT DRD1 MOUSE 5HT5B RAT 5HT5B MOUSE 5HT5A HUMAN 5HT5A RAT 5HT5A MOUSE Q8BGS4 MOUSE 5HT1A MOUSE Q8BZP1 MOUSE 5HT1A RAT 5HT1A HUMAN 5HT1F RAT 5HT1F MOUSE HT1F HUMAN 5HT1E HUMAN 5HT1B RAT 5HT1B MOUSE 5HT1B HUMAN HT1D HUMAN 5HT1D RAT Q8BUW7 MOUSE 5HT1D MOUSE

5HT7R RAT 5HT7R MOUSE P97842 RAT Q5VX04 HUMAN 5HT7R HUMAN Q8IXE5 HUMAN 5HT6R RAT 5HT6R HUMAN Q63004 RAT 5HT6R MOUSE Q13167 HUMAN DRD3 RAT DRD3 MOUSE 3PBL DRD2 HUMAN DRD2 RAT DRD2 MOUSE DRD4 RAT DRD4 MOUSE DRD4 HUMAN Q8NGM5 HUMAN Q8BZI5 MOUSE 5HT2C MOUSE 5HT2C RAT 5HT2C HUMAN 5HT2A HUMAN 5HT2A RAT 5HT2A MOUSE 5HT2B RAT Q8JZK5 MOUSE 5HT2B RAT Q8JZK5 MOUSE 5HT2B MOUSE Q6P523 HUMAN 5HT2B HUMAN Q925K6 MOUSE Q6NXV9 MOUSE ADA2B MOUSE ADA2B RAT ADA2B HUMAN ADA2C RAT ADA2C MOUSE ADA2C HUMAN ADA2A HUMAN Q5VZT1 HUMAN ADA2A RAT ADA2A MOUSE

Q8VBU7 RAT ADRB2 RAT Q8BH38 MOUSE ADRB2 MOUSE Q6GMT4 HUMAN ADRB2 HUMAN 2R4R ADRB1 HUMAN ADRB1 RAT ADRB1 MOUSE ADRB3 HUMAN ADRB3 RAT ADRB3 MOUSE ADA1D RAT ADA1D MOUSE ADA1D HUMAN Q9DBL0 MOUSE ADA1B MOUSE Q6IRH4 RAT ADA1B RAT ADA1B HUMAN Q96RE8 HUMAN ADA1A HUMAN ADA1A RAT Q8BUE5 MOUSE Q8BV77 MOUSE ADA1A MOUSE

Q8WY00 HUMAN Q8NI49 HUMAN HRH3 RAT HRH3 MOUSE Q8WXZ9 HUMAN Q8WY01 HUMAN Q5PPG3 RAT HRH4 MOUSE HRH4 RAT Q96LD9 HUMAN HRH4 HUMAN ACM4 RAT ACM4 MOUSE Q96RG8 HUMAN ACM4 HUMAN Q96RH0 HUMAN ACM2 HUMAN ACM2 RAT ACM2 MOUSE Q6NUM3 HUMAN ACM5 HUMAN Q8IVW0 HUMAN ACM5 RAT ACM5 MOUSE ACM3 RAT ACM3 MOUSE ACM3 HUMAN ACM1 RAT ACM1 MOUSE Q96RH1 HUMAN ACM1 HUMAN

3SN6 Q16538 HUMAN O88835 MOUSE Q8K0Z9 MOUSE Q6NV75 HUMAN Q5TGR5 HUMAN

Q8TDV5 HUMAN Q7TQN8 RAT Q7TQP3 MOUSE Q8BXB7 MOUSE Q8BZL4 MOUSE GPR22 HUMAN 3RZE Q6P9E5 HUMAN HRH1 HUMAN HRH1 RAT HRH1 MOUSE Q91V66 RAT Q91V49 RAT

Cluster 25

Cluster 26

Cluster 27

Cluster 28

Cluster 29

Cluster 30

NPY5R MOUSE NPY5R RAT NPY5R HUMAN

Q8K1V9 MOUSE EDNRB MOUSE EDNRB RAT Q9UD23 HUMAN Q5W0G9 HUMAN EDNRB HUMAN EDNRA HUMAN EDNRA RAT EDNRA MOUSE GPR37 MOUSE GPR37 RAT GPR37 HUMAN ETBR2 RAT O88313 MOUSE Q5SXP7 HUMAN ETBR2 HUMAN Q8K418 RAT

CCKAR RAT CCKAR MOUSE CCKAR HUMAN Q8BKF6 MOUSE GASR MOUSE GASR RAT Q9NYK7 HUMAN Q96LC6 HUMAN Q92492 HUMAN GASR HUMAN

Q6VLX3 MOUSE OX2R MOUSE OX2R RAT Q5VTM0 HUMAN OX2R HUMAN Q8BV78 MOUSE Q6VNS3 MOUSE OX1R RAT Q9HBV6 HUMAN OX1R HUMAN QRFPR RAT QRFPR MOUSE QRFPR HUMAN Q8BHH0 MOUSE Q924G9 RAT NPFF1 RAT NPFF1 HUMAN

Q8BWV1 MOUSE NPY2R MOUSE Q9ERC0 RAT NPY2R HUMAN Q6VMN6 MOUSE GPR10 RAT Q5VXR9 HUMAN GPR10 HUMAN Q6NWR4 HUMAN GPR83 HUMAN Q6NWR3 HUMAN Q8VHD7 RAT GPR83 MOUSE PKR1 RAT PKR1 MOUSE PKR1 HUMAN PKR2 HUMAN

Q86VU0 HUMAN Q6UY15 HUMAN Q5T512 HUMAN LGR6 HUMAN LGR5 MOUSE LGR5 HUMAN Q8BZR7 MOUSE LGR4 RAT TSHR MOUSE Q9D697 MOUSE TSHR RAT TSHR HUMAN FSHR RAT FSHR MOUSE FSHR HUMAN LSHR HUMAN LSHR RAT

73

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96 Table 1 (continued) Cluster 25

Cluster 26

Cluster 27

BRS3 MOUSE BRS3 HUMAN GRPR RAT GRPR MOUSE GRPR HUMAN Q5VUK8 HUMAN NMBR HUMAN NMBR RAT NMBR MOUSE

Cluster 28

Cluster 29

Cluster 30

NPFF2 HUMAN NPFF2 RAT Q924N0 MOUSE NPFF2 MOUSE

Q8BZV9 MOUSE NK2R MOUSE NK2R RAT Q9UDE6 HUMAN Q8NGQ8 HUMAN NK2R HUMAN Q6NXX1 MOUSE NK3R MOUSE NK3R RAT NK3R HUMAN NK1R HUMAN NK1R RAT Q8BYR7 MOUSE NK1R MOUSE

LSHR MOUSE Q99MX9 MOUSE/Gpr84 Q8CIM5 MOUSE/Gpr84 Q9NQS5 HUMAN/Gpr84 MTR1L HUMAN MTR1L MOUSE MTR1A MOUSE MTR1A HUMAN MTR1B MOUSE MTR1B HUMAN

Cluster 31

Cluster 32

Cluster 33

Cluster 34

Cluster 35

Cluster 36

Q8BZF9 MOUSE NPY4R MOUSE NPY4R RAT NPY4R HUMAN NPY1R HUMAN NPY1R RAT NPY1R MOUSE

Q6NWR2 HUMAN O95800 HUMAN Q8BXP3 MOUSE Q6X632 MOUSE Q921A8 MOUSE Q7TQW1 MOUSE Q6NXF6 HUMAN Q14439 HUMAN Q80WT4 MOUSE Q7TMA4 MOUSE Q7Z605 HUMAN Q5VY25 HUMAN Q5VY26 HUMAN

TA2R RAT TA2R MOUSE TA2R HUMAN PE2R1 HUMAN PE2R1 RAT PE2R1 MOUSE Q6RYQ6 HUMAN PF2R HUMAN PF2R RAT Q9D627 MOUSE PF2R MOUSE PE2R3 RAT PE2R3 MOUSE Q6PDF2 MOUSE Q6TTN3 HUMAN Q5TH86 HUMAN PE2R3 HUMAN Q5TH88 HUMAN O00326 HUMAN O00325 HUMAN Q91VE4 MOUSE PE2R4 MOUSE PE2R4 HUMAN PI2R RAT PI2R MOUSE PI2R HUMAN PE2R2 MOUSE PE2R2 RAT Q8BZ75 MOUSE PE2R2 HUMAN PD2R HUMAN Q8CCM3 MOUSE PD2R MOUSE PD2R RAT PD2RL RAT Q8TDV2 HUMAN Q86U87 HUMAN

EDG2 RAT EDG2 HUMAN Q9H228 HUMAN EDG2_MOUSE Q9QY79 RAT Q9JKM5 RAT EDG4 HUMAN EDG4 MOUSE Q6P290_MOUSE Q6GPG7_HUMAN EDG7_HUMAN Q9NRB8_HUMAN EDG7_RAT EDG7_MOUSE Q6P290 MOUSE Q6NSY0 HUMAN Q6B0G7 HUMAN CNR2 HUMAN CNR2 RAT CNR2 MOUSE Q5UB37 HUMAN CNR1 HUMAN CNR1 RAT CNR1 MOUSE GPR12 RAT GPR12 MOUSE GPR12 HUMAN GPR6 MOUSE GPR6 RAT GPR6 HUMAN GPR3 MOUSE GPR3 RAT Q80T02 RAT Q8TDU6 HUMAN Q80SS6 MOUSE

Q8BGU7 MOUSE AA1R MOUSE Q8CAH1 MOUSE Q6AXD8 MOUSE O08766 RAT AA1R RAT AA1R HUMAN AA3R RAT AA3R MOUSE Q6UWU0 HUMAN AA3R HUMAN Q8BXI2 MOUSE AA2BR MOUSE 3EML AA2BR RAT AA2BR HUMAN AA2AR HUMAN AA2AR RAT AA2AR MOUSE

Q7M4L8 HUMAN ACTHR MOUSE ACTHR HUMAN MC4R RAT MC4R MOUSE MC4R HUMAN MC5R HUMAN MC5R MOUSE MC5R RAT MC3R HUMAN MC3R RAT MC3R MOUSE Q75NA2 MOUSE Q6UR95 HUMAN Q6UR94 HUMAN Q6UR93 HUMAN Q6UR92 HUMAN MSHR HUMAN Q6UR98 HUMAN Q86YW1 HUMAN Q6URA0 HUMAN Q6UR96 HUMAN Q6UR99 HUMAN Q6UR97 HUMAN

Fig. 2. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 4 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 346 positions in the final dataset.

like peptide receptor/ Relaxin family peptide receptors. These receptors are widespread in the male and female reproductive system (IUPHAR database, Summers et al., 2009). Our analysis was in agreement with experimental results where APJ subfamily and relaxin subfamily levels were shown to be important in cardiovascular regulation (Papadopoulos et al., 2013). Angiotensin 2 and bradykinin receptors were closely related (63% bootstrap replicates). The known agonists of angiotensin 2 receptor are angiotensin I, II, III and antagonist saralasin, PD123177, PD123319, etc. (IUPHAR database, Alexander et al., 2012). Bradykinin is the endogenous ligand of bradykinin receptor and icatibant, chroman 28,

[Leu9,des-Arg10] kallidin, etc. are the antagonists (IUPHAR database: Coulson et al., 2013). Our analysis suggests that these two receptors may show binding affinity for the ligands of both angiotensin type and bradykinin receptors. Our results confirm the earlier finding where bradykinin receptors were shown to respond to angiotensin-(1–7) [Ang-(1–7)] (Fernandes et al., 2001) (Fig. 7). Cluster 7 consists of LT4R2/BLT2 and LT4R1/BLT1 receptors. The endogenous ligand, LTA4, is the only available agonist of BLT1 receptor, whereas 12 (S)-HETE, 12 (S)-HpETE, and 15 (S)-HETE, 12-HHT have been identified as ligands for BLT2 receptor (IUPHAR database, Bäck et al., 2013) (Fig. 8).

74

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

Fig. 3. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 10 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 363 positions in the final dataset.

Fig. 4. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 29 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 155 positions in the final dataset.

Cluster 8 consists of receptors of peptide family and subfamily C5a/C3a anaphylatoxin, chemokine receptor like 1 and Fmetleu-phe along with Class A orphan receptors of subfamily GPR. C3a/ C5a anaphylatoxins are also known as complement peptide receptors. These receptors are activated with peptide (74–77 aminoacid) fragments produced through activation of complement cascade. Only three receptors for complement peptides have been reported in mammals (C5a1, C5a2 & C3a) (IUPHAR database, Monk et al., 2013). YSFKPMPLaR is the peptide agonist of both C5a and C3a, while NDT9520492, JPE1375, RPR121154C089, FLTChaAR, etc. act as antagonists of C5a and C3a (IUPHAR database, Hawksworth et al., 2013). Interestingly, GPR32 HUMAN and Q6NWS5 HUMAN, which belong to chemokine receptor-like 1, clustered with Fmet-leu-phe subfamily may possibly share ligands. Our results agree with reports where GPR32 was reported to be related to the chemotaxic formyl peptide receptors (Marchese et al., 1998). The other interesting result was with respect to GPR44_Human. Our result shows it as a separate clade, although it is considered as belonging to chemokine class 1 receptor by GLIDA database (Okuno et al., 2008). Recent studies have suggested that PGD (2) acts as an antagonist to GPR44 and this PGD (2) – GPR44 pathway is a potential target for treatment of

androgenetic alopecia (Garza et al., 2012). The DP2 receptor is structurally distinct from all of the other known prostanoid receptors, being more closely related to chemoattractant receptors such as FMet-Leu-Phe (fMLP) and BLT receptors (IUPHAR database, Robert et al., 2013). Our results brings out very elegantly this relatedness. FMet-leu-phe receptor is also known as the N-formyl peptide receptor, and its endogenous ligand is Annexin I (Walther et al., 2000). Although distantly related, orphan receptors GPR1_Human, GPR_Rat, GPCR_Mouse and putative class A/unclassified receptor GPR39_Human may interact with ligands of C5a/C3a anaphylatoxin, chemokine receptor like 1 and Fmet-leu-phe subfamily as reported (Metpally and Sowdhamini, 2005b) (Fig. 9). Cluster 9 consists of Class A Orphan receptors of Mas Protooncogene or SSNR (sensory nerve specific receptor) type. MrgA, B, C and D subfamilies and Human MRGX2, 5 & 7 are expressed in sensory neurons with nociceptive phenotypes (IUPHAR database, Walther et al., 2000). It is hypothesized that sensory neuroactive mediators are released from mast cells that may stimulate these receptors and also MrgC11 receptors are stimulated by the RFamide, neuropeptide FF, etc. (Lee et al., 2008). Angiotensin metabolites were suggested to stimulate receptors of the Mas-related

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

75

Fig. 5. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 85 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 171 positions in the final dataset.

76

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

Fig. 6. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2): the analysis involved 12 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 340 positions in the final dataset.

Fig. 7. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 22 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 292 positions in the final dataset.

Fig. 8. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2): the analysis involved 6 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 265 positions in the final dataset.

gene family (Jackson et al., 1988). An endogenous peptide with a high degree of sequence similarity to ang-(1–7), alamandine, was shown to promote NO release in MrgD-transfected cells. The antagonists known are D-Pro7-angiotensin-(1–7), b-alanine and PD123319 (IUPHAR database, Davenport et al., 2013a). Our initial analysis using Neighbour Joining method suggested distant relationship between these Orphan receptors and chemokine type 1 receptors, Fmet-leu-phe receptors. However, putative/unclassified Class A receptor GPR152 was associated with CRTH2/FPR in a study which has associated nine new members with Rhodopsin family (Gloriam et al., 2005) (Fig. 10). Cluster 10 was represented by P2Y receptors P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11, which are one of the three families of extracellular receptors for purine and pyrimidine nucleotides involved in signaling (Burnstock, 1997). P2Y1, P2Y2, P2Y4, P2Y6 were suggested to be closely related compared to P2Y12, 13 & 14 (IUPHAR

database, Burnstock et al., 2013). They are activated by adenine and uracil nucleotide di- or tri- phosphates. Our results also have brought out this distinction very clearly, showcasing the significance of this work. Apart from these receptors, interestingly, P2Y11 clustered with Class A Orphan receptors Q8TDS5 HUMAN, Q86WP7 HUMAN, Q8NGW4 HUMAN, GPR31 HUMAN, Q9JLS1 MOUSE and putative/unclassified Class A receptors GPR109B HUMAN, Q8NGE4 HUMAN, Q8TDS4 HUMAN, Q80Z39 RAT, Q9EP66 MOUSE, GPR81 HUMAN, GPR81 MOUSE (61% bootstrap replicates). Our results suggest that these receptors may get activated by ligands of nucleotide receptor subtype P2Y11like ATP, adenosine 5’-O-(3-thiotriphosphate), Uridine Triphosphate, etc. Our results have separated P2Y11 from the rest of the nucleotide receptors, agreeing with the IUPHAR database description. However, these receptors were named as the hydroxycarboxylic acid (HCA) receptors (Offermanns et al., 2011), although the affinity to the endogenous

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

77

Fig. 9. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 39 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 282 positions in the final dataset.

ligand HCA3 (3-hydroxyoctanoic acid) is reported to be low. The other putative unclassified receptors which are in this group are Q76L88 HUMAN, GPR82 HUMAN, Q8BZR0 MOUSE/GPR82, GPR18 HUMAN, Q8K1Z6 MOUSE/GPR18, Q9H2L2 HUMAN, Q8BLT7 MOUSE, Q7Z602 HUMAN/GPR141, Q7TQN5 RAT/GPR141, Q7TQP0 MOUSE. Studies have identified N-arachidonylglycine as the endogenous ligand for orphan G-protein-coupled receptor GPR18 (Kohno et al., 2006). Our results suggest that N-arachidonylglycine may activate putative/unclassified Class A receptors along with ligands of nucleotide receptors which are clustered in this group. The other distantly related GPCR is Duffy receptor. This receptor is classified as a separate class in Peptide subfamily. Our classification suggests that it may bind to ligands of nucleotide receptors and N-arachidonylglycin (Fig. 11). Cluster 11 represents a highly heterogeneous group comprising nucleotide, platelet activating factor, putative/unclassified class A and Class A Orphan receptors. Our results show that putative/ unclassified Class A receptors Q8IV06 HUMAN, GP171 HUMAN, Q8CIF3 MOUSE, Q8BY85 MOUSE, Q8BTN1 MOUSE, Q8BG55 MOUSE, GPR34 HUMAN, Q6XCE7 RAT, Q8BYI1 MOUSE, GPR34 MOUSE, are grouped with purinoceptors of type P2Y12 and P2Y14 (90% bootstrap replicates). Treefam tree for family TF330969 (http://www. pfam.janelia.org/protein/O14626#tabview=tab5) also depicts that the receptor GPR 171_Human is related phylogenetically to purino-

ceptors P2Y12 and P2Y14. Our results show that P2Y12 and P2Y14 are closely related, to GPR171, GPR85 and GPR55 (96% bootstrap replicates). P2Y12 are activated by ATP and ADP while clopidogrel and ticlopidine act as potent antagonists (Savi et al., 2001). The agonists of P2Y14 receptor are UDP-glucose, UDP-galactose, UDPglucuronic acid and UDP-N-acetylglucosamine (Han et al., 2011), while there are no known antagonists of P2Y14 receptor. Thus, these putative/unclassified Class A receptors may also bind to ligands of P2Y12 & P2Y14 recspectively. Cysteinyl leukotrienes of Class A Orphan receptors, protease activated receptors, platelet activating factor receptors also are part of this cluster. Protease activated receptors are involved in many pathophysiological conditions including cancer (Han et al., 2011). They are activated by proteases, resulting in exposure of N-terminal end which acts as a ligand resulting in activation of the receptor. There are no endogenous ligands identified for these receptors yet. Our results suggest possible cognate ligands for this receptor i.e., leukotrienes, ATP, UDP etc. However further studies are required to test the binding affinity of the ligands of this group with protease activated receptor subfamily (Fig. 12). Cluster 12 comprises receptors of the peptide family; opioid receptors, somatostatin receptors, melanin concentrating hormone receptors and urotensin receptor. Opioid receptors, somatostatin receptors, and melanin concentrating hormone receptors grouped

78

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

Fig. 10. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 50 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 251 positions in the final dataset.

together (94% bootstrap replicates) indicating similar structure and function. The ligands of the opioid receptor are a-neoendorphin, b-neoendorphin, [Met] enkephalin (Borsodi et al., 2013). Our results showed that GPR7_Human and GPR8_Human, belonging to the class A Orphan may bind to ligands of Opiod receptor like are a-neoendorphin, b-neoendorphin, [Met]enkephalin, etc. (IUPHAR database, Borsodi et al., 2013). Our results agree with previous findings that GPR7_Human and GPR8_Human receptors bind to neuropeptide ligands (Tanaka et al., 2003). The other receptors are somatostatin receptors that bind with high affinity to the endogenous polypeptides somatostatin-14, somatostatin28 and cortistatin, as well as different synthetic ligands

(Krulich and Dhariwal, 1968; Pradayrol et al., 1980). The melanin-concentrating hormone (MCH) receptors were reported to be involved in the regulation of processes like sleep and emotionality (Gehlert et al., 2009). Melanin concentrating hormone is the natural ligand for melanin concentrating hormone and the agonist reported are S36057, p-guanidinobenzoyl-MCH(7–17) and antagonist SNAP-7941, [3H] SNAP-7941, ATC0175, etc. (IUPHAR database, Audinot and Boutin, 2013). Our results have shown that urotensin receptors are distantly related to opioid, somatotostatin, and melanin concentrating hormone receptors. Experimental studies have also proved that urotensin-II is an endogenous ligand for urotensin receptor and is not similar

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

79

Fig. 11. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 44 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 170 positions in the final dataset.

to somatostatin or cortisan as cited in the IUPHAR database (Mori et al., 1999) (Fig. 13). Cluster 13 Our results suggests that receptors belonging to peptide family namely neurotensin, neuromedin U like and thyrotropin-releasing hormone & secretagogue family form a group of closely related receptors (82% bootstrap replicates). Therefore, these receptors may bind to ligands like neurotensin, anabolic hormone ghrelin, etc. Thyrotropin-releasing hormone & secretagogue formed a separate group (80% bootstrap replicates). The known ligand of this receptor is thyrotropin-releasing hormone (Yamada et al., 1995). Neuromedin S and neuromedin U were identified as endogenous ligands for the same receptors (Mori et al., 2005). Our classification matches with earlier findings where neuromedin-1 (NM1) was reported to be homologous with the growth hormone secretagogue receptor (ghrelin receptor; 33% homology) and the neurotensin receptor (29% homology) (Tan et al., 1998; Metpally and Sowdhamini, 2005a) and the classification reported earlier (Holst and Sivertsen (2013)). The putative/unclassified Class A receptors, Q6DWJ6 HUMAN/GPR139, Q7Z601 HUMAN/GPR142,

Q7TQN9 MOUSE/GPR142, clustered with receptors like neuromedin, ghrelin, neuromedin U and thyrotropin-releasing hormone and secretagogue/hormone receptor (81% bootstrap replications) hence may resemble in structure and also may bind with these ligands with varying affinity. This finding may aid in understanding the structure and function of these putative/unclassified Class A receptors. Q6DWJ6 HUMAN/GPR139 is an orphan GPCR, mainly expressed in central nervous system and was proposed to play an important role in the control of locomotor activity and affective behavior (Matsuo et al., 2005). Although the information on synthetic agonist and antagonist is available (Hu et al., 2009) there is no information on natural ligands and hence from our analysis it is possible to test binding activity of endogenous ligands of the receptors which clustered along with it. The other putative/ unclassified Class A receptors which may be distantly related to other members of the group are Q8BHU4 MOUSE/GPR146, Q8BHR6 MOUSE/GPR146, Q99LE2 MOUSE/GPR146 and Q96CH1 HUMAN/GPR146. Recently GPR146 was reported to bind to proinsulin C peptide (Yosten et al., 2013) (Fig. 14).

80

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

Fig. 12. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 81 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 224 positions in the final dataset.

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

81

Fig. 13. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 66 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 233 positions in the final dataset.

82

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

Fig. 14. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 39 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 269 positions in the final dataset.

Cluster 14 represents receptors of peptide family and galanin subfamily. Members of galanin subfamily clustered with kiss receptors (54% bootstrap replicates). Galanin and kisspeptins or cyclic peptides are their endogenous ligands. This analysis has shown that the galanin receptor subtypes 2, 3 and kisspeptins are related closely in comparison to galanin subtype-1. Our results show that putative/unclassified Class A receptors GP151_Human,

GP151_RAT and GP151_MOUSE are distantly related to galanin and kisspeptin receptors. These putative receptors may interact with galanin and kisspeptins (Fig. 15). Cluster 15 represents a rhodopsin family of GPCRs. 11-cis-retinal is the reported natural ligand (Del Valle et al., 2003) of this family. Our results show that putative/unclassified Class A receptors (Q8NH39 HUMAN/GPR135, Q8IZ08 HUMAN/GPR135, Q7TQP2

Fig. 15. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2): the analysis involved 16 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 318 positions in the final dataset.

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

MOUSE/GPR135, Q7TQN7RAT/GPR135, Q9ESP4RAT/GPR88, Q9EPB7 MOUSE/GPR88, Q9GZN0 HUMAN/GPR88, Q6VN48 HUMAN/GPR88, Q5TGK2 HUMAN/GPR161, Q5TGK0 HUMAN/ GPR161, GP161 HUMAN, GP101 HUMAN) and receptors from Class A Orphan GPCRs (Q6NWS4 HUMAN, GPR45 HUMAN, Q6NXU6 HUMAN, O43898 HUMAN, Q8CA29 MOUSE, GPR45 MOUSE, Q8BZ93 MOUSE, GPR63 MOUSE, Q6NWR7 HUMAN, Q6NWR9 HUMAN, Q6NWR8 HUMAN, GPR63 HUMAN) are distantly related to rhodopsin family and hence may interact with 11-cis-retinal (Fig. 16). Cluster 16 represents GPCRs of Class A Orphan and putative/ unclassified Class A receptors. Our results show that these putative/unclassified Class A receptors: GPR26 RAT, GPR26 MOUSE, GPR26 HUMAN, GPR78 HUMAN, Q8TAM0 HUMAN, GPR62 HUMAN, Q8C010 MOUSE and Class A Orphan receptors GPR61 HUMAN, Q8TDV4 HUMAN, Q6NWS0 HUMAN could not be associated with any of the family/subfamily of our dataset. However, our initial analysis using Neighbor-Joining indicates these unassociated putative/unclassified Class A receptors may be related to rhodopsin family distantly (Fig. 17). Cluster 17 consists of aminergic receptors belonging to trace amine and 5-Hydroxytryptamine receptors (5 HT) (serotonin)

83

subfamily. These trace amine receptors were discovered during the search for subtypes of 5 HT receptor. Trace amines (para-tyramine, b-phenylethylamine (b-PEA) and octopamine are endogenous ligands for these receptors, but not all the receptors share high affinity with trace amines. The order of potency is as follows: tyramine > b-phenylethylamine > octopamine = dopamine, whereas EPPTB (N-(3-ethoxyphenyl)-4-(pyrrolidin-1-yl)-3-(trifluoromethyl) benzamide acts as a synthetic antagonist (IUPHAR database, Maguire and Davenport, 2013). Our results show that 5HT4 receptors (5HT4R HUMAN, Q8IXH9 HUMAN, Q712M9 HUMAN, 5HT4R RAT, 5HT4R MOUSE) are distantly related to trace amine receptors. Association of trace amine receptors with – hydroxytryptamine receptors was reported by earlier studies also (Metpally and Sowdhamini, 2005a) (Fig. 18). Cluster18 represents histamine receptor subtype 2 of amine family. Our results show that human histamine subtype 2 may be distantly related to rat and mouse histamine subtype 2 receptors. Histamine is the endogenous ligand for these receptors, while burimamide, cimetidine, ranitidine and tiotidine, etc. act as H2 subtype specific antagonist (IUPHAR database, Chazot et al., 2013) (Fig. 19).

Fig. 16. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 47 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 92 positions in the final dataset.

Fig. 17. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2): the analysis involved 10 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 303 positions in the final dataset.

84

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

Fig. 18. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 46 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 295 positions in the final dataset.

Cluster 19 consists of 5-Hydroxytryptamine receptors (serotonin), Dopamine receptors. Our results show that 5-Hydroxytryptamine type 1 receptors are related to dopamine receptors 1 & 5. However, 5-Hydroxytryptamine receptor subtype 5 appears to be distantly related to type 1 as reported (IUPHAR database, Barnes et al., 2012). The endogenous ligands of this cluster are 5-Hydroxy Tryptamine/Dopamine (Fig. 20).

Cluster 20 comprised of receptors from 5-hydroxytryptamine subfamily type 2,6,7, a-adrenoceptors type 2 A, B, C (a2) and dopamine receptor type 2,3 & 4. Our result show that 5-hydroxytryptamine subtype 2 and 6 are closely related to a adrenoceptors type 2 A, B, C (56% bootstrap replicates) and within a adrenoceptor type 2 receptors, A subtype is closely related to C subtype. From our analysis it is clear that a 2A/B/C adrenergic receptors are related

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

phylogenetically more closely to Dopamine receptors rather than a1-adrenoceptor and b-adrenoceptor. This analysis also confirms our previous publication where the homology model was developed using dopamine D3 crystal structure (3PBL:PDBID) as a template rather than available b2 adrenergic receptor structure (Kakarala et al., 2012; Jayaraman et al., 2013). Our results also show that the 5-Hydroxytryptamine receptor 7 (5-HT7) is distantly related to other receptors of the cluster. Our result is further confirmed by the fact that 5-HT7 displays less than 39% amino acid sequence identity to other 5-HT receptors (Barnes et al., 2012) (Fig. 21). The endogenous ligands with which these receptors could show cross reactivity are 5-Hydroxytryptamine, adrenaline, noradrenaline, dopamine (Fig. 21). Cluster 21 represents b-adrenoceptors (b1, b2, and b3 subtype), 2R4R (crystal structure of the human b2 receptor) and a1 type adrenergic receptor. Adrenaline and noradrenaline are the major endogenous ligands of these receptors, the known agonists being xamoterol, isoprenaline, T-0509, denopamine, etc. while cicloprolol, metoprolol, atenolol, etc. are the antagonists (IUPHAR database, Bond et al., 2013). Our results show that ADA1D_human (a1D) is

85

distantly related to rest of the a1 adrenergic receptors as it forms a separate clade. Interestingly, experimental studies have also confirmed that a1D signal less effectively when compared to other receptors. This is one more example showcasing the accuracy of this approach (Fig. 22). Cluster 22 represents two subfamilies-muscarinic acetylcholine and histamine type 3 & 4. There are five different types of muscarinic receptors (M1–M5), out of which M2 and M4-muscarinic receptors are able to couple to Gi/o-proteins and M1, 2M3 and M5 couple to Gq/11-proteins. However, they are reported to couple to a wide range of diverse signaling pathways. Muscarinic receptors respond to the endogenous ligand acetylcholine and are involved in numerous central and peripheral physiological responses (IUPHAR database, Challiss and Tobin, 2009). Our results also have classified M2 and M4 separately and M1, M3 and M5 separately. The agonists and antagonists known for this receptor are oxotremorine, acrecoline, sabcomeline, xanomeline, ipratropium, scopolamine, etc. (IUPHAR database: Challiss and Tobin (2009). Furthermore, our results show that muscarinic receptors are distantly related to histamine 3 and 4 type. Selective H3 receptor ago-

Fig. 19. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 6 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 358 positions in the final dataset.

Fig. 20. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 27 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 135 positions in the final dataset.

86

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

Fig. 21. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 45 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 151 positions in the final dataset.

Fig. 22. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). Analysis involved 24 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 322 positions in the final dataset.

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

87

Fig. 23. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 31 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 217 positions in the final dataset.

nists (e.g. R-a-methylhistamine, immetit) and antagonists (e.g. thioperamide, clobenpropit, iodoproxyfan and ciproxifan) and H4 selective agonists (VUF 8430) and antagonist (e.g. JNJ 7777120) (IUPHAR database: Greig et al., 2013; Chazot et al., 2013) are reported (Fig. 23). Cluster 23 comprises of putative/unclassified Class A receptors and the sequence corresponding to the crystal structure of b2 adrenergic receptor (PDB ID:3SN6). 3SN6 represents the first high-resolution structure of b2 adrenergic receptor (Rasmussen et al., 2011). It is interesting to observe that our classification has grouped high and low resolution crystal structure of the human b2 adrenergic receptor differently. Our results show that the putative/unclassified class A receptors Q16538 HUMAN/

GPR162, O88835 MOUSE, Q8K0Z9 MOUSE, Q6NV75 HUMAN/ GPR153, Q5TGR5 HUMAN may be related to amine family. Work done by Sreedharan et al. (2011), has identified that the homolog of Q6NV75 HUMAN/GPR153 and Q16538 HUMAN/GPR162 are closely related, and they share a common ancestor that split most likely through a duplication event before the divergence of the tetrapods and the teleost lineage (Sreedharan et al., 2011). Q6NV75 HUMAN/GPR153 was reported to share 20% sequence similarity with serotonin receptors (Gloriam et al., 2005). However, our results suggest that these putative/unclassified Class A receptors are closely related to b2 adrenergic receptor and hence may respond to adrenaline and noradrenaline (Fig. 24).

Fig. 24. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 7 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 277 positions in the final dataset.

88

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

Fig. 25. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 13 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 273 positions in the final dataset.

Fig. 26. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2) analysis involved 10 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 406 positions in the final dataset.

Fig. 27. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 10 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 406 positions in the final dataset.

Cluster 24 comprises histamine subtype 1, putative/unclassified Class A and Class A Orphan receptors. The entry corresponding to the crystal structure of the human histamine H1 (PDBID:3RZE) was also grouped in this cluster. Our results show that the

putative/unclassified Class A receptors Q8TDV5 HUMAN/GPR119, Q7TQN8 RAT/GPR119, Q7TQP3 MOUSE/119 and Class A Orphan receptor, Q8BXB7 MOUSE, Q8BZL4 MOUSE, GPR22 HUMAN may be related distantly to histamine type 1. However, some studies

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

89

Fig. 28. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 22 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 272 positions in the final dataset.

Fig. 29. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 30 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 320 positions in the final dataset.

have suggested the role of GPR119_HUMAN in diabetes (Chu et al., 2008). GPR119 was also reported to get activated by endogenous ligands, like oleoyl lysophosphatidylcholine and oleoylethanol amide (IUPHAR database, Ansarullah et al., 2013). GPR119 was also proved to be a target for obesity and related metabolic diseases (Overton et al., 2006). Our classification supports the report that

histamine, the endogenous ligand of histamine receptor may be involved in regulating glucose and lipid metabolism (Wang et al., 2010) as it is evident that they may share similar ligands (Fig. 25). Cluster 25 is represented by neuropeptide Y5 subtype receptors, which are named so because of large no of tyrosine residues (Larhammar, 1996). They are activated by endogenous ligand neu-

90

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

Fig. 30. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 31 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 266 positions in the final dataset.

Fig. 31. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 9 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 282 positions in the final dataset.

Fig. 32. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 13 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 215 positions in the final dataset.

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

ropeptide Y. Our results prove that NPY5 subtype is different from other neuropeptide receptors (IUPHAR database, Martin et al., 2008). Cluster 26 comprises receptors from bombesin, endothelin and GPR37/endothelin B-like receptors of peptide family. Neuromedin B, gastrin-releasing peptide and bombesin are endogenous ligands of bombesin receptor (Spindel, 2013). Endothelin is the endogenous ligand for endothelin receptors (Saeki et al., 1991) while, prosaposin was proved to be the endogenous ligand for GPR37 (Meyer et al., 2013). Our results are in agreement with previous studies reporting relatedness of bombesin and endothelin (Joost and Methner, 2002; Metpally and Sowdhamini, 2005a) (Fig. 26). Cluster 27 represents receptors for peptide hormones namely cholecystokinin and gastrin (Q8BKF6 MOUSE, GASR MOUSE, GASR RAT, Q9NYK7 HUMAN, Q96LC6 HUMAN, Q92492 HUMAN, GASR HUMAN). Cholecystokinin-4 (CCK-4), gastrin-17 are the endogenous ligands (IUPHAR database, Chen et al., 2008) (Fig. 27). Cluster 28 is composed of peptide receptors from orexin, neuropeptides and orexiigenic neuropeptide subfamily. Orexin receptors are suggested to be involved in various functions including food intake during daytime, in promoting wakefulness and stabilizing vigilance state. Orexin-A and orexin-B are endogenous ligands of this subfamily (IUPHAR database, Winrow et al., 2012). Neuropeptides FF 1 and 2 subtypes are involved in the regulation of pain, cardiovascular functions, appetite, thirst and body temperature. The endogenous ligands are Neuropeptide AF, neuropeptide AF, neuropeptide SF (IUPHAR database, Mollereau-Manaute et al., 2013) (Fig. 28).

91

Cluster 29 is composed of tacykinin and neuropeptide Y of peptide family, Class A Orphan receptors and prokinectin receptors. Our results show that the tachykinin and neuropeptide Y receptors are closely related (75% bootstrap replicates). These receptors are of three types with different ligand preference NK1 (substance PSP), NK2 (neurokinin B-NKB) and NK3 neurokinin B (NKB). Ligand information of GPR83_Human and its orthologues is not known yet. From our analysis, it is predicted that these receptors may bind to tachykinin, endogenous ligand of tachykinin receptors. Our results also show that the class A orphan receptors Q6VMN6 MOUSE, GPR10 RAT, Q5VXR9 HUMAN, GPR10 HUMAN are distantly related to tachykinin receptors and hence may bind to tachykinin. However, in an independent study, human prolactin-releasing peptide (PrRP) has been identified as an endogenous ligand for GPR10 (Marchese et al., 1994). Our results show that neuropeptide Y of type 2 is closely related with prokineticin receptors (76% bootstrap replicates). Neuropeptide Y is the principal endogenous agonist and BIE0246, BIIE0246 ANS JNJ-5207787 are the principal antagonists of neuropeptide receptors (IUPHAR database, Martin et al., 2008). Prokineticin-2 is the principle endogenous ligand while 1,3,5-triazin-4,6-diones and their derivatives are nonpeptidic prokineticin receptor antagonists (Balboni et al., 2008) (Fig. 29). Cluster30 comprises glycoprotein hormone receptors (GpHRs), putative/unclassified Class A receptors and melatonin receptors. These receptors also have nine leucine-rich repeats (LRRs) (Hsu et al., 2000), hence they clustered with the Class A Orphan /LGR like receptors. Earlier studies have also proved that these receptors are similar to receptors for gonadotrophins and thyroid stimulating hormone (TSH) (Ascoli et al., 2002). In mammals LGR4, LGR5 and

Fig. 33. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 34 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 211 positions in the final dataset.

92

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

LGR6 are still orphan receptors, whereas LGR7 and LGR8 function as relaxin and InsL3 receptors, respectively (Hsu, 2003). Our results are similar to previous studies for these receptors (Metpally and Sowdhamini, 2005a,b). Our results show that peptide hormone melatonin is closely related to putative/unclassified Class A receptors Q99MX9 MOUSE/GPR84, Q8CIM5 MOUSE/GPR84, Q9NQS5 HUMAN/GPR84 (100% bootstrap replicates). The various agonists tested are melatonin, ramelteon, 6-hydroxymelatonin, 6-Cl-MLT, IIK7, 5-HEAT, S24014, S24014 and the antagonist are S26131,

S22153, K185, 4P-PDOT, S20928, Luzindole, etc. (Dubocovich, 1988) (Fig. 30). Cluster 31 The receptor clustering obtained from our study show that neuropeptide Y subtype1(NPY1) and neuropeptide subtype 6 (NPY6) are closely related phylogenetically, whereas the neuropeptideY subtype 4 (NPY4) is distantly related. Experimental evidence has shown that NPY1 and NPY6 binds preferentially to neuropeptide Y (NPY) and pancreatic polypeptide (PP), while NPY4 preferentially binds to pancreatic polypeptide (PP). Subtype

Fig. 34. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 36 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 258 positions in the final dataset.

Fig. 35. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2). The analysis involved 19 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 240 positions in the final dataset.

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

specific antagonist were reported for NPY1 (BIBP3226, GR231118, SR120819A, etc.) and NPY4 (GR231118) (Beck-Sickinger et al., 2013) (Fig. 31). Cluster 32 comprises of putative/unclassified Class A receptors, which could not be associated with any of the annotated GPCRs. However GPR120 was reported to act as receptor for medium and long-chain free fatty acids (FFAs), omega-3 fatty acids mediating anti-inflammatory effects, mainly in macrophages and fat cells http://www.uniprot.org/uniprot/Q7TMA4 (Hirasawa et al., 2005). Q7TMA4 MOUSE, Q7Z605 HUMAN, Q5VY25 HUMAN, Q5VY26 HUMAN (GPR120), Q14439 HUMAN/GPR176 and Q80WT4 MOUSE/ GPR176, may also bind to fatty acids, as our results show that these receptors are closely related (58% bootstrap replicates). The other receptors Q921A8 MOUSE, Q7TQW1 MOUSE, Q6NWR2 HUMAN/ GPR75, Q8BXP3 MOUSE/75, Q6X632 MOUSE/GPR75 and O95800 HUMAN (GPR75) may be distantly related to neuropeptide Y (receptor of neighboring cluster). Our results agree with previous studies where human GPR75 was reported to be closely related to a putative Caenorhabditis elegans neuropeptide Y receptor (24% homology), the rat galanin receptor type 3 (25% homology) and the porcine growth hormone secretagogue receptor type 1b (25% homology) (Tarttelin et al., 1999). RANTES is an inflammatory chemokine belonging to the CC-chemokine subfamily (CCL5) was shown to activate GPR75 (Schall et al., 1988; Ignatov et al., 2006), however, binding could not be established experimentally (Fig. 32). Cluster 33 consists of prostanoid and putative/unclassified Class A receptors. Prostanoids (PGD2, PGE2, PGF2a, PGI2) are derived from prostaglandin H2, and these are the natural ligands for the prostanoid receptors. The other known ligands reported are cloprostenol, U46619, iloprost, carbacyclin, indomethacin, bimatoprost, sulprostone and antagonist laropiprant, BWA868C, AH6809, ramatroban, ONO_8713,ONO-8711, AH6809, AS604872, etc. (IUPHAR database, Giembycz et al., 2013; Jones et al., 2013). Our results show that putative/unclassified class A receptors, Q8TDV2 HUMAN (GPR148) and Q86U87 HUMAN (GPR148) may be distantly related

93

to prostanoid receptors hence may respond to ligands of prostanoid receptors (Fig. 33). Cluster 34 consists of lysosphingolipid and LPA (EDG)/sphingosine 1-phosphate, class A orphan receptors and cannabinoid receptors. Lysophosphatidic acid and sphingosine 1-phosphate (S1P) are the endogenous ligands for these receptors. EDG2, EDG4 and EDG7 receptors hence are named as LPA1, LPA2 and LPA3 as reviewed in (Chun et al., 2010). Our results show that LPA receptors are closely related to class A orphan receptors GPR3, GPR6 and GPR12 (GPR12 RAT, GPR12 MOUSE, GPR12 HUMAN, GPR6 MOUSE, GPR6 RAT, GPR6 HUMAN, GPR3 MOUSE, GPR3 RAT). Earlier studies have reported that orphan receptors share greater than 50% identity and 65% similarity at the amino acid level (Marchese et al., 1994; Iismaa et al., 1994; Song et al., 1995; Heiber et al., 1995). These orphan receptors were also reported to be involved in up-regulation of cyclic AMP levels, promoting neuritic outgrowth (Tanaka et al., 2007). G protein-coupled receptor 3 (GPR3) is a newly discovered sphingosine 1-phosphate receptor, which directly or indirectly takes part in regulating the processes of the nervous system and follicle development in the vertebrates (Davenport et al., 2013b). GPR3 is a new therapeutic drug target for neurological diseases and premature ovarian failure. However, the information is limited partly because of the absence of information on ligands (Zhang et al., 2013). Our classification agrees with the IUPHAR database information (http://www.iuphar-db.org/DATABASE/GPCRListForward) where Spingosine 1 posphate was reported as cognate ligand for GPR3, GPR6 and GPR12. From our results, it is apparent that cannabinoid receptors are distantly related to LPA and orphan receptors GPR 3, 6 and 12. Cannabinoid receptors are activated by ‘endocannabinoids’ like N-arachidonoylethanolamine (anandamide) and 2-arachidonoyl glycerol, N-dihomo-c-linolenoylethanolamine, N-docosatetraenoylethanolamine, O-arachidonoylethanolamine (virodhamine), oleamide, N-arachidonoyl dopamine and N-oleoyl dopamine (IUPHAR database, Pertwee et al., 2011). We propose that LPA receptors, orphan receptors GPR3, GPR6, GPR12 together with cannabinoids receptors may be involved in lipid mediated signaling.

Fig. 36. Molecular Phylogenetic analysis by Maximum Likelihood and evolutionary analysis was conducted in MEGA 5 (as described in Section 2): the analysis involved 25 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 277 positions in the final dataset.

94

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

Interestingly, according to present classification cannabinoid receptors are subfamily of under peptide family whereas LPA receptors are separate family itself (Fig. 34). Cluster 35 is composed of nucleotide family and adenosine subfamily receptors. They are known to get activated by the endogenous ligand adenosine, which is present in all body fluids. Adenosine receptors are divided into A1, A2A, A2B and A3. Binodenoson, regadenoson, 2-chloroadenosine, PENECA, NECA, MRS5151, BAY60-6583 are the synthetic agonists and PSB36, DPCPX, CPFPX are some of the antagonists (IUPHAR database, Ijzerman et al., 2013) (Fig. 35). Cluster 36 is composed of peptide receptors from melanocortin subfamily. There are five melanocortin subtypes of receptors known at present. The current IUPHAR nomenclature for these receptors is MC1, MC2, MC3, MC4 and MC5. Our results show that MC1 receptors (former name-MSHR) also known as melanocyte-stimulating hormone is distantly related to the rest of the melanocortin receptors. MC2 (originally termed ACTH-R) forms a separate group in comparison with MC3, MC4. Thus MC3, MC4 and MC5 may share similar binding affinity. Adrenocorticotropic hormone and melanocyte stimulating hormone are the known endogenous ligands of this subfamily (IUPHAR database, Fong et al., 2013) (Fig. 36). 4. Conclusions Our study suggests the possible cognate ligands for the orphan receptors with no ligand information yet. The results of our study also hints at the need of regrouping of the receptors based on the sequence structure homologs identified, as this would reflect the binding probability towards a particular ligand. Our classification suggests the subtype relatedness and thus may prove useful in designing subtype specific drugs. Furthermore, we suggest that this classification would be useful in selection of templates for homology modeling, identifying endogenous ligands for GPCRs, enhancing our understanding to the concept of one receptor– multiple ligand and vice versa thereby supporting structure based drug discovery. Funding sources This work is supported by the Department of Science and Technology (SERC), Government of India, Women Scientist Program (WOS-A) Ref. No. SR/WOS-A/LS-408/2011. Acknowledgment We thank Ms. Archana Jayaraman for helping us in formatting the paper. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ympev.2014.01. 022. References Alexander, W., Bernstein, K.E., Catt, K.J, et al., 2012. Angiotensin Receptors: AT2 receptorIUPHAR Database (IUPHAR-DB). (last modified 26.11.12). An, S., Bleu, T., Hallmark, O.G., Goetzl, E.J., 1998. Characterization of a novel subtype of human G protein-coupled receptor for lysophosphatidic acid. J. Biol. Chem. 273, 7906–7910. Ansarullah, Lu, Y., Holstein, M., DeRuyter, B., Rabinovitch, A., Guo, Z., 2013. Stimulating b-cell regeneration by combining a GPR119 agonist with a DPP-IV inhibitor. PLoS One 8, e53345. Ascoli, M., Fanelli, F., Segaloff, D.L., 2002. The lutropin/choriogonadotropin receptor, a 2002 perspective. Endocr. Rev. 23, 141–174.

Attwood, T.K., Findlay, J.B., 1994. Fingerprinting G-protein-coupled receptors. Protein Eng. 7, 195–203. Audinot, V., Boutin, J.A., 2013. Melanin-Concentrating Hormone Receptors: MCH1 Receptor IUPHAR Database (IUPHAR-DB). (last modified 31.01.13, accessed 21/09/2013. Bäck, M., Shimizu, T., Yokomizo, T, et al., 2013. Leukotriene Receptors, Introductory Chapter. IUPHAR Database (IUPHAR-DB). (last modified 26.07.13). Balboni, G., Lazzari, I., Trapella, C., et al., 2008. Triazine compounds as antagonists at Bv8-prokineticin receptors. J. Med. Chem. 51, 7635–7639. Barnes, N.M., Andrade, R., Bockaert, J., et al., 2012. 5-Hydroxytryptamine Receptors, Introduction. IUPHAR Database (IUPHAR-DB). (last modified 10.01.12). Beck-Sickinger, A., Colmers, W.F., Cox, H.M, et al., 2013. Neuropeptide Y Receptors: Y2 Receptor. IUPHAR Database (IUPHAR-DB). (last modified 15.02.13). Bond, R.A., Bylund, D.B., Eikenburg, D.C, et al., 2013. Adrenoceptors: b1Adrenoceptor. IUPHAR Database (IUPHAR-DB). (last modified 23.07.13). Borsodi, A., Caló G., Chavkin, C., et al., 2013. Opioid Receptors: j Receptor. IUPHAR Database (IUPHAR-DB). (last modified 14.06.13). Burnstock, G., 1997. The past, present and future of purine nucleotides as signalling molecules. Neuropharmacology 36, 1127–1139. Burnstock, G., Abbracchio, M.P., Boeynaems, J.M., et al., 2013. P2Y Receptors, Introductory Chapter. IUPHAR Database (IUPHAR-DB). (last modified 10.05.13). Challiss, R.A.J., Tobin, A.B., 2009. Acetylcholine Receptors (Muscarinic), Introductory Chapter. IUPHAR Database (IUPHAR-DB). (last modified 13.10.09). Chazot, P., Fukui, H., Ganellin, C.R., et al., 2013. Histamine Receptors: H3 Receptor. IUPHAR Database (IUPHAR-DB). (last modified 28.02.13). Chen, Q., Gao, F., Miller, L.J., Cholecystokinin Receptors, Introductory Chapter. IUPHAR Database (IUPHAR-DB). (last modified 09.10.08). Chu, Z.L., Carroll, C., Alfonso, J., et al., 2008. A role for intestinal endocrine cellexpressed g protein-coupled receptor 119 in glycemic control by enhancing glucagon-like peptide-1 and glucose-dependent insulinotropic peptide release. Endocrinology 149, 2038–2047. Chun, J., Hla, T., Lynch, K.R., Spiegel, S., Moolenaar, W.H., 2010. International union of basic and clinical pharmacology. LXXVIII. Lysophospholipid receptor nomenclature. Pharmacol. Rev. 62, 579–587. Coulson, J., Faussner, A., Leeb-Lundberg, F., et al., 2013. Bradykinin Receptors: B1 Receptor. IUPHAR Database (IUPHAR-DB). (last modified 06.03.13). Davenport, A.P., Alexander, S., Sharman, J.L., Pawson, A.J., Benson, H.E., Monaghan, A.E., Liew, W.C., et al., 2013a. Class A Orphans: MAS1. IUPHAR Database (IUPHAR-DB). (last modified 03.07.13, accessed 19.10.13). Davenport, A.P., Alexander, S., Sharman, J.L., et al., 2013b. Class A Orphans: GPR17. IUPHAR Database (IUPHAR-DB). (last modified 24.07.13c). Del Valle, L.J., Ramon, E., Bosch, L., Manyosa, J., Garriga, P., 2003. Specific isomerization of rhodopsin-bound 11-cis-retinal to all-trans-retinal under thermal denaturation. Cell Mol. Life Sci. 60, 2532–2537. Dubocovich, M.L., 1988. Pharmacology and function of melatonin receptors. FASEB J. 2, 2765–2773. Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. Fernandes, L., Fortes, Z.B., Nigro, D., Tostes, R.C., Santos, R.A., Catelli, De Carvalho, M.H., 2001. Potentiation of bradykinin by angiotensin-(1–7) on arterioles of spontaneously hypertensive rats studied in vivo. Hypertension 37, 703–709. Fitch, W.M., 1971. Rate of change of concomitantly variable codons. J. Mol. Evol. 1, 84–96. Fong, T., Schiöth, H., Gantz, I., Cone, R.D., et al., Melanocortin Receptors: MC3 Receptor. IUPHAR Database (IUPHAR-DB). (last modified 07.02.13). Fredriksson, R., Schioth, H.B., 2005. The repertoire of G-protein-coupled receptors in fully sequenced genomes. Mol. Pharmacol. 67, 1414–1425. http://dx.doi.org/ 10.1124/mol.104.009001. Fredriksson, R., Lagerstrom, M.C., Lundin, L.-G., Schioth, H.B., 2003. The Gprotein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol. 63, 1256–1272. Gardner, J., Wu, S., Ling, L., et al., 2012. G-protein-coupled receptor GPR21 knockout mice display improved glucose tolerance and increased insulin response. Biochem. Biophys. Res. Commun. 418, 1–5. Garza, L.A., Liu, Y., Yang, Z., et al., 2012. Prostaglandin D2 inhibits hair growth and is elevated in bald scalp of men with androgenetic alopecia. Sci. Transl. Med. 4 (126ra34). Gehlert, D.R., Rasmussen, K., Shaw, J., et al., 2009. Preclinical evaluation of melanin concentrating hormone receptor 1 (MCHR1) antagonism for the treatment of obesity and depression. J. Pharmacol. Exp. Ther. 329, 429–438.

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96 Gether, U., 2000. Uncovering molecular mechanisms involved in activation of G protein-coupled receptors. Endocr. Rev. 21, 90–113. Giembycz, M., Jones, R.L., Narumiya, S., et al., 2013. Prostanoid Receptors: EP2 Receptor. IUPHAR Database (IUPHAR-DB). (last modified 08.07.13). Gloriam, D.E., Schiöth, H.B., Fredriksson, R., 2005. Nine new human Rhodopsin family G-protein coupled receptors: identification, sequence characterisation and evolutionary relationship. Biochim. Biophys. Acta. 1722, 235–246. Gloriam, D.E., Foord, S.M., Blaney, F.E., Garland, S.L., 2009. Definition of the G protein coupled receptor transmembrane bundle binding pocket and calculation of receptor similarities for drug design. J. Med. Chem. 52, 4429–4442. Greig, N.H., Reale, M., Tata, A.M., 2013. New pharmacological approaches to the cholinergic system: an overview on muscarinic receptor ligands and cholinesterase inhibitors. Recent Pat. CNS Drug Discov. 8, 123–141. Han, N., Jin, K., He, K., Cao, J., Teng, L., 2011. Protease-activated receptors in cancer: a systematic review. Oncol. Lett. 2, 599–608. Hawksworth, O., Coulthard, L., Woodruff, T., 2013. Complement Peptide Receptors: C3a Receptor. IUPHAR Database (IUPHAR-DB). (last modified 30.01.13). Heiber, M., Docherty, J.M., Shah, G., et al., 1995. Isolation of three novel human genes encoding G protein-coupled receptors. DNA Cell. Biol. 14, 25–35. Hirasawa, A., Tsumaya, K., Awaji, T., et al., 2005. Free fatty acids regulate gut incretin glucagon-like peptide-1 secretion through GPR120. Nat. Med. 11, 90–94. Holst, B., Sivertsen, B.B., 2013. Ghrelin Receptor, Introductory Chapter. IUPHAR Database (IUPHAR-DB). (last modified 10.07.13). Hsu, S.Y., 2003. New insights into the evolution of the relaxin-LGR signaling system. Trends Endocrinol. Metab. 14, 303–309. Hsu, S.Y., Kudo, M., Chen, T., et al., 2000. The three subfamilies of leucine-rich repeat-containing G protein-coupled receptors (LGR): identification of LGR6 and LGR7 and the signaling mechanism for LGR7. Mol. Endocrinol. 14, 1257– 1271. Hu, L.A., Tang, P.M., Eslahi, N.K., Zhou, T., Barbosa, J., Liu, Q., 2009. Identification of surrogate agonists and antagonists for orphan G-protein-coupled receptor GPR139. J. Biomol. Screen 14, 789–797. Hubbard, T.J., Blundell, T.L., 1987. Comparison of solvent-inaccessible cores of homologous proteins: definitions useful for protein modelling. Protein Eng. 1, 159–171. Ignatov, A., Robert, J., Gregory-Evans, C., Schaller, H.C., 2006. RANTES stimulates Ca2+ mobilization and inositol trisphosphate (IP3) formation in cells transfected with G protein-coupled receptor 75. Br. J. Pharmacol. 149, 490–497. Iismaa, T.P., Kiefer, J., Liu, M.L., Baker, E., Sutherland, G.R., Shine, J., 1994. Isolation and chromosomal localization of a novel human G-protein-coupled receptor (GPR3) expressed predominantly in the central nervous system. Genomics 24, 391–394. Ijzerman, A.P., Fredholm, B.B., Jacobson, K.A., Linden, J., Müeller, C.E., 2013. Adenosine Receptors: A1 Receptor. IUPHAR Database (IUPHAR-DB). (last modified 18.06.13). Im, D.S., 2010. Pharmacological tools for lysophospholipid GPCRs: development of agonists and antagonists for LPA and S1P receptors. Acta Pharmacol. Sin. 31, 1213–1222. Jackson, T.R., Blair, L.A., Marshall, J., Goedert, M., Hanley, M.R., 1988. The mas oncogene encodes an angiotensin receptor. Nature 335, 437–440. Jard, S., Gaillard, R.C., Guillon, G., et al., 1986. Vasopressin antagonists allow demonstration of a novel type of vasopressin receptor in the rat adenohypophysis. Mol. Pharmacol. 30, 171–177. Jayaraman, A., Jamil, K., Kakarala, K.K., 2013. Homology modelling and docking studies of human a2-adrenergic receptor subtypes. J. Comput. Sci. Syst. Biol. 6, 136–149. Jones, D.T., Taylor, W.R., Thornton, J.M., 1992. The rapid generation of mutation data matrices from protein sequences. Comput. Appl. Biosci. 8, 275–282. Jones, R.L., Shuh, N., Woodward, D.F., Giembycz, M., Xavier, N., 2013. Prostanoid Receptors, Introduction. IUPHAR Database (IUPHAR-DB). (last modified 18.09.13). Joost, P., Methner, A., 2002. Phylogenetic analysis of 277 human G-protein-coupled receptors as a tool for the prediction of orphan receptor ligands. Genome Biol. 3 (RESEARCH0063). Josefsson, L.G., 1999. Evidence for kinship between diverse G-protein coupled receptors. Gene 239, 333–340. Kakarala, K.K., Jayaraman, A., Jamil, K., 2012. In silico analysis of Human and Zebra fish a-2 adrenergic receptors. J. Biol. Agric. Health 2, 11–31. Kapas, S., Clark, A.J., 1995. Identification of an orphan receptor gene as a type 1 calcitonin gene-related peptide receptor. Biochem. Biophys. Res. Commun. 217, 832–838. Kapas, S., Catt, K.J., Clark, A.J., 1995. Cloning and expression of cDNA encoding a rat adrenomedullin receptor. J. Biol. Chem. 270, 25344–25347. Kohno, M., Hasegawa, H., Inoue, A., et al., 2006. Identification of Narachidonylglycine as the endogenous ligand for orphan G-protein-coupled receptor GPR18. Biochem. Biophys. Res. Commun. 347, 827–832. Kolakowski Jr., L.F., 1994. GCRDb: a G-protein-coupled receptor database. Recept. Channel. 2, 1–27. Krulich, L., Dhariwal, A.P., McCann, S.M., 1968. Stimulatory and inhibitory effects of purified hypothalamic extracts on growth hormone release from rat pituitary in vitro. Endocrinology 83, 783–790.

95

Larhammar, D., 1996. Evolution of neuropeptide Y, peptide YY and pancreatic polypeptide. Regul. Pept. 62, 1–11. Lee, M.G., Dong, X., Liu, Q., et al., 2008. Agonists of the MAS-related gene (Mrgs) orphan receptors as novel mediators of mast cell-sensory nerve interactions. J. Immunol. 180, 2251–2255. Maguire, J.J., Davenport, A.P., 2013. Trace Amine Receptor, Introductory Chapter. IUPHAR Database (IUPHAR-DB). (last modified 19.04.13). Marchese, A., Docherty, J.M., Nguyen, T., et al., 1994. Cloning of human genes encoding novel G protein-coupled receptors. Genomics 23, 609–618. Marchese, A., Nguyen, T., Malik, P., et al., 1998. Cloning genes encoding receptors related to chemoattractant receptors. Genomics 50, 281–286. Martin, C., Michel., Annette Beck-Sickinger, William, F., Colmers, Helen M., Cox, Henri, N., Doods., Herbert, Herzog., Dan, Larhammar., Remi, Quirion., Thue, Schwartz., Thomas Westfall., 2008. Neuropeptide Y Receptors, Introduction. IUPHAR database (IUPHAR-DB). (last modified 27.10.08). Matsumoto, M., Saito, T., Takasaki, J., et al., 2000. An evolutionarily conserved Gprotein coupled receptor family, SREB, expressed in the central nervous system. Biochem. Biophys. Res. Commun. 272, 576–582. Matsuo, A., Matsumoto, S., Nagano, M., et al., 2005. Molecular cloning and characterization of a novel Gq-coupled orphan receptor GPRg1 exclusively expressed in the central nervous system. Biochem. Biophys. Res. Commun. 331, 363–369. Metpally, R.P., Sowdhamini, R., 2005a. Cross genome phylogenetic analysis of human and Drosophila G protein-coupled receptors: application to functional annotation of orphan receptors. BMC Genom. 6, 1–20. Metpally, R.P.R., Sowdhamini, R., 2005b. Genome wide survey of G protein coupled receptors in Tetraodon nigroviridis. BMC Evol. Biol. 5, 41. Meyer, R.C., Giddens, M.M., Schaefer, S.A., Hall, R.A., 2013. GPR37 and GPR37L1 are receptors for the neuroprotective and glioprotective factors prosaptide and prosaposin. Proc. Natl. Acad. Sci., USA 110, 9529–9534. Mollereau-Manaute, C., Roumy, M., Zajac, J.M., 2013. Neuropeptide FF/ Neuropeptide AF Receptors: NPFF1 Receptor. IUPHAR Database (IUPHAR-DB). (last modified 11.03.13). Monk, P., Woodruff, T., Coulthard, L., Hawksworth, O., 2013. Complement Peptide Receptors, Introductory Chapter. IUPHAR Database (IUPHAR-DB). (last modified 15.02.13). Mori, K., Miyazato, M., Ida, T., et al., 2005. Identification of neuromedin S and its possible role in the mammalian circadian oscillator system. EMBO J. 2005 (24), 325–335. Mori, M., Sugo, T., Abe, M., et al., 1999. Urotensin II is the endogenous ligand of a Gprotein-coupled orphan receptor, SENR (GPR14). Biochem. Biophys. Res. Commun. 265, 123–129. Mouillac, B., Bichet, D., Bouvier, M., Chini, B., Gimpl, G., et al., 2012. Vasopressin and oxytocin receptors, introduction. (last modified 06.06.12). Murphy, P.M., Charo, I.F., Horuk, R., Matsushima, K., Oppenheim, J.J., 2013. Chemokine Receptors, Introductory Chapter. IUPHAR Database (IUPHAR-DB). (last modified 10.07.13). Narumiya, S., Sugimoto, Y., Ushikubi, F., 1999. Prostanoid receptors: structures, properties, and functions. Physiol. Rev. 79, 1193–1226. Njuki, F., Nicholl, C.G., Howard, A., et al., 1993. A new calcitonin-receptor-like sequence in rat pulmonary blood vessels. Clin. Sci. (Lond.) 85, 385–388. O’Dowd, B.F., Nguyen, T., Marchese, A., et al., 1998. Discovery of three novel Gprotein-coupled receptor genes. Genomics 1998 (47), 310–313. Offermanns, S., Colletti, S.L., Lovenberg, T.W., Semple, G., Wise, A., IJzerman, A.P., 2011. International union of basic and clinical pharmacology. LXXXII: nomenclature and classification of hydroxy-carboxylic acid receptors (GPR81, GPR109A, and GPR109B). Pharmacol. Rev. 63, 269–290. Okuno, Y., Tamon, A., Yabuuchi, H., et al., 2008. GLIDA: GPCR – ligand database for chemical genomics drug discovery – database and tools update. Nucleic Acids Res. 36 (Database issue) (D907-12). Overton, H.A., Babbs, A.J., Doel, S.M., et al., 2006. Deorphanization of a G proteincoupled receptor for oleoylethanolamide and its use in the discovery of smallmolecule hypophagic agents. Cell. Metab. 3, 167–175. Papadopoulos, D.P., Mourouzis, I., Faselis, C., et al., 2013. Masked hypertension and atherogenesis: the impact of apelin and relaxin plasma levels. J. Clin. Hypertens. (Greenwich) 15, 333–336. Pei, J., Grishin, N.V., 2007. PROMALS: towards accurate multiple sequence alignments of distantly related proteins. Bioinformatics 23, 802–808. Pei, J., Tang, M., Grishin, N.V., 2008. PROMALS3D web server for accurate multiple protein sequence and structure alignments. Nucleic Acids Res. 36 (Web Server issue) (W30-4). Pelé, J., Abdi, H., Moreau, M., Thybert, D., Chabbert, M., 2011. Multidimensional scaling reveals the main evolutionary pathways of class A G-protein-coupled receptors. PLoS One 6, e19094. Pertwee, R.G., Howlett, A.C., Abood, M., et al., 2011. Cannabinoid Receptors, Introductory Chapter IUPHAR Database (IUPHAR-DB). (last modified 07.12.11). Pitkin, S.L., Maguire, J.J., Bonner, T.I., Davenport, A.P., 2010. International union of basic and clinical pharmacology. LXXIV. Apelin receptor nomenclature, distribution, pharmacology, and function. Pharmacol. Rev. 62, 331–342.

96

K.K. Kakarala, K. Jamil / Molecular Phylogenetics and Evolution 74 (2014) 66–96

Pradayrol, L., Jornvall, H., Mutt, V., Ribet, A., 1980. N-terminally extended somatostatin: the primary structure of somatostatin-28. FEBS Lett. 109, 55–58. Rasmussen, S.G., DeVree, B.T., Zou, Y., et al., 2011. Crystal structure of the b2 adrenergic receptor-Gs protein complex. Nature 477, 549–555. Robert, L.J, Shuh, N., David, F., Woodward., Mark, Giembycz., Xavier, Norel., 2013. Prostanoid Receptors, Introduction. IUPHAR Database (IUPHAR-DB). (last modified 18.09.13). Russell, R.B., Barton, G.J., 1994. Structural features can be unconserved in proteins with similar folds. An analysis of side-chain to side-chain contacts secondary structure and accessibility. J. Mol. Biol. 244, 332–350. Saeki, T., Ihara, M., Fukuroda, T., Yamagiwa, M., Yano, M., 1991. [Ala1,3,11,15] endothelin-1 analogs with ETB agonistic activity. Biochem. Biophys. Res. Commun. 179, 286–292. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Savi, P., Labouret, C., Delesque, N., Guette, F., Lupker, J., Herbert, J.M., 2001. P2y(12), a new platelet ADP receptor, target of clopidogrel. Biochem. Biophys. Res. Commun. 283, 379–383. Schall, T.J., Jongstra, J., Dyer, B.J., et al., 1988. A human T cell-specific molecule is a member of a new gene family. J. Immunol. 141, 1018–1025. Song, Z.H., Modi, W., Bonner, T.I., 1995. Molecular cloning and chromosomal localization of human genes encoding three closely related G protein-coupled receptors. Genomics 28, 347–349. Southern, C., Cook, J.M., Neetoo-Isseljee, Z., et al., 2013. Screening b-Arrestin recruitment for the identification of natural ligands for orphan G-proteincoupled receptors. J. Biomol. Screen 18, 599–609. Spindel, E.R., 2013. Bombesin peptides. In: Kastin, A.J. (Ed.), Handbook of Biologically Active Peptides. Elsevier, pp. 325–330, ISBN: 9780123694423. Sreedharan, S., Almén, M.S., Carlini, V.P., et al., 2011. The G protein coupled receptor Gpr153 shares common evolutionary origin with Gpr162 and is highly expressed in central regions including the thalamus, cerebellum and the arcuate nucleus. FEBS J. 278, 4881–4894. Studer, R.A., Robinson-Rechavi, M., 2010. Large-scale analysis of orthologs and paralogs under covarion-like and constant-but-different models of amino acid evolution. Mol. Biol. Evol. 27, 2618–2627. Summers, R., Bathgate, R., Ivell, R., Sanborn, B., Sherwood, D., 2009. Relaxin Family Peptide Receptors, Introductory Chapter. IUPHAR Database (IUPHAR-DB). (last modified 13.10.09). Surgand, J.S., Rodrigo, J., Kellenberger, E., Rognan, D., 2006. A chemogenomic analysis of the transmembrane binding cavity of human G-protein-coupled receptors. Proteins 62, 509–538.

Szekeres, P.G., Muir, A.I., Spinage, L.D., et al., 2000. Neuromedin U is a potent agonist at the orphan G protein-coupled receptor FM3. J. Biol. Chem. 275, 20247–20250. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739. Tan, C.P., McKee, K.K., Liu, Q., et al., 1998. Cloning and characterization of a human and murine T-cell orphan G-protein-coupled receptor similar to the growth hormone secretagogue and neurotensin receptors. Genomics 52, 223–229. Tanaka, H., Yoshida, T., Miyamoto, N., et al., 2003. Characterization of a family of endogenous neuropeptide ligands for the G protein-coupled receptors GPR7 and GPR8. Proc. Natl. Acad. Sci. USA 100, 6251–6256. Tanaka, S., Ishii, K., Kasai, K., Yoon, S.O., Saeki, Y., 2007. Neural expression of G protein-coupled receptors GPR3, GPR6, and GPR12 up-regulates cyclic AMP levels and promotes neurite outgrowth. J. Biol. Chem. 282, 10506–10515. Tarttelin, E.E., Kirschner, L.S., Bellingham, J., et al., 1999. Cloning and characterization of a novel orphan G-protein-coupled receptor localized to human chromosome 2p16. Biochem. Biophys. Res. Commun. 260, 174–180. Tatemoto, K., Hosoya, M., Habata, Y., et al., 1998. Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor. Biochem. Biophys. Res. Commun. 251, 247. Tuffley, C., Steel, M., 1998. Modeling the covarion hypothesis of nucleotide substitution. Math. Biosci. 147, 63–91. van der Horst, E., Peironcely, J.E., Ijzerman, A.P., et al., 2010. A novel chemogenomics analysis of G protein-coupled receptors (GPCRs) and their ligands: a potential strategy for receptor de-orphanization. BMC Bioinformat. 11, 316. Walther, A., Riehemann, K., Gerke, V., 2000. A novel ligand of the formyl peptide receptor: annexin I regulates neutrophil extravasation by interacting with the FPR. Mol. Cell. 5, 831–840. Wang, K.Y., Tanimoto, A., Yamada, S., et al., 2010. Histamine regulation in glucose and lipid metabolism via histamine receptors: model for nonalcoholic steatohepatitis in mice. Am. J. Pathol. 177, 713–723. Winrow, C.J., Coleman, P., de Lecea L., et al., 2012. Orexin Receptors, Introductory Chapter. IUPHAR Database (IUPHAR-DB). (last modified 28.02.12). Yamada, M., Iwasaki, T., Satoh, T., et al., 1995. Activation of the thyrotropinreleasing hormone (TRH) receptor by a direct precursor of TRH, TRH-Gly. Neurosci. Lett. 196, 109–112. Yosten, G.L., Kolar, G.R., Redlinger, L.J., Samson, W.K., 2013. Evidence for an interaction between proinsulin C-peptide and GPR146. J. Endocrinol. 218, B1–B8. Zhang, B.L., Gao, D.S., Xu, Y.X., 2013. G protein-coupled receptor 3: a key factor in the regulation of the nervous system and follicle development. Yi. Chuan. 35, 578–586.