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Identification of one capa and two pyrokinin receptors from the malaria mosquito Anopheles gambiae
Biochemical and Biophysical Research Communications 362 (2007) 245–251 www.elsevier.com/locate/ybbrc
Identification of one capa and two pyrokinin receptors from the malaria mosquito Anopheles gambiae q Stine S. Olsen, Giuseppe Cazzamali, Michael Williamson, Cornelis J.P. Grimmelikhuijzen, Frank Hauser * Center for Functional and Comparative Insect Genomics, Department of Cell Biology and Comparative Zoology, Institute of Biology, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen, Denmark Received 28 June 2007 Available online 27 July 2007
Abstract We cloned the cDNA of three evolutionarily related G protein-coupled receptors from the malaria mosquito Anopheles gambiae and functionally expressed them in Chinese hamster ovary cells. One receptor, Ang-Capa-R, was only activated by the two Anopheles capa neuropeptides Ang-capa-1 (GPTVGLFAFPRVamide) and Ang-capa-2 (pQGLVPFPRVamide) with EC50 values of 8.6 · 109 M and 3.3 · 109 M, respectively, but not by any other known mosquito neuropeptide. The second receptor, Ang-PK-1-R, was selectively activated by the Anopheles pyrokinin-1 peptides Ang-PK-1-1 (AGGTGANSAMWFGPRLamide) and Ang-PK-1-2 (AAAMWFGPRLamide) with EC50 values of 3.3 · 108 M and 2.5 · 108 M, respectively, but not by mosquito capa or pyrokinin-2 peptides. For the third receptor, Ang-PK-2-R, the most potent ligands were the pyrokinin-2 peptides Ang-PK-2-1 (DSVGENHQRPPFAPRLamide) and Ang-PK-2-2 (NLPFSPRLamide) with EC50 values of 5.2 · 109 M and 6.4 · 109 M, respectively. However, this receptor could also be activated by the two pyrokinins-1, albeit with lower potency (EC50: 2–5 · 108 M). Because Ang-capa-1 and -2 and Ang-PK-1-1 are located on one preprohormone and the other peptides on another prohormone, these results imply a considerable crosstalk between the capa, pyrokinin-1 and pyrokinin-2 systems. Gene structure and phylogenetic tree analyses showed that Ang-Capa-R was the orthologue of the Drosophila capa receptor CG14575, Ang-PK-1-R the orthologue of the Drosophila pyrokinin-1 receptor CG9918, and Ang-PK-2R the orthologue of the Drosophila pyrokinin-2 receptors CG8784 and CG8795. This is the first report on the functional characterization and crosstalk properties of capa and pyrokinin receptors in mosquitoes. 2007 Elsevier Inc. All rights reserved. Keywords: Malaria; Mosquito; Insect; GPCR; Neuropeptide; Capa; Pyrokinin; PBAN; Ecdysis-triggering hormone; Neuromedin U
Malaria is the most important parasitic disease in the world, causing over 300 million cases of illness and about two million deaths each year [1]. The disease is caused by an infection with Plasmodium falciparum, a parasite that is transmitted by mosquitoes belonging to the genus Anopheles during a blood meal [2]. Any knowledge on
q The sequence data from this paper have been submitted to the GenBank database under Accession Nos. AY900217, AY900218 and AY900219. * Corresponding author. Fax: +45 3532 1200. E-mail address: [email protected] (F. Hauser). URL: http://www.bi.ku.dk/staff/staff-vip-details.asp?ID=91 (F. Hauser).
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.06.190
the behavior and physiology of Anopheles, therefore, might help to interfere with the transmission of malaria. In the recently sequenced Anopheles gambiae genome, 35 preprohormone genes encoding about 70 neuropeptides, and 25 neuropeptide receptor genes belonging to the large family of G protein-coupled receptors (GPCRs) have been annotated [3–5]. These neuropeptide receptors steer important physiological processes such as development, reproduction and behavior. During the last six years, we and others have identified the endogenous ligands for about 25 neuropeptide receptors from Drosophila melanogaster [6,7], the insect with the first sequenced genome [8]. Examples of them are the fly receptors for capa and pyrokinin neuropeptides schematically presented in Fig. 1A.
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A
Capa-1
Capa-2
Drm-Capa-R
B
Capa-1
Drm-PK-1-R
Capa-2
Ang-Capa-R
PK-1
PK-1-1
Ang-PK-1-R
Hug-γ
Drm-PK-2-R1 PK-1-2
PK-2-1
PK-2
Drm-PK-2-R2 PK-2-2
PK-RP
Ang-PK-2-R
Fig. 1. Schematic representation of the capa/pyrokinin signalling system in Drosophila (A) and Anopheles (B). The capa and pyrokinin preprohormones are drawn as long open boxes, the neuropeptides contained therein are indicated by black boxes (not drawn to scale). (A) The four Drosophila capa/ pyrokinin receptors are Drm-Capa-R (CG14575), Drm-PK-1-R (CG9918), Drm-PK-2-R1 (CG8784), and Drm-PK-2-R2 (CG8795). (B) The three Anopheles capa/pyrokinin receptors are Ang-Capa-R, Ang-PK-1-R, and Ang-PK-2-R (this paper). A thick, red arrow indicates a peptide activating a receptor with an EC50 value of <1 · 108 M. A black arrow indicates a peptide activating a receptor with an EC50 value of 1–15 · 108 M. Stippled black arrows indicate peptides activating a receptor with an EC50 value of 15–100 · 108 M. All the indicated ligand/receptor interactions are derived from our functional assay data, but might not necessarily reflect the in vivo situation due to anatomical restrictions.
In Drosophila, there are two preprohormones (Fig. 1A), one yielding a capa-1, a capa-2, and a pyrokinin-1 (PK-1) peptide [9], and one yielding a pyrokinin-2 (PK-2) and a Hug-c peptide [10]. All these peptides are individual members of the PRL/I/Vamide neuropeptide family. Capa-1 and capa-2 have the C-terminal capa consensus sequence FPRVamide [9,11]. PK-1 has the C-terminal pyrokinin-1 consensus sequence WFGPRLamide [12], and PK-2 has the C-terminal pyrokinin-2 consensus sequence PFXPRLamide [10,12,13]. Hug-c has the C-terminal sequence PRLamide, but is not a pyrokinin senso stricto, because a pyrokinin has been defined by the C-terminal sequence FXPRLamide [10,14]. Drosophila capa-1 and capa-2 specifically activate their own capa receptor (Fig. 1A) [15]. Drosophila PK-1 only activates its PK-1 receptor [12], whereas PK-2 and Hug-c activate the two Drosophila PK-2 receptors (Fig. 1A) [13]. There is crosstalk between these ligand/receptor couples, because PK-2 and Hug-c slightly interact with the PK-1 receptor (Fig. 1A). Also, PK-1 is located on the same prohormone as the two capa peptides, implying that the PK-1 and capa receptors are simultaneously stimulated (Fig. 1A). Because the Drosophila capa/pyrokinin system is rather complex, we wanted to investigate the capa/ pyrokinin system in mosquitoes to see whether our findings in Drosophila are a general phenomenon in insects.
In Anopheles, the capa preprohormone has a similar organization as in Drosophila with two capa and a pyrokinin-1 (PK-1-1) neuropeptide (Fig. 1B) [4]. The pyrokinin-2 preprohormone has been expanded, containing two pyrokinins-2 (PK-2-1 and PK-2-2), one pyrokinin-1 (PK1-2), and one pyrokinin-related peptide (PK-RP). The structures of these Anopheles neuropeptides are given in Table 1. Table 1 also contains the structures of two Anopheles ecdysis-triggering hormones (ETHs). These peptides with the C-terminal sequence VPRIamide resemble capa and pyrokinin neuropeptides but have their own preprohormones [16] and, in Drosophila, their own specific receptors [17]. In the present paper, we report the molecular cloning and functional characterization of one capa and two pyrokinin receptors from the mosquito A. gambiae and the crosstalk that occurs between the receptor/ligand couples. Materials and methods Total RNA from adult A. gambiae (strain KWA) was isolated using TRIzol reagent (Invitrogen). The isolated total RNA was treated with DNase using the DNA-free kit (Ambion, Inc.). cDNA was synthesized using the SMART RACE cDNA Amplification kit (Clontech). PCR was performed using the Herculase Hotstart DNA polymerese (Stratagene).
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Table 1 Amino acid sequences of structurally related Anopheles neuropeptides and their potencies on the indicated receptors Name Ang-Capa-1 Ang-Capa-2 Ang-PK-1-1 Ang-PK-1-2 Ang-PK-2-1 Ang-PK-2-2 Ang-PK-RP Ang-ETH-1 Ang-ETH-2
Structure GPTVGLFAFPRVamide pQGLVPFPRVamide AGGTGANSAMWFGPRLamide AAAMWFGPRLamide DSVGENHQRPPFAPRLamide NLPFSPRLamide KPQPIFYHTTSPRLamide SESPGFFIKLSKSVPRIamide GDLENFFLKQSKSVPRIamide
EC50 (M) Ang-CapaR 9
8.6 · 10 3.3 · 109 na na na na na na na
EC50 (M) Ang-PK-1R
EC50 (M) Ang-PK-2R
na na 3.3 · 108 2.5 · 108 >106 >106 na na na
na na 5.0 · 108 2.5 · 108 5.2 · 109 6.4 · 109 1.6 · 107 >106 na
na, not active in concentrations up to 105 M. Bold highlights the pyrokinin consensus sequence (FXPRLamide) or the C-terminal PRL/I/Vamide sequence.
The sequences of previously identified capa and pyrokinin receptors from D. melanogaster [12,13,15] were used for BLAST searching of the genomic DNA database from A. gambiae (http://www.ncbi.nlm.nih.gov/ genome/seq/BlastGen/BlastGen.cgi?taxid=7165). Based on the identified sequences, PCR primers were designed as described in Supplementary Materials and methods. All PCR products were cloned into pCR4-TOPO plasmids (Invitrogen) using the TOPO TA cloning kit and sequenced. PCR products of the coding regions were subcloned into the pIRES2EGFP expression vector (Clontech) using the Rapid DNA Ligation Kit (Roche Applied Science). CHO cells stably expressing the human G-protein G16 (CHO/G16) were grown and transfected as described previously [18], the bioluminescence assay was performed as described in [19]. The Anopheles peptides shown in Table 1 were synthesized by GeneMed (San Francisco, CA). DNA sequence comparisons were carried out using the Lasergene software package (DNASTAR, Inc.). Protein sequence alignments were carried out using ClustalW (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_clustalw.html). Prediction of transmembrane helices of the receptor protein was done using the TMHMM server (http:// www.cbs.dtu.dk/services/TMHMM/). EC50 values were calculated using Prism software.
Results By BLAST searching of the A. Gambiae DNA database [3] with Drosophila capa and pyrokinin receptor sequences [12,13,15], we identified several DNA fragments coding for putative Anopheles orthologues. Using this information, we cloned the cDNAs coding for an Anopheles capa receptor (Ang-Capa-R) and two Anopheles pyrokinin receptors (Ang-PK-1-R and Ang-PK-2-R, Fig. S1–S3, supporting material). All three cDNAs contained at least one in-frame stop codon preceding the ATG start codon in their 5 0 -untranslated regions and two of them (Ang-PK-1-R and Ang-PK-2-R) contained a polyadenylation signal in their 3 0 -untranslated regions. Compared to the genomic DNA sequences, our cloned cDNA sequences showed 1–2% nucleotide differences (Tables S1–S3, supporting material). Some of these differences, which are probably due to allelic variations, also changed individual amino acid residues of the encoded protein sequences (Table S1– S3, supporting material). These comparisons also revealed that the Ang-Capa-R gene contained six introns, the AngPK-1-R gene two introns and the Ang-PK-2-R gene five introns (Tables S4–S6, supporting material).
The amino acid sequences of the three encoded proteins are shown in Fig. S1–S3 (supporting material). Fig. 2 gives an alignment of these three receptors together with the Drosophila capa receptor CG14575 [15], the Drosophila pyrokinin-1 receptor CG9918 [12] and the two Drosophila pyrokinin-2 receptors CG8784 and CG8795 [13]. This alignment shows large blocks of identical amino acid residues conserved between the two Diptera species, i.e., conserved over an evolutionary distance of about 250 million years [20]. The three Anopheles receptors show typical features of rhodopsin-like (family A or 1) GPCRs, such as seven transmembrane a-helices containing highly conserved sequence motifs and the canonical D/ERF/Y (Asp/Glu-Arg-Phe/Tyr) motif immediately following the third transmembrane a-helix (Fig. 2). In order to identify and characterize the endogenous ligands for these three receptors, we stably expressed their coding regions individually in CHO–G16 cells. We tested these transfected cell lines with our library of eight biogenic amines and 33 invertebrate neuropeptides [21] together with the nine Anopheles neuropeptides shown in Table 1 using our bioluminescence assay [18,19]. Ang-Capa-R showed very high selectivity and was only activated by the two Anopheles peptides Capa-1 and Capa2 (EC50 values of 8.6 · 109 M and 3.3 · 109 M, respectively; Table 1 and Fig. 3). Ang-PK-1-R was activated by low concentrations of the two pyrokinins Ang-PK-1-1 and Ang-PK-1-2 (EC50 values of 3.3 · 108 M and 2.5 · 108 M, respectively; see Table 1 and Fig. S4). Ang-PK-2-R was rather promiscuous. The most potent ligands were Ang-PK-2-1 and Ang-PK-2-2 (EC50 values of 5.2 · 109 M and 6.4 · 109 M, respectively; see Table 1 and Fig. S5). However, this receptor was also activated by the two Anopheles pyrokinins-1, Ang-PK-1-1, and Ang-PK-1-2, although 4- to 10-fold higher concentrations of these peptides were required (EC50 values of 5.0 · 108 M and 2.5 · 108 M, respectively; see Table 1). Also the pyrokinin-related peptide Ang-PK-RP could activate Ang-PK-2-R, but only at rather high concentrations (EC50 value of 1.6 · 107 M; see Table 1). All the other tested ligands showed no activity on this receptor.
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S.S. Olsen et al. / Biochemical and Biophysical Research Communications 362 (2007) 245–251 Ang-Capa-R Drm-Capa-R Ang-PK-1-R Drm-PK-1-R Ang-PK-2-R Drm-PK-2-R1 Drm-PK-2-R2
Ang-Capa-R Drm-Capa-R Ang-PK-1-R Drm-PK-1-R Ang-PK-2-R Drm-PK-2-R1 Drm-PK-2-R2
METVVPNATALAWPMSSTVPAMLLPDSSYASFPTG---GVTMSLDVLLG------LGLFDANGTGTENATGDPLEPPHPCDPSSDQFD------MN--------SSTDPTFSELNASFTNTP-----------DT-----------LFATS------VSSDPSHGFGEEDYACGTFN-------------------------------------------MSTSLKN--------MPYEL------LE----ILTTDNESILSDG----------------------------------------------MSAGN--------MSHD-------L--------------------MMELSPTVGSSGSLLDAL----------ITN-----------GTNGRTG--------LLHDI------LDGHRTLLHQSQQQQQQHYNHP MLQGVAITIANDSNDDGLNQSFMAHVSPSPNQSPSIGVGIGIASSTMANPSESPEMLLLKNDKF----LTHVAHLLNITTENLSNLLG-MLP--------------------------TN-----------SSGVLAT-----DLQLFHNEKF----LLNLTQVLNISADNLTSLL--TM I TM II -------------CSVDEFLVYARGPQRMPLFTAMLVTVLFVGILVTGVIGNLIVCLVIVRHPSMRTATNYYLFSLAVSDLIFLLLGLPY -------------CSPKEFVAFVLGPQTLPLYKAVLITIIFGGIFITGVVGNLLVCIVIIRHSAMHTATNYYLFSLAVSDLLYLLFGLPT ----------------VESLTEMYGPKRDPLYVVIPITIIYLLIFITGVVGNISTCIVIARNRSMHTATNYYLFSLAVSDFLLLVSGVPQ ------------------------GPPRDPLAIVIPVTVVYSLIFITGVVGNISTCIVIKKNRSMHTATNYYLFSLAISDFLLLLSGVPQ HHQQYQPQYLYANESLAGSVPESASYGTDSICIILPITIFYCFIFVAGIVGNLSICVVIAKNKSMHTATNYYLFNLAVSDFLLLLFGMPQ -----------STNGTNASTMAADSPVDESLTLRTALTVCYALIFVAGVLGNLITCIVISRNNFMHTATNFYLFNLAVSDLILLVSGIPQ -------------QGLEPEELLPTVIPMTPLSLLATLSVGYALIFIAGVLGNLITCIVISRNNFMHTATNFYLFNLAISDMILLCSGMPQ TM III TM IV EISLYWHQYPYNLGLPFCKIRALISEASTYVSVLTIVAFSMERFLAICHPLHLYTMSGLQRPVRIIAGLWVVSLLSAVPFAVFTDIDYIA EVFLYWHQYPDLFGMPFCKIRAFISEACTYVSVFTIVAFSMERFLAICHPLHLYAMVGFKRAIRIITALWIVSFISAIPFGLLSDIQYLN EIYFIWSKYPYVFGETFCVLRGIAAEMSANATVLTITAFTIERYFAICHPFLSHTMSKLSRAVRFICVIWLIAIVSAIPQALQFGVTNQG EVSYIWSKYPYVFGEYICIGRGLLAETSANATVLTITAFTVERYIAICHPFLGQAMSKLSRAIRIIVLVWIMAIVTAIPQAAQFGIEHYS ELYGTWNPFAYPFNQIACIIMGLLSETAANATVLTITSFTVERYIAICHPFRSHTMSKLSRAIKFVIVIWLVAFGLATPQALQFGVVETN ELYNLWYPDMYPFTDAMCIMGSVLSEMAANATVLTITAFTVERYIAICHPFRQHTMSKLSRAIKFIFAIWLAAFLLALPQAMQFSVVYQN DLYNLWHPDNYPFSDSICILESVLSETAANATVLTITAFTVERYIAICHPFRQHTMSKLSRAVKFIFAIWIAALLLALPQAIQFSVVMQG TM V YPPTNEKILDSAFCAMLSNPE-GFPLWELSTCLFFAFPMLIMVVLYGRMGMQIRSRTQRTAELGVRNGS--------------------YPLDHSRIEESAFCSMSPKIVNEIPVFEVSFCIFFVIPMILIILLYGRMGAKIRSRTN--QKLGVQQGT--------------------G---------IDQCVVKRIII--QHSFELSTFLFFFAPMTMITILYALIGLKLRTSTLMQRDGTLQRR---------------------G---------VEQCGIVRVIV--KHSFQLSTFIFFLAPMSIILVLYLLIGVHLYRSTLVEGPASVARRQQLKSVPSDTILYRYGGSGTAM T---------TRLCTIKNEHF--SHAFEVSSFLFFVGPMTLIAVLYVLIGIKLRKSKLLQGVKRASSD---------------------E---------GYSCTMENDFY--AHVFAVSGFIFFGGPMTAICVLYVLIGVKLKRSRLLQSLPRRTFD---------------------M---------GTSCTMKNDFF--AHVFAVSGFLFFGGPMTAICVLYVLIGVKLKRSRLLQALPRRCYD---------------------TM VI ------------------------VNGPSRISQSRK-AIIRMLAAVVITFFVCWAPFHAQRLLFLYAR-------DWQHFNT-VNTWLFS ------------------------NNRETRNSQMRKKTVIRMLAAVVITFFVCWFPFHLQRLIFLYAK-------NMDNYLD-INEALFS -------------------TQPSPRQSFANSQGSRR--VLKMLVAVVVAFFICWAPFHAQRLVYIYGVNT--NHQPSDPLILKLFIITTY SFNGGGSGAGTAGLMGGSGAQLSSVRGRLNHYGTRR--VLRMLVAVVVCFFLCWAPFHAQRLIAIYAPAR--GAKLRDQHEF-VYTVMTY --------------------YTTPPRGIS--AQSR---VIRMLVAVVATFFICWAPFHAQRLMAVYGVFS----KTENVFFYKVYMVLNY -----------------------ANRGLN--AQGR---VIRMLVAVAVAFFLCWAPFHAQRLMAVYGLNLIN-IGISRDAFNDYFRILDY -----------------------VNRGIS--AQTR---VIRMLVAVAVAFFICWAPFHAQRLMAVYGS---T-SGIESQWFNDVFSILDY TM VII VAGWLYYVSCTINPILYNVMSHRYRVAFRETLCGRRRG-----FGAGFARDQSSFRET-------------------------------IAGFAYYVSCTVNPIVYSVMSRRYRVAFRELLCGKAVGAY---YNSGFARDHSSFRESS---AYDRVH---------------------ISGILYYLSTCINPLLYNIMSNKFRQAFKNMLT--------------------------------------------------------VSGVLYYLSTCINPLLYNIMSHKFREAFKAVLFGKKVS-----KGSLNSRNNIESRRLR--RALT------------------------TSGILYFLSTCINPLLYNIMSHKFRDASRHTLKMSCCGGK---RSKSDGQHHTYSAVSR--YGVTGCGSFKVNSNNMACIGAGIGLGTGG TSGVLYFLSTCINPLLYNIMSHKFREAFKITLTRQFGLARNHHHQQSQHHQHNYSALLR-QNGSMRLQPASCSVNNNALEPYGSYRVVQF TSGVLYFLSTCINPLLYNIMSHKFREAFKVTLARHFGLGG---KNQGRGLPHTYSALRRNQTGSLRLHTTD-----SVRTT-----MTSM
Ang-Capa-R Drm-Capa-R Ang-PK-1-R Drm-PK-1-R Ang-PK-2-R Drm-PK-2-R1 Drm-PK-2-R2
----------------TIDVNTAGGGNGYGTG-----------------------------------YESSRLVSVDCTTQAQNSR-------------------SVHVRASQHPNKFETDS-----------SSANRVLIKKT----YSLPLPKNADSTVLSTTDIVIVLENSHTVCE -------------------------------------------VRSAR-------------KPPWQTSYGASPPHGSTWTG--------------------------------NSSQTQRFS----------IESAEQ-----------PKPSIMQNPTNKPPVAAQYAMIGVQVN--GNAASKQQHQQPGNGTAATPNQCQQESNMSLLA----------NESCNRGTMPPI----AIRPTFTRADSHCISISSSQSTTGTNVSSNG RCRDANHQLSLQDSIRTTTTTTTINSNSMAAGNGVGGGAGGGGRRLRKQELYGPVPGTAVPHRMLQAQVSQLSSLGDANSLLEAEVVDRH ATTTTGLNGSANGSGNGTTTGQSVRLNRVSLDS-----VQMQGQNRSRQDLFD------NPRRMLQTQISQLSSVGDAHSLLEEDLQFPG
459 460 373 430 514 550 479
Ang-Capa-R Drm-Capa-R Ang-PK-1-R Drm-PK-1-R Ang-PK-2-R Drm-PK-2-R1 Drm-PK-2-R2
PPGGGKWIGLVVEQ---------------------------------------------------------------------------EPKVENDIWIENEETCI----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------KLSRSSNGSLRHGGSALEQPTDHAAGGGGGGGRLST---IAEKLRRGTKKVLAFSKSPTGTPSRSIVSGACAEGTNGRRKAYRKRNSCDS YASGRAKRALLATKSGALLVTPPQSGDPSEVSQPATRLKLTRVISRRDEVANASTPPFCGSHS----------------LPDPETCQSAS EPLQRQPTMCSIDELTDDLAISRSRLKLTRITRPPG--GVTGGVAGGSTTVAAGSGGVSGDESSGKV-----------RKAKVKVLKSSS
473 477
Ang-Capa-R Drm-Capa-R Ang-PK-1-R Drm-PK-1-R Ang-PK-2-R Drm-PK-2-R1 Drm-PK-2-R2
--------------------------------------------------------------------------------------------------------------------------------------------------------VDTNTISNSSLKEFDEEEFSSAELARFMGEVNSEIR--VAGRSSRKFPWRKRRQKTEDPSSEGLTYGSPKSQ----PFKGLRTKFNWRARRKGSHKPHEKGATVNGGDTEERAAF
Ang-Capa-R Drm-Capa-R Ang-PK-1-R Drm-PK-1-R Ang-PK-2-R Drm-PK-2-R1 Drm-PK-2-R2 Ang-Capa-R Drm-Capa-R Ang-PK-1-R Drm-PK-1-R Ang-PK-2-R Drm-PK-2-R1 Drm-PK-2-R2 Ang-Capa-R Drm-Capa-R Ang-PK-1-R Drm-PK-1-R Ang-PK-2-R Drm-PK-2-R1 Drm-PK-2-R2 Ang-Capa-R Drm-Capa-R Ang-PK-1-R Drm-PK-1-R Ang-PK-2-R Drm-PK-2-R1 Drm-PK-2-R2
79 47 27 10 55 84 41 156 124 101 76 145 163 118 246 214 191 166 235 253 208 314 281 248 245 292 310 265 371 339 315 330 353 371 323 424 401 348 388 438 460 400
601 624 556
637 658 595
Fig. 2. Protein sequence alignment of the Anopheles capa receptor (Ang-Capa-R; Accession No. AY900217), the Drosophila capa receptor (Drm-Capa-R; CG14575), the Anopheles pyrokinin-1 receptor (Ang-PK-1-R; Accession No. AY900218), the Drosophila pyrokinin-1 receptor (Drm-PK-1-R; CG9918), the Anopheles pyrokinin-2 receptor (Ang-PK-2-R, Accession No. AY900219) and the two Drosophila pyrokinin-2 receptors (Drm-PK-2-R1; CG8784 and Drm-PK-2-R2; CG8795). Spaces are introduced to optimize alignments. The seven transmembrane a-helices are indicated by TM1–TM7. Amino acid residues that are identical in at least four of the seven receptor sequences are highlighted in grey. Intron positions are indicated by boxes.
As seen in the amino acid sequence alignment (Fig. 2), the three Anopheles receptors are structurally and evolutionarily closely related to their Drosophila orthologues.
There is 45% sequence identity (59% similarity) between Ang-Capa-R and the Drosophila capa receptor CG14575 [15] and all six introns found in the mosquito gene are con-
S.S. Olsen et al. / Biochemical and Biophysical Research Communications 362 (2007) 245–251
1.0
0 -5 sec. 5 -10 sec. 10 -15 sec.
10000
L/L max
Luminescence
15000
249
Capa-1 Capa-2
0.5
5000
0.0 -13 -12 -11 -10 -9 -8 -7 -6 -5 Log M
0 A
B
C
D
Fig. 3. Functional assay of the Ang-Capa-R. The left panel shows the bioluminescence responses of non-transfected CHO–G16 cells (A,B) and of CHO– G16 cells expressing Ang-Capa-R (C,D), 0–5 s (black), 5–10 s (grey), and 10–15 s (white) after addition of 106 M Ang-capa-1 (A,C) and Ang-capa-2 (B,D). The right panel shows the dose–response curve of a cloned CHO–G16 cell line expressing Ang-Capa-R to Ang-Capa-1 (EC50, 8.6 · 109 M) and Ang-Capa-2 (EC50, 3.3 · 109 M) using the same assay. In all panels, the SEMs are given as vertical bars, which are sometimes smaller than the symbols used. In these cases only the symbols are given.
Drm-PK-2-R1 (CG8784) Drm-PK-2-R2 (CG8795) Ang-PK-2-R Hez-PBAN-R Bom-PBAN-R Ang-PK-1-R Drm-PK-1-R (CG9918) Ang-Capa-R Drm-Capa-R (CG14575) Mum-NMU-R1 Ang-ETH-R Drm-ETH-R (CG5911) Drm-MS-R1 (CG13803)
178.4 160
140
120
100
80
60
40
20
0
Fig. 4. A phylogenetic tree analysis of the seven receptors from Fig. 1, as well as the PBAN receptors from Helicoverpa zea (Hez-PBAN-R) [19] and Bombyx mori (Bom-PBAN-R) [20], the eclosion triggering hormone receptor from Drosophila (Drm-ETH-R; CG5911) [13], the orthologue receptor from Anopheles (Ang-ETH-R) [Andersen et al., unpublished] and the mouse neuromedin U receptor 1 (Mum-NMU-R1) [21]. The Drosophila myosuppressin receptor (Drm-MS-R1; CG13803) [23] was used as an outgroup.
served in fly (Fig. 2). Ang-PK-1-R shows 46% sequence identity (57% similarity) to the Drosophila pyrokinin-1 receptor CG9918 [12], and these two receptor genes have both introns present in their coding regions in common (Fig. 2). Finally, Ang-PK-2-R has 35% and 38% sequence identity (46% and 49% similarity) with the Drosophila pyrokinin-2 receptors CG8784 and CG8795, respectively [13], and these three receptor genes share all five introns found in the mosquito gene. These close relationships are further supported by a phylogenetic tree analysis using the seven receptors from Fig. 2 together with the characterized pheromone biosynthesis activating neuropeptide (PBAN) receptors from Helicoverpa zea [22] and Bombyx mori [23], the ETH receptor from Drosophila [17] and its orthologue from Anopheles [Andersen et al., unpublished], and the mouse neuromedin U receptor 1 [24] (Fig. 4). The insect PK-1 and PK-2 receptors clearly form two separate clusters. These clusters show, together with the cluster of PBAN receptors, higher homologies to each other than to the capa receptors, which
form a separate branch. Another, even more distantly related branch is formed by the insect ETH receptors. Finally, the mouse neuromedin U receptor is clearly evolutionary related to the insect capa and pyrokinin receptors as observed earlier [25]. Discussion It is very important to understand the biology of the malaria mosquito A. gambiae to be able to prevent transmission of malaria. Important aspects of the mosquito’s biology, such as behavior, reproduction and development, are steered by neurohormones and their receptors. Selective interference with these endocrine systems, for example, by the development of highly selective non-peptide neurohormone receptor antagonists or agonists, could greatly reduce the populations of A. gambiae. This would be an important contribution to the fight against malaria. The first step for such a project is the characterization of A. gambiae neurohormone GPCRs and an understanding of
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their physiology. We and other research groups have previously characterized the A. gambiae GPCRs for myosuppressin [26], adipokinetic hormone [21], corazonin [21], neuropeptide F [27], and short neuropeptide F [28]. The present paper describes the cloning and functional expression of three other, closely related, GPCRs from Anopheles. Based on our functional analysis (Fig. 3; Figs. S4, S5, supporting material) we named the three receptors Ang-CapaR, Ang-PK-1-R, and Ang-PK-2-R. A summary of our results is given in Table 1 and Fig. 1B. It is interesting that these results from Anopheles strongly resemble the situation in Drosophila. Both in Drosophila (Fig. 1A) and in Anopheles (Fig. 1B) there is one capa prohormone, each containing two copies of a capa peptide (capa-1 and capa-2) that stimulate a specific capa receptor that does not react with other neuropeptides. In both Drosophila and Anopheles the capa preprohormone contains a pyrokinin with the C-terminal WFGPRLamide sequence, that we call pyrokinin-1 (PK-1; [12]). In both insects this PK-1 activates a specific PK-1 receptor that is different from the capa receptor (Fig. 1). In addition to the capa preprohormone, both insects have a pyrokinin preprohormone. In Drosophila, this precursor protein contains a pyrokinin with the C-terminal PFXPRLamide sequence that we call pyrokinin-2 (PK-2; [12,13]). In Anopheles, the gene region coding for the pyrokinin peptides has apparently been duplicated twice, giving rise to two pyrokinins-2 (PK-2-1 and PK-2-2) and one pyrokinin-1 (PK-12). In both insects the pyrokinins-2 activate selectively and at very low concentrations (indicated by red arrows in Fig. 1) their own PK-2 receptors, which are two receptors in Drosophila and one in Anopheles (Fig. 1). There is apparently more crosstalk in Anopheles than in Drosophila between the two peptide cocktails derived from the two precursor proteins and the three receptor subtypes. The two Anopheles pyrokinins-1 (PK-1-1 and PK-1-2), derived from two different preprohormones, activate the pyrokinin-1 and pyrokinin-2 receptors equally well, while this cross-reactivity of pyrokinin-1 on the pyrokinin-2 receptor is absent in Drosophila (Fig. 1). Interestingly, the two Anopheles pyrokinins-1 share the C-terminal eight amino acid sequence (Table 1). As mentioned above, PK1-2 apparently originated from a gene region coding for a PK-2 peptide. This suggests that there must have been evolutionary pressure in the evolutionary line leading to mosquitoes to convert a PK-2 into a PK-1 sequence and to integrate signalling from both preprohormones on the PK-1 receptor. An additional crosstalk might be mediated by the pyrokinin-related peptide, Hug-c, in Drosophila (Fig. 1A). Hug-c has a high affinity to the pyrokinin-2 receptors, but also low affinity to the pyrokinin-1 receptor. This last interaction might be a substitute for the crosstalk mediated by the two pyrokinins-1 in Anopheles, which is lacking in Drosophila. The same is true for the Drosophila pyrokinin-2, which has a low affinity for the Drosophila pyrokinin-1 receptor.
Many neurohormone systems in insects are constructed in such a way that there is one preprohormone, of which the products (the peptide cocktail) activate one or more receptors [6,7]. Already the presence of two or more receptors suggests that several physiological processes are synchronized or coordinated. Such an extreme crosstalk as illustrated in Fig. 1, however, has never been seen, so far, in insects. The only system that comes close to it, is the myosuppressin/FMRFamide system in Drosophila, where one copy of the neuropeptide myosuppressin activates the two myosuppressin receptors and, with a somewhat lower affinity, the FMRFamide receptor [29,30]. The numerous FMRFamide peptides, however, do not activate the two myosuppressin receptors [30]. What potential contributions could our current results have for malaria control? Female mosquitoes need one or more blood meals to collect enough proteins for egg formation. During this blood meal, their total body weight increases several times. However, already during and immediately after blood feeding, large amounts of liquid are secreted by the animal to allow for proper flying and escape. Insect capa peptides and capa receptors are controlling fluid secretion by the Malpighian tubes, also in mosquitoes [31]. Capa receptors are, therefore, interesting targets for the development of a selective and environmentally friendly anti-mosquito insecticide. The same might be true for the pyrokinin receptors, because the pyrokinin (PK-1-1) release is physiologically connected to the release of the capa peptides (Fig. 1B). A disrupted liquid homeostasis in mosquitoes will stop malaria mosquito reproduction and disrupt the transmission of malaria. Acknowledgments We thank the Danish Research Agency (Research Council for Nature and Universe), and Novo Nordisk Foundation for financial support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2007. 06.190. References [1] R.W. Snow, C.A. Guerra, A.M. Noor, H.Y. Myint, S.I. Hay, The global distribution of clinical episodes of Plasmodium falciparum malaria, Nature 434 (2005) 214–217. [2] A.F. Cowman, B.D. Crabb, Invasion of red blood cells by malaria parasites, Cell 124 (2006) 755–766. [3] R.A. Holt et al., The genome sequence of the malaria mosquito Anopheles gambiae, Science 298 (2002) 129–149. [4] M.A. Riehle, S.F. Garczynski, J.W. Crim, C.A. Hill, M.R. Brown, Neuropeptides and peptide hormones in Anopheles gambiae, Science 298 (2002) 172–175. [5] C.A. Hill, A.N. Fox, R.J. Pitts, L.B. Kent, P.L. Tan, M.A. Chrystal, A. Cravchik, F.H. Collins, H.M. Robertson, L.J. Zwiebel, G proteincoupled receptors in Anopheles gambiae, Science 298 (2002) 176–178.
S.S. Olsen et al. / Biochemical and Biophysical Research Communications 362 (2007) 245–251 [6] F. Hauser, M. Williamson, G. Cazzamali, C.J.P. Grimmelikhuijzen, Identifying neuropeptide and protein hormone receptors in Drosophila melanogaster by exploiting genomic data, Brief. Funct. Genomics Protomics 4 (2006) 321–330. [7] F. Hauser, G. Cazzamali, M. Williamson, W. Blenau, C.J.P. Grimmelikhuijzen, A review of neurohormone GPCRs present in the fruitfly Drosophila melanogaster and the honey bee Apis mellifera, Prog. Neurobiol. 80 (2006) 1–19. [8] M.D. Adams et al., The genome sequence of Drosophila melanogaster, Science 287 (2000) 2185–2195. [9] L. Kean, W. Cazenave, L. Costes, K.E. Broderick, S. Graham, V.P. Pollock, S.A. Davies, J.A. Veenstra, J.A. Dow, Two nitridergic peptides are encoded by the gene capability in Drosophila melanogaster, Am. J. Physiol. Regul. Integr. Comp. Physiol. 282 (2002) R1297– R1307. [10] X. Meng, G. Wahlstro¨m, T. Immonen, M. Kolmer, M. Tirronen, R. Predel, N. Kalkkinen, T.I. Heino, H. Sariola, C. Roos, The Drosophila hugin gene codes for myostimulatory and ecdysis-modifying neuropeptides, Mech. Dev. 117 (2002) 5–13. [11] R. Predel, C. Wegener, Biology of the CAPA peptides in insects, Cell. Mol. Life Sci. 63 (2006) 2477–2490. [12] G. Cazzamali, M. Torp, F. Hauser, M. Williamson, C.J.P. Grimmelikhuijzen, The Drosophila gene CG9918 codes for a pyrokinin-1 receptor, Biochem. Biophys. Res. Commun. 335 (2005) 14–19. [13] C. Rosenkilde, G. Cazzamali, M. Williamson, F. Hauser, L. Søndergaard, R. DeLotto, C.J.P. Grimmelikhuijzen, Molecular cloning, functional expression, and gene silencing of two Drosophila receptors for the Drosophila neuropeptide pyrokinin-2, Biochem. Biophys. Res. Commun. 309 (2003) 485–494. [14] R.J. Nachman, V.A. Roberts, H.J. Dyson, G.M. Holman, J.A. Tainer, Active conformation of an insect neuropeptide family, Proc. Natl. Acad. Sci. USA 88 (1991) 4518–4522. [15] A. Iversen, G. Cazzamali, M. Williamson, F. Hauser, C.J.P. Grimmelikhuijzen, Molecular cloning and functional expression of a Drosophila receptor for the neuropeptides capa-1 and -2, Biochem. Biophys. Res. Commun. 299 (2002) 628–633. [16] D. Zitnan, T.G. Kingan, J.L. Hermesman, M.E. Adams, Identification of ecdysis-triggering hormone from an epitracheal endocrine system, Science 271 (1996) 88–91. [17] A. Iversen, G. Cazzamali, M. Williamson, F. Hauser, C.J.P. Grimmelikhuijzen, Molecular identification of the first insect ecdysis triggering hormone receptors, Biochem. Biophys. Res. Commun. 299 (2002) 924–931. [18] J. Stables, A. Green, F. Marshall, N. Fraser, E. Knight, M. Sautel, G. Milligan, M. Lee, S. Rees, A bioluminescent assay for agonist activity at potentially any G-protein-coupled receptor, Anal. Biochem. 252 (1997) 115–126.
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Identification of one capa and two pyrokinin receptors from the malaria mosquito Anopheles gambiae q Stine S. Olsen, Giuseppe Cazzamali, Michael Williamson, Cornelis J.P. Grimmelikhuijzen, Frank Hauser * Center for Functional and Comparative Insect Genomics, Department of Cell Biology and Comparative Zoology, Institute of Biology, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen, Denmark Received 28 June 2007 Available online 27 July 2007
Abstract We cloned the cDNA of three evolutionarily related G protein-coupled receptors from the malaria mosquito Anopheles gambiae and functionally expressed them in Chinese hamster ovary cells. One receptor, Ang-Capa-R, was only activated by the two Anopheles capa neuropeptides Ang-capa-1 (GPTVGLFAFPRVamide) and Ang-capa-2 (pQGLVPFPRVamide) with EC50 values of 8.6 · 109 M and 3.3 · 109 M, respectively, but not by any other known mosquito neuropeptide. The second receptor, Ang-PK-1-R, was selectively activated by the Anopheles pyrokinin-1 peptides Ang-PK-1-1 (AGGTGANSAMWFGPRLamide) and Ang-PK-1-2 (AAAMWFGPRLamide) with EC50 values of 3.3 · 108 M and 2.5 · 108 M, respectively, but not by mosquito capa or pyrokinin-2 peptides. For the third receptor, Ang-PK-2-R, the most potent ligands were the pyrokinin-2 peptides Ang-PK-2-1 (DSVGENHQRPPFAPRLamide) and Ang-PK-2-2 (NLPFSPRLamide) with EC50 values of 5.2 · 109 M and 6.4 · 109 M, respectively. However, this receptor could also be activated by the two pyrokinins-1, albeit with lower potency (EC50: 2–5 · 108 M). Because Ang-capa-1 and -2 and Ang-PK-1-1 are located on one preprohormone and the other peptides on another prohormone, these results imply a considerable crosstalk between the capa, pyrokinin-1 and pyrokinin-2 systems. Gene structure and phylogenetic tree analyses showed that Ang-Capa-R was the orthologue of the Drosophila capa receptor CG14575, Ang-PK-1-R the orthologue of the Drosophila pyrokinin-1 receptor CG9918, and Ang-PK-2R the orthologue of the Drosophila pyrokinin-2 receptors CG8784 and CG8795. This is the first report on the functional characterization and crosstalk properties of capa and pyrokinin receptors in mosquitoes. 2007 Elsevier Inc. All rights reserved. Keywords: Malaria; Mosquito; Insect; GPCR; Neuropeptide; Capa; Pyrokinin; PBAN; Ecdysis-triggering hormone; Neuromedin U
Malaria is the most important parasitic disease in the world, causing over 300 million cases of illness and about two million deaths each year [1]. The disease is caused by an infection with Plasmodium falciparum, a parasite that is transmitted by mosquitoes belonging to the genus Anopheles during a blood meal [2]. Any knowledge on
q The sequence data from this paper have been submitted to the GenBank database under Accession Nos. AY900217, AY900218 and AY900219. * Corresponding author. Fax: +45 3532 1200. E-mail address: [email protected] (F. Hauser). URL: http://www.bi.ku.dk/staff/staff-vip-details.asp?ID=91 (F. Hauser).
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.06.190
the behavior and physiology of Anopheles, therefore, might help to interfere with the transmission of malaria. In the recently sequenced Anopheles gambiae genome, 35 preprohormone genes encoding about 70 neuropeptides, and 25 neuropeptide receptor genes belonging to the large family of G protein-coupled receptors (GPCRs) have been annotated [3–5]. These neuropeptide receptors steer important physiological processes such as development, reproduction and behavior. During the last six years, we and others have identified the endogenous ligands for about 25 neuropeptide receptors from Drosophila melanogaster [6,7], the insect with the first sequenced genome [8]. Examples of them are the fly receptors for capa and pyrokinin neuropeptides schematically presented in Fig. 1A.
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A
Capa-1
Capa-2
Drm-Capa-R
B
Capa-1
Drm-PK-1-R
Capa-2
Ang-Capa-R
PK-1
PK-1-1
Ang-PK-1-R
Hug-γ
Drm-PK-2-R1 PK-1-2
PK-2-1
PK-2
Drm-PK-2-R2 PK-2-2
PK-RP
Ang-PK-2-R
Fig. 1. Schematic representation of the capa/pyrokinin signalling system in Drosophila (A) and Anopheles (B). The capa and pyrokinin preprohormones are drawn as long open boxes, the neuropeptides contained therein are indicated by black boxes (not drawn to scale). (A) The four Drosophila capa/ pyrokinin receptors are Drm-Capa-R (CG14575), Drm-PK-1-R (CG9918), Drm-PK-2-R1 (CG8784), and Drm-PK-2-R2 (CG8795). (B) The three Anopheles capa/pyrokinin receptors are Ang-Capa-R, Ang-PK-1-R, and Ang-PK-2-R (this paper). A thick, red arrow indicates a peptide activating a receptor with an EC50 value of <1 · 108 M. A black arrow indicates a peptide activating a receptor with an EC50 value of 1–15 · 108 M. Stippled black arrows indicate peptides activating a receptor with an EC50 value of 15–100 · 108 M. All the indicated ligand/receptor interactions are derived from our functional assay data, but might not necessarily reflect the in vivo situation due to anatomical restrictions.
In Drosophila, there are two preprohormones (Fig. 1A), one yielding a capa-1, a capa-2, and a pyrokinin-1 (PK-1) peptide [9], and one yielding a pyrokinin-2 (PK-2) and a Hug-c peptide [10]. All these peptides are individual members of the PRL/I/Vamide neuropeptide family. Capa-1 and capa-2 have the C-terminal capa consensus sequence FPRVamide [9,11]. PK-1 has the C-terminal pyrokinin-1 consensus sequence WFGPRLamide [12], and PK-2 has the C-terminal pyrokinin-2 consensus sequence PFXPRLamide [10,12,13]. Hug-c has the C-terminal sequence PRLamide, but is not a pyrokinin senso stricto, because a pyrokinin has been defined by the C-terminal sequence FXPRLamide [10,14]. Drosophila capa-1 and capa-2 specifically activate their own capa receptor (Fig. 1A) [15]. Drosophila PK-1 only activates its PK-1 receptor [12], whereas PK-2 and Hug-c activate the two Drosophila PK-2 receptors (Fig. 1A) [13]. There is crosstalk between these ligand/receptor couples, because PK-2 and Hug-c slightly interact with the PK-1 receptor (Fig. 1A). Also, PK-1 is located on the same prohormone as the two capa peptides, implying that the PK-1 and capa receptors are simultaneously stimulated (Fig. 1A). Because the Drosophila capa/pyrokinin system is rather complex, we wanted to investigate the capa/ pyrokinin system in mosquitoes to see whether our findings in Drosophila are a general phenomenon in insects.
In Anopheles, the capa preprohormone has a similar organization as in Drosophila with two capa and a pyrokinin-1 (PK-1-1) neuropeptide (Fig. 1B) [4]. The pyrokinin-2 preprohormone has been expanded, containing two pyrokinins-2 (PK-2-1 and PK-2-2), one pyrokinin-1 (PK1-2), and one pyrokinin-related peptide (PK-RP). The structures of these Anopheles neuropeptides are given in Table 1. Table 1 also contains the structures of two Anopheles ecdysis-triggering hormones (ETHs). These peptides with the C-terminal sequence VPRIamide resemble capa and pyrokinin neuropeptides but have their own preprohormones [16] and, in Drosophila, their own specific receptors [17]. In the present paper, we report the molecular cloning and functional characterization of one capa and two pyrokinin receptors from the mosquito A. gambiae and the crosstalk that occurs between the receptor/ligand couples. Materials and methods Total RNA from adult A. gambiae (strain KWA) was isolated using TRIzol reagent (Invitrogen). The isolated total RNA was treated with DNase using the DNA-free kit (Ambion, Inc.). cDNA was synthesized using the SMART RACE cDNA Amplification kit (Clontech). PCR was performed using the Herculase Hotstart DNA polymerese (Stratagene).
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Table 1 Amino acid sequences of structurally related Anopheles neuropeptides and their potencies on the indicated receptors Name Ang-Capa-1 Ang-Capa-2 Ang-PK-1-1 Ang-PK-1-2 Ang-PK-2-1 Ang-PK-2-2 Ang-PK-RP Ang-ETH-1 Ang-ETH-2
Structure GPTVGLFAFPRVamide pQGLVPFPRVamide AGGTGANSAMWFGPRLamide AAAMWFGPRLamide DSVGENHQRPPFAPRLamide NLPFSPRLamide KPQPIFYHTTSPRLamide SESPGFFIKLSKSVPRIamide GDLENFFLKQSKSVPRIamide
EC50 (M) Ang-CapaR 9
8.6 · 10 3.3 · 109 na na na na na na na
EC50 (M) Ang-PK-1R
EC50 (M) Ang-PK-2R
na na 3.3 · 108 2.5 · 108 >106 >106 na na na
na na 5.0 · 108 2.5 · 108 5.2 · 109 6.4 · 109 1.6 · 107 >106 na
na, not active in concentrations up to 105 M. Bold highlights the pyrokinin consensus sequence (FXPRLamide) or the C-terminal PRL/I/Vamide sequence.
The sequences of previously identified capa and pyrokinin receptors from D. melanogaster [12,13,15] were used for BLAST searching of the genomic DNA database from A. gambiae (http://www.ncbi.nlm.nih.gov/ genome/seq/BlastGen/BlastGen.cgi?taxid=7165). Based on the identified sequences, PCR primers were designed as described in Supplementary Materials and methods. All PCR products were cloned into pCR4-TOPO plasmids (Invitrogen) using the TOPO TA cloning kit and sequenced. PCR products of the coding regions were subcloned into the pIRES2EGFP expression vector (Clontech) using the Rapid DNA Ligation Kit (Roche Applied Science). CHO cells stably expressing the human G-protein G16 (CHO/G16) were grown and transfected as described previously [18], the bioluminescence assay was performed as described in [19]. The Anopheles peptides shown in Table 1 were synthesized by GeneMed (San Francisco, CA). DNA sequence comparisons were carried out using the Lasergene software package (DNASTAR, Inc.). Protein sequence alignments were carried out using ClustalW (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_clustalw.html). Prediction of transmembrane helices of the receptor protein was done using the TMHMM server (http:// www.cbs.dtu.dk/services/TMHMM/). EC50 values were calculated using Prism software.
Results By BLAST searching of the A. Gambiae DNA database [3] with Drosophila capa and pyrokinin receptor sequences [12,13,15], we identified several DNA fragments coding for putative Anopheles orthologues. Using this information, we cloned the cDNAs coding for an Anopheles capa receptor (Ang-Capa-R) and two Anopheles pyrokinin receptors (Ang-PK-1-R and Ang-PK-2-R, Fig. S1–S3, supporting material). All three cDNAs contained at least one in-frame stop codon preceding the ATG start codon in their 5 0 -untranslated regions and two of them (Ang-PK-1-R and Ang-PK-2-R) contained a polyadenylation signal in their 3 0 -untranslated regions. Compared to the genomic DNA sequences, our cloned cDNA sequences showed 1–2% nucleotide differences (Tables S1–S3, supporting material). Some of these differences, which are probably due to allelic variations, also changed individual amino acid residues of the encoded protein sequences (Table S1– S3, supporting material). These comparisons also revealed that the Ang-Capa-R gene contained six introns, the AngPK-1-R gene two introns and the Ang-PK-2-R gene five introns (Tables S4–S6, supporting material).
The amino acid sequences of the three encoded proteins are shown in Fig. S1–S3 (supporting material). Fig. 2 gives an alignment of these three receptors together with the Drosophila capa receptor CG14575 [15], the Drosophila pyrokinin-1 receptor CG9918 [12] and the two Drosophila pyrokinin-2 receptors CG8784 and CG8795 [13]. This alignment shows large blocks of identical amino acid residues conserved between the two Diptera species, i.e., conserved over an evolutionary distance of about 250 million years [20]. The three Anopheles receptors show typical features of rhodopsin-like (family A or 1) GPCRs, such as seven transmembrane a-helices containing highly conserved sequence motifs and the canonical D/ERF/Y (Asp/Glu-Arg-Phe/Tyr) motif immediately following the third transmembrane a-helix (Fig. 2). In order to identify and characterize the endogenous ligands for these three receptors, we stably expressed their coding regions individually in CHO–G16 cells. We tested these transfected cell lines with our library of eight biogenic amines and 33 invertebrate neuropeptides [21] together with the nine Anopheles neuropeptides shown in Table 1 using our bioluminescence assay [18,19]. Ang-Capa-R showed very high selectivity and was only activated by the two Anopheles peptides Capa-1 and Capa2 (EC50 values of 8.6 · 109 M and 3.3 · 109 M, respectively; Table 1 and Fig. 3). Ang-PK-1-R was activated by low concentrations of the two pyrokinins Ang-PK-1-1 and Ang-PK-1-2 (EC50 values of 3.3 · 108 M and 2.5 · 108 M, respectively; see Table 1 and Fig. S4). Ang-PK-2-R was rather promiscuous. The most potent ligands were Ang-PK-2-1 and Ang-PK-2-2 (EC50 values of 5.2 · 109 M and 6.4 · 109 M, respectively; see Table 1 and Fig. S5). However, this receptor was also activated by the two Anopheles pyrokinins-1, Ang-PK-1-1, and Ang-PK-1-2, although 4- to 10-fold higher concentrations of these peptides were required (EC50 values of 5.0 · 108 M and 2.5 · 108 M, respectively; see Table 1). Also the pyrokinin-related peptide Ang-PK-RP could activate Ang-PK-2-R, but only at rather high concentrations (EC50 value of 1.6 · 107 M; see Table 1). All the other tested ligands showed no activity on this receptor.
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S.S. Olsen et al. / Biochemical and Biophysical Research Communications 362 (2007) 245–251 Ang-Capa-R Drm-Capa-R Ang-PK-1-R Drm-PK-1-R Ang-PK-2-R Drm-PK-2-R1 Drm-PK-2-R2
Ang-Capa-R Drm-Capa-R Ang-PK-1-R Drm-PK-1-R Ang-PK-2-R Drm-PK-2-R1 Drm-PK-2-R2
METVVPNATALAWPMSSTVPAMLLPDSSYASFPTG---GVTMSLDVLLG------LGLFDANGTGTENATGDPLEPPHPCDPSSDQFD------MN--------SSTDPTFSELNASFTNTP-----------DT-----------LFATS------VSSDPSHGFGEEDYACGTFN-------------------------------------------MSTSLKN--------MPYEL------LE----ILTTDNESILSDG----------------------------------------------MSAGN--------MSHD-------L--------------------MMELSPTVGSSGSLLDAL----------ITN-----------GTNGRTG--------LLHDI------LDGHRTLLHQSQQQQQQHYNHP MLQGVAITIANDSNDDGLNQSFMAHVSPSPNQSPSIGVGIGIASSTMANPSESPEMLLLKNDKF----LTHVAHLLNITTENLSNLLG-MLP--------------------------TN-----------SSGVLAT-----DLQLFHNEKF----LLNLTQVLNISADNLTSLL--TM I TM II -------------CSVDEFLVYARGPQRMPLFTAMLVTVLFVGILVTGVIGNLIVCLVIVRHPSMRTATNYYLFSLAVSDLIFLLLGLPY -------------CSPKEFVAFVLGPQTLPLYKAVLITIIFGGIFITGVVGNLLVCIVIIRHSAMHTATNYYLFSLAVSDLLYLLFGLPT ----------------VESLTEMYGPKRDPLYVVIPITIIYLLIFITGVVGNISTCIVIARNRSMHTATNYYLFSLAVSDFLLLVSGVPQ ------------------------GPPRDPLAIVIPVTVVYSLIFITGVVGNISTCIVIKKNRSMHTATNYYLFSLAISDFLLLLSGVPQ HHQQYQPQYLYANESLAGSVPESASYGTDSICIILPITIFYCFIFVAGIVGNLSICVVIAKNKSMHTATNYYLFNLAVSDFLLLLFGMPQ -----------STNGTNASTMAADSPVDESLTLRTALTVCYALIFVAGVLGNLITCIVISRNNFMHTATNFYLFNLAVSDLILLVSGIPQ -------------QGLEPEELLPTVIPMTPLSLLATLSVGYALIFIAGVLGNLITCIVISRNNFMHTATNFYLFNLAISDMILLCSGMPQ TM III TM IV EISLYWHQYPYNLGLPFCKIRALISEASTYVSVLTIVAFSMERFLAICHPLHLYTMSGLQRPVRIIAGLWVVSLLSAVPFAVFTDIDYIA EVFLYWHQYPDLFGMPFCKIRAFISEACTYVSVFTIVAFSMERFLAICHPLHLYAMVGFKRAIRIITALWIVSFISAIPFGLLSDIQYLN EIYFIWSKYPYVFGETFCVLRGIAAEMSANATVLTITAFTIERYFAICHPFLSHTMSKLSRAVRFICVIWLIAIVSAIPQALQFGVTNQG EVSYIWSKYPYVFGEYICIGRGLLAETSANATVLTITAFTVERYIAICHPFLGQAMSKLSRAIRIIVLVWIMAIVTAIPQAAQFGIEHYS ELYGTWNPFAYPFNQIACIIMGLLSETAANATVLTITSFTVERYIAICHPFRSHTMSKLSRAIKFVIVIWLVAFGLATPQALQFGVVETN ELYNLWYPDMYPFTDAMCIMGSVLSEMAANATVLTITAFTVERYIAICHPFRQHTMSKLSRAIKFIFAIWLAAFLLALPQAMQFSVVYQN DLYNLWHPDNYPFSDSICILESVLSETAANATVLTITAFTVERYIAICHPFRQHTMSKLSRAVKFIFAIWIAALLLALPQAIQFSVVMQG TM V YPPTNEKILDSAFCAMLSNPE-GFPLWELSTCLFFAFPMLIMVVLYGRMGMQIRSRTQRTAELGVRNGS--------------------YPLDHSRIEESAFCSMSPKIVNEIPVFEVSFCIFFVIPMILIILLYGRMGAKIRSRTN--QKLGVQQGT--------------------G---------IDQCVVKRIII--QHSFELSTFLFFFAPMTMITILYALIGLKLRTSTLMQRDGTLQRR---------------------G---------VEQCGIVRVIV--KHSFQLSTFIFFLAPMSIILVLYLLIGVHLYRSTLVEGPASVARRQQLKSVPSDTILYRYGGSGTAM T---------TRLCTIKNEHF--SHAFEVSSFLFFVGPMTLIAVLYVLIGIKLRKSKLLQGVKRASSD---------------------E---------GYSCTMENDFY--AHVFAVSGFIFFGGPMTAICVLYVLIGVKLKRSRLLQSLPRRTFD---------------------M---------GTSCTMKNDFF--AHVFAVSGFLFFGGPMTAICVLYVLIGVKLKRSRLLQALPRRCYD---------------------TM VI ------------------------VNGPSRISQSRK-AIIRMLAAVVITFFVCWAPFHAQRLLFLYAR-------DWQHFNT-VNTWLFS ------------------------NNRETRNSQMRKKTVIRMLAAVVITFFVCWFPFHLQRLIFLYAK-------NMDNYLD-INEALFS -------------------TQPSPRQSFANSQGSRR--VLKMLVAVVVAFFICWAPFHAQRLVYIYGVNT--NHQPSDPLILKLFIITTY SFNGGGSGAGTAGLMGGSGAQLSSVRGRLNHYGTRR--VLRMLVAVVVCFFLCWAPFHAQRLIAIYAPAR--GAKLRDQHEF-VYTVMTY --------------------YTTPPRGIS--AQSR---VIRMLVAVVATFFICWAPFHAQRLMAVYGVFS----KTENVFFYKVYMVLNY -----------------------ANRGLN--AQGR---VIRMLVAVAVAFFLCWAPFHAQRLMAVYGLNLIN-IGISRDAFNDYFRILDY -----------------------VNRGIS--AQTR---VIRMLVAVAVAFFICWAPFHAQRLMAVYGS---T-SGIESQWFNDVFSILDY TM VII VAGWLYYVSCTINPILYNVMSHRYRVAFRETLCGRRRG-----FGAGFARDQSSFRET-------------------------------IAGFAYYVSCTVNPIVYSVMSRRYRVAFRELLCGKAVGAY---YNSGFARDHSSFRESS---AYDRVH---------------------ISGILYYLSTCINPLLYNIMSNKFRQAFKNMLT--------------------------------------------------------VSGVLYYLSTCINPLLYNIMSHKFREAFKAVLFGKKVS-----KGSLNSRNNIESRRLR--RALT------------------------TSGILYFLSTCINPLLYNIMSHKFRDASRHTLKMSCCGGK---RSKSDGQHHTYSAVSR--YGVTGCGSFKVNSNNMACIGAGIGLGTGG TSGVLYFLSTCINPLLYNIMSHKFREAFKITLTRQFGLARNHHHQQSQHHQHNYSALLR-QNGSMRLQPASCSVNNNALEPYGSYRVVQF TSGVLYFLSTCINPLLYNIMSHKFREAFKVTLARHFGLGG---KNQGRGLPHTYSALRRNQTGSLRLHTTD-----SVRTT-----MTSM
Ang-Capa-R Drm-Capa-R Ang-PK-1-R Drm-PK-1-R Ang-PK-2-R Drm-PK-2-R1 Drm-PK-2-R2
----------------TIDVNTAGGGNGYGTG-----------------------------------YESSRLVSVDCTTQAQNSR-------------------SVHVRASQHPNKFETDS-----------SSANRVLIKKT----YSLPLPKNADSTVLSTTDIVIVLENSHTVCE -------------------------------------------VRSAR-------------KPPWQTSYGASPPHGSTWTG--------------------------------NSSQTQRFS----------IESAEQ-----------PKPSIMQNPTNKPPVAAQYAMIGVQVN--GNAASKQQHQQPGNGTAATPNQCQQESNMSLLA----------NESCNRGTMPPI----AIRPTFTRADSHCISISSSQSTTGTNVSSNG RCRDANHQLSLQDSIRTTTTTTTINSNSMAAGNGVGGGAGGGGRRLRKQELYGPVPGTAVPHRMLQAQVSQLSSLGDANSLLEAEVVDRH ATTTTGLNGSANGSGNGTTTGQSVRLNRVSLDS-----VQMQGQNRSRQDLFD------NPRRMLQTQISQLSSVGDAHSLLEEDLQFPG
459 460 373 430 514 550 479
Ang-Capa-R Drm-Capa-R Ang-PK-1-R Drm-PK-1-R Ang-PK-2-R Drm-PK-2-R1 Drm-PK-2-R2
PPGGGKWIGLVVEQ---------------------------------------------------------------------------EPKVENDIWIENEETCI----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------KLSRSSNGSLRHGGSALEQPTDHAAGGGGGGGRLST---IAEKLRRGTKKVLAFSKSPTGTPSRSIVSGACAEGTNGRRKAYRKRNSCDS YASGRAKRALLATKSGALLVTPPQSGDPSEVSQPATRLKLTRVISRRDEVANASTPPFCGSHS----------------LPDPETCQSAS EPLQRQPTMCSIDELTDDLAISRSRLKLTRITRPPG--GVTGGVAGGSTTVAAGSGGVSGDESSGKV-----------RKAKVKVLKSSS
473 477
Ang-Capa-R Drm-Capa-R Ang-PK-1-R Drm-PK-1-R Ang-PK-2-R Drm-PK-2-R1 Drm-PK-2-R2
--------------------------------------------------------------------------------------------------------------------------------------------------------VDTNTISNSSLKEFDEEEFSSAELARFMGEVNSEIR--VAGRSSRKFPWRKRRQKTEDPSSEGLTYGSPKSQ----PFKGLRTKFNWRARRKGSHKPHEKGATVNGGDTEERAAF
Ang-Capa-R Drm-Capa-R Ang-PK-1-R Drm-PK-1-R Ang-PK-2-R Drm-PK-2-R1 Drm-PK-2-R2 Ang-Capa-R Drm-Capa-R Ang-PK-1-R Drm-PK-1-R Ang-PK-2-R Drm-PK-2-R1 Drm-PK-2-R2 Ang-Capa-R Drm-Capa-R Ang-PK-1-R Drm-PK-1-R Ang-PK-2-R Drm-PK-2-R1 Drm-PK-2-R2 Ang-Capa-R Drm-Capa-R Ang-PK-1-R Drm-PK-1-R Ang-PK-2-R Drm-PK-2-R1 Drm-PK-2-R2
79 47 27 10 55 84 41 156 124 101 76 145 163 118 246 214 191 166 235 253 208 314 281 248 245 292 310 265 371 339 315 330 353 371 323 424 401 348 388 438 460 400
601 624 556
637 658 595
Fig. 2. Protein sequence alignment of the Anopheles capa receptor (Ang-Capa-R; Accession No. AY900217), the Drosophila capa receptor (Drm-Capa-R; CG14575), the Anopheles pyrokinin-1 receptor (Ang-PK-1-R; Accession No. AY900218), the Drosophila pyrokinin-1 receptor (Drm-PK-1-R; CG9918), the Anopheles pyrokinin-2 receptor (Ang-PK-2-R, Accession No. AY900219) and the two Drosophila pyrokinin-2 receptors (Drm-PK-2-R1; CG8784 and Drm-PK-2-R2; CG8795). Spaces are introduced to optimize alignments. The seven transmembrane a-helices are indicated by TM1–TM7. Amino acid residues that are identical in at least four of the seven receptor sequences are highlighted in grey. Intron positions are indicated by boxes.
As seen in the amino acid sequence alignment (Fig. 2), the three Anopheles receptors are structurally and evolutionarily closely related to their Drosophila orthologues.
There is 45% sequence identity (59% similarity) between Ang-Capa-R and the Drosophila capa receptor CG14575 [15] and all six introns found in the mosquito gene are con-
S.S. Olsen et al. / Biochemical and Biophysical Research Communications 362 (2007) 245–251
1.0
0 -5 sec. 5 -10 sec. 10 -15 sec.
10000
L/L max
Luminescence
15000
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Capa-1 Capa-2
0.5
5000
0.0 -13 -12 -11 -10 -9 -8 -7 -6 -5 Log M
0 A
B
C
D
Fig. 3. Functional assay of the Ang-Capa-R. The left panel shows the bioluminescence responses of non-transfected CHO–G16 cells (A,B) and of CHO– G16 cells expressing Ang-Capa-R (C,D), 0–5 s (black), 5–10 s (grey), and 10–15 s (white) after addition of 106 M Ang-capa-1 (A,C) and Ang-capa-2 (B,D). The right panel shows the dose–response curve of a cloned CHO–G16 cell line expressing Ang-Capa-R to Ang-Capa-1 (EC50, 8.6 · 109 M) and Ang-Capa-2 (EC50, 3.3 · 109 M) using the same assay. In all panels, the SEMs are given as vertical bars, which are sometimes smaller than the symbols used. In these cases only the symbols are given.
Drm-PK-2-R1 (CG8784) Drm-PK-2-R2 (CG8795) Ang-PK-2-R Hez-PBAN-R Bom-PBAN-R Ang-PK-1-R Drm-PK-1-R (CG9918) Ang-Capa-R Drm-Capa-R (CG14575) Mum-NMU-R1 Ang-ETH-R Drm-ETH-R (CG5911) Drm-MS-R1 (CG13803)
178.4 160
140
120
100
80
60
40
20
0
Fig. 4. A phylogenetic tree analysis of the seven receptors from Fig. 1, as well as the PBAN receptors from Helicoverpa zea (Hez-PBAN-R) [19] and Bombyx mori (Bom-PBAN-R) [20], the eclosion triggering hormone receptor from Drosophila (Drm-ETH-R; CG5911) [13], the orthologue receptor from Anopheles (Ang-ETH-R) [Andersen et al., unpublished] and the mouse neuromedin U receptor 1 (Mum-NMU-R1) [21]. The Drosophila myosuppressin receptor (Drm-MS-R1; CG13803) [23] was used as an outgroup.
served in fly (Fig. 2). Ang-PK-1-R shows 46% sequence identity (57% similarity) to the Drosophila pyrokinin-1 receptor CG9918 [12], and these two receptor genes have both introns present in their coding regions in common (Fig. 2). Finally, Ang-PK-2-R has 35% and 38% sequence identity (46% and 49% similarity) with the Drosophila pyrokinin-2 receptors CG8784 and CG8795, respectively [13], and these three receptor genes share all five introns found in the mosquito gene. These close relationships are further supported by a phylogenetic tree analysis using the seven receptors from Fig. 2 together with the characterized pheromone biosynthesis activating neuropeptide (PBAN) receptors from Helicoverpa zea [22] and Bombyx mori [23], the ETH receptor from Drosophila [17] and its orthologue from Anopheles [Andersen et al., unpublished], and the mouse neuromedin U receptor 1 [24] (Fig. 4). The insect PK-1 and PK-2 receptors clearly form two separate clusters. These clusters show, together with the cluster of PBAN receptors, higher homologies to each other than to the capa receptors, which
form a separate branch. Another, even more distantly related branch is formed by the insect ETH receptors. Finally, the mouse neuromedin U receptor is clearly evolutionary related to the insect capa and pyrokinin receptors as observed earlier [25]. Discussion It is very important to understand the biology of the malaria mosquito A. gambiae to be able to prevent transmission of malaria. Important aspects of the mosquito’s biology, such as behavior, reproduction and development, are steered by neurohormones and their receptors. Selective interference with these endocrine systems, for example, by the development of highly selective non-peptide neurohormone receptor antagonists or agonists, could greatly reduce the populations of A. gambiae. This would be an important contribution to the fight against malaria. The first step for such a project is the characterization of A. gambiae neurohormone GPCRs and an understanding of
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their physiology. We and other research groups have previously characterized the A. gambiae GPCRs for myosuppressin [26], adipokinetic hormone [21], corazonin [21], neuropeptide F [27], and short neuropeptide F [28]. The present paper describes the cloning and functional expression of three other, closely related, GPCRs from Anopheles. Based on our functional analysis (Fig. 3; Figs. S4, S5, supporting material) we named the three receptors Ang-CapaR, Ang-PK-1-R, and Ang-PK-2-R. A summary of our results is given in Table 1 and Fig. 1B. It is interesting that these results from Anopheles strongly resemble the situation in Drosophila. Both in Drosophila (Fig. 1A) and in Anopheles (Fig. 1B) there is one capa prohormone, each containing two copies of a capa peptide (capa-1 and capa-2) that stimulate a specific capa receptor that does not react with other neuropeptides. In both Drosophila and Anopheles the capa preprohormone contains a pyrokinin with the C-terminal WFGPRLamide sequence, that we call pyrokinin-1 (PK-1; [12]). In both insects this PK-1 activates a specific PK-1 receptor that is different from the capa receptor (Fig. 1). In addition to the capa preprohormone, both insects have a pyrokinin preprohormone. In Drosophila, this precursor protein contains a pyrokinin with the C-terminal PFXPRLamide sequence that we call pyrokinin-2 (PK-2; [12,13]). In Anopheles, the gene region coding for the pyrokinin peptides has apparently been duplicated twice, giving rise to two pyrokinins-2 (PK-2-1 and PK-2-2) and one pyrokinin-1 (PK-12). In both insects the pyrokinins-2 activate selectively and at very low concentrations (indicated by red arrows in Fig. 1) their own PK-2 receptors, which are two receptors in Drosophila and one in Anopheles (Fig. 1). There is apparently more crosstalk in Anopheles than in Drosophila between the two peptide cocktails derived from the two precursor proteins and the three receptor subtypes. The two Anopheles pyrokinins-1 (PK-1-1 and PK-1-2), derived from two different preprohormones, activate the pyrokinin-1 and pyrokinin-2 receptors equally well, while this cross-reactivity of pyrokinin-1 on the pyrokinin-2 receptor is absent in Drosophila (Fig. 1). Interestingly, the two Anopheles pyrokinins-1 share the C-terminal eight amino acid sequence (Table 1). As mentioned above, PK1-2 apparently originated from a gene region coding for a PK-2 peptide. This suggests that there must have been evolutionary pressure in the evolutionary line leading to mosquitoes to convert a PK-2 into a PK-1 sequence and to integrate signalling from both preprohormones on the PK-1 receptor. An additional crosstalk might be mediated by the pyrokinin-related peptide, Hug-c, in Drosophila (Fig. 1A). Hug-c has a high affinity to the pyrokinin-2 receptors, but also low affinity to the pyrokinin-1 receptor. This last interaction might be a substitute for the crosstalk mediated by the two pyrokinins-1 in Anopheles, which is lacking in Drosophila. The same is true for the Drosophila pyrokinin-2, which has a low affinity for the Drosophila pyrokinin-1 receptor.
Many neurohormone systems in insects are constructed in such a way that there is one preprohormone, of which the products (the peptide cocktail) activate one or more receptors [6,7]. Already the presence of two or more receptors suggests that several physiological processes are synchronized or coordinated. Such an extreme crosstalk as illustrated in Fig. 1, however, has never been seen, so far, in insects. The only system that comes close to it, is the myosuppressin/FMRFamide system in Drosophila, where one copy of the neuropeptide myosuppressin activates the two myosuppressin receptors and, with a somewhat lower affinity, the FMRFamide receptor [29,30]. The numerous FMRFamide peptides, however, do not activate the two myosuppressin receptors [30]. What potential contributions could our current results have for malaria control? Female mosquitoes need one or more blood meals to collect enough proteins for egg formation. During this blood meal, their total body weight increases several times. However, already during and immediately after blood feeding, large amounts of liquid are secreted by the animal to allow for proper flying and escape. Insect capa peptides and capa receptors are controlling fluid secretion by the Malpighian tubes, also in mosquitoes [31]. Capa receptors are, therefore, interesting targets for the development of a selective and environmentally friendly anti-mosquito insecticide. The same might be true for the pyrokinin receptors, because the pyrokinin (PK-1-1) release is physiologically connected to the release of the capa peptides (Fig. 1B). A disrupted liquid homeostasis in mosquitoes will stop malaria mosquito reproduction and disrupt the transmission of malaria. Acknowledgments We thank the Danish Research Agency (Research Council for Nature and Universe), and Novo Nordisk Foundation for financial support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2007. 06.190. References [1] R.W. Snow, C.A. Guerra, A.M. Noor, H.Y. Myint, S.I. Hay, The global distribution of clinical episodes of Plasmodium falciparum malaria, Nature 434 (2005) 214–217. [2] A.F. Cowman, B.D. Crabb, Invasion of red blood cells by malaria parasites, Cell 124 (2006) 755–766. [3] R.A. Holt et al., The genome sequence of the malaria mosquito Anopheles gambiae, Science 298 (2002) 129–149. [4] M.A. Riehle, S.F. Garczynski, J.W. Crim, C.A. Hill, M.R. Brown, Neuropeptides and peptide hormones in Anopheles gambiae, Science 298 (2002) 172–175. [5] C.A. Hill, A.N. Fox, R.J. Pitts, L.B. Kent, P.L. Tan, M.A. Chrystal, A. Cravchik, F.H. Collins, H.M. Robertson, L.J. Zwiebel, G proteincoupled receptors in Anopheles gambiae, Science 298 (2002) 176–178.
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