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Focus on the splicing of Secretin GPCRs transmembrane-domain 7
Review
Focus on the splicing of Secretin GPCRs transmembrane-domain 7 Danijela Markovic§ and Dimitris K. Grammatopoulos GPCR Pathophysiology Group, Division of Endocrinology and Metabolism, Clinical Sciences Research Institute, Warwick Medical School, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK
The family of G-protein coupled receptors (GPCRs) is one of the largest protein families in the mammalian genome with a fundamental role in cell biology. GPCR activity is finely tuned by various transcriptional, post-transcriptional and post-translational mechanisms. Alternative pre-mRNA splicing is now emerging as a crucial process regulating GPCR biological function. Intriguingly, this mechanism appears to extensively target the Secretin family of GPCRs, especially the exon that encodes a 14 amino acid sequence that forms the distal part of 7th transmembrane helix, and exhibits an unusually high level of sequence conservation among most Secretin GPCRs. Do the ‘‘TMD7-short’’ receptor variants have a role as novel regulators of GPCR signallng and, if so, what are the implications for hormonal actions and physiology? Alternative pre-mRNA splicing and GPCR superfamily diversity Mammalian evolution is tightly linked to highly conserved as well as divergent aspects of gene function. An important evolutionary mechanism generating considerable gene divergence and protein complexity is alternative pre-mRNA splicing [1,2]. This mechanism, discovered 30 years ago, allows production of multiple transcript isoforms from a single gene through exon skipping, alternative exon insertions, use of alternative 50 splice site and 30 splice site, and intron retention. These novel exonic combinations offer considerable plasticity toward changing environmental pressures, or stage of development. Although alternative splicing (AS) was originally considered to be an unusual event occurring in only 5% of human genes [3], this view has now been considerably revised. Indeed, computational analysis of AS has estimated that more than 90% of genes in humans are alternatively spliced [4]. Genome-wide analysis of expressed sequence tags (ESTs), datasets for discovering tissue-specific AS mechanisms, suggests that the brain contains 18% of all tissue-specific AS (TS-AS) events. Peripheral tissues with the highest number of TSAS forms are the retina, lung, liver, pancreas, placenta, ovary, uterus, testis, lymph, muscle and skin [5]. In addition, AS can provide a functional on/off switch for gene regulation because as many as one-third of AS events insert premature termination codons that usually result in mRNA degradation by nonsense-mediated decay [6]. Corresponding author: Grammatopoulos, D.K. ([email protected]) § Current address: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester LE1 9HN, UK..
A gene family extensively targeted by AS mechanisms is the superfamily of G-protein coupled receptors (GPCRs). These cell-surface proteins are the largest family of signaling molecules, with more than 1000 members, and fundamental roles in cell biology. AS appears to have a major role in the cellular machinery regulating GPCRs expression and activity through generation of functionally distinct receptor variants. AS of noncoding exons present in the 5’-untranslated region (UTR) of GPCR genes might be important for the efficient translation into protein [7]. The first GPCR splice variants were reported in 1989 for the rat D2 dopamine receptor [8], and subsequently a growing number of other GPCR genes have been identified exhibiting sequence diversity. An important prerequisite for generation of multiple GPCR splice variants is the presence of intronic sequences in their open reading frames (ORFs). Interestingly, it is believed that most mammalian GPCRs (>90%) lack introns in their protein-coding regions [9], a property found in less than 5% of human genes. At present, we do not understand why an intronless genomic architecture is preferred among mammalian GPCRs. It is possible that intronless GPCR genes are evolutionarily advantageous in complex eukaryotes [9], because of higher transcriptional fidelity and translational efficiency, although in a number of cases introns increase gene expression levels [10]. It is certainly intriguing that retaining introns allows some GPCRs to enrich their molecular diversity and potential functionality through encoded GPCR variants. Given the fundamental role of AS in mammalian biology, it is not surprising that disruption of this mechanism might contribute to the pathogenic mechanisms of disease or disease susceptibility. In fact, an unexpectedly large number of exonic mutations can cause disease by disrupting the splicing code. It is now well documented that certain types of cancer are associated with splicing abnormalities resulting in abnormal regulation of various proteins [11,12]. In this context, aberrant AS events affecting GPCRs function and hormonal responses might be a major determinant of disease development. The PraderWilli syndrome is the first example of a genetic disorder where a defect in pre-mRNA splicing and processing of the serotonin receptor 2C has been identified [13]. Furthermore, GPCR splicing might be important in the pathogenesis of various polygenic disorders, such as asthma, schizophrenia, obesity and stress-induced heart disease. [14–18]. A multitude of AS events modulating GPCR structural characteristics and function have been described that can
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generate distinct GPCR protein variants. Although the relationship between AS and protein structural modification appears to be unique for each GPCR, depending on its genomic architecture, recent evidence suggests that in some GPCR families, highly conserved structural motifs offer the potential for common patterns of specific AS events that might affect the structure of GPCRs and signaling potency of a plethora of hormonal signals. This previously unrecognised splicing potential highlights the multifaceted regulation of GPCRs in mammalian cells. Impact of alternative splicing on GPCR structure and function According to the GRAFS classification system [19], the superfamily of mammalian GPCRs can be broadly subdivided into five families according to their protein sequence homology, the ligand structure, and receptor function. These are: the Glutamate (G) family, Rhodopsin (R) family, Adhesion (A) family, Frizzled/Taste2 (F) family, and the Secretin (S) family [19]. AS variants have been described for more than 50 GPCRs [20–22]. Many GPCRs have multiple variants and some are species-specific [23] or exhibit tissue-specific distribution. A survey of GPCR splicing patterns suggests that AS-related structural variations can be divided into three main groups: (a) variations in the intracellular Cterminal tail length and sequence; (b) variations in the third intracellular (IC3) loop [24]; and (c) variations in the extracellular N terminus. By contrast, splice variants in the putative extracellular loops (EC) or transmembrane domains (TMD) are less frequent. Ultimately, the structural alterations resulting from the splice variations would determine the impact on receptor function [for detailed reviews see Refs 20 and 22].
The Secretin family of GPCRs: biology and splicing patterns The Secretin (or ‘‘brain-gut’’ neuropeptide) GPCRs (Table 1) have been identified throughout mammalian genomes, but not in plants, fungi or prokaryotes. All Secretin GPCRs have a complex genomic architecture comprising multiple exons and introns which appear to be extensively targeted by AS events. The Secretin GPCRs In humans, the Secretin receptors, encoded by 15 genes, and the Adhesion receptors, encoded by 33 genes, represent two distinct but related subfamilies of GPCR [25]. Evolution studies suggest that the Secretin GPCRs family could have descended from a subgroup of Adhesion GPCRs that contains receptors including GPR133 and GPR144 [26]. Secretin GPCRs bind large polypeptide hormones that share a high degree of amino acid identity and most often act in a paracrine or autocrine manner. Phylogenetic relationship analysis between the members of this family according to TMD1 to TMD7 sequence identities showed that the tree has four main subgroups: the corticotropinreleasing hormone (CRH) and Rs/calcitonin (CALC)-Rs, which are the two most ancient GPCR groups within the family [26], the parathyroid hormone (PTH)-Rs, glucagonlike peptide (GLP)-Rs/glucagon (GCG)-R/gastric inhibitory polypeptide (GIP)-R and the subgroup including Secretin and four other receptors [19] (Figure 1). Alternatively spliced variants of Secretin GPCRs Secretin GPCRs exhibit a diverse splicing pattern and a plethora of mRNA splice variants has been identified (Figure 2) in a wide range of tissues and cell types in
Table 1. Alternative splicing in secretin GPCRs. Receptor name
Gene name
Ligands
Calcitonin receptor
CALCR
Calcitonin receptor-like receptor
CALCRL
Corticotropin releasing hormone 1 receptor (CRH-R1) Corticotropin releasing hormone 2 receptor (CRH-R2) Gastric inhibitory polypeptide (GIP) receptor Glucagon receptor Glucagon-related peptide 1 (GLP-1) receptor Glucagon-related peptide 2 (GLP-2) receptor Growth hormone releasing hormone (GHRH) receptor PAC1 receptor
CRHR1
Calcitonin, amylin, calcitonin gene-related peptide Calcitonin gene-related peptide, adrenomedullin CRH, urocortin (UCN)
CRHR2
Parathyroid hormone 1 (PTH1) receptor Parathyroid hormone 2 (PTH2) receptor Secretin receptor VPAC1 receptor
PTHR1
VPAC2 receptor
VIPR2
444
Length of wild type receptor (amino acids) 474
Number of exons 12
461
12
415
14
UCN, UCN II, UCN III, CRH
411
12
GIPR
GIP
466
13
GCGR GLP1R
Glucagon GLP-1
477 463
13 13
GLP2R
GLP-2
553
13
GHRHR
GHRH
423
13
ADCYAP1R1
Pituitary adenylate cyclase-activating polypeptide (PACAP) Parathyroid hormone, PTH-related peptide Tuberoinfundibular peptide of 39 residues (TIP39) secretin Vasoactive intestinal peptide (VIP), PACAP VIP, PACAP
468
15
593
14
550
13
440 457
13 13
438
13
PTHR2 SCTR VIPR1
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Figure 1. Phylogenetic relationships of the Secretin family of GPCRs. These relationships are based on the sequence similarity of TMD1 and TMD7 according to Fredriksson et al. [19]. The tree consists of four main subgroups as indicated by the colored ovals.
human and other mammals. The resulting structural modifications might alter ligand binding and/or receptor signaling properties. For example, AS resulting in modifications of IC1 amino acid length of the CRH-R1 [27] and CALC-R receptors [28] appears to modulate G-protein coupling and post-translational modification [29]. Recent data have implicated some of these receptor variants in
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mammalian development and physiology as well as the pathophysiological mechanisms of disease [30,31]. Although the specific biological function of these receptor variants remains to be elucidated, it appears that some might act as dominant negative (DN) regulators of hormonal responses by interfering with wild type receptor cell membrane expression or activation by its cognate agonists [32–34]. The relevance of these receptor variants for mammalian physiology has been questioned, primarily because the lack of variant-specific antibodies prevents conclusive demonstration of protein expression in native tissues. Although aberrant or ‘‘random’’ splicing should always be considered as a potential source of receptor sequence variation, it is now evident that specific mechanisms have been developed or retained during mammalian evolution that allow the generation of discrete mRNAs and potentially encode functionally diverse protein isoforms. This idea is supported by the results of sequence comparison studies, which suggest that the overall complexity and splicing patterns are highly conserved across all members of Secretin GPCRs. A number of well conserved splice sites have been identified in sequences encoding amino acids present in EC1 and EC2, TMDs 4, 5, and 7 and within IC1 and IC3 (the latter is absent from the CALC-R/calcitonin-gene related peptide (CGRP)-R group of receptors) [26] (Figure 2). These conserved splice sites are present also in Adhesion GPCRs; in particular, the vertebrate orthologs of GPR144 and GPR133 have most (six) or all (seven) of the conserved splice sites found in Secretin GPCRs. This in silico prediction analysis is supported by in vitro studies that identified several predicted mRNA splice variants in mammalian tissues and cells; for example, mRNA variants at the
Figure 2. Examples of Secretin GPCR splice variants. A splicing map depicting examples of the different types of mRNA splice variants identified, involving members of the Secretin family of GPCRs, and their potential structural/functional consequences. Stars indicate the position of splice sites that are conserved in Secretin and Adhesion V GPCRs according to Nordstro¨m et al. [26].
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Figure 3. The conservation of exons in the Secretin family GPCRs. (a) Alignment of Secretin GPCRs according to individual exon nucleotide sequence length combinations demonstrating conservation of the -61 (pink) and 42 bp (red) exons across all members of Secretin GPCRs. (b) Amino acid sequence alignment as encoded by exon 12 (42 bp) in Secretin GPCRs. A pink background indicates residues conserved in all GPCRs; a blue background indicates residues conserved in 10 out of 15 GPCRs.
IC1 conserved splicing site, which permits insertion of variable length amino acid sequences and extension of IC1, have been described for the CRH-R1 and CALC-R [27,28]. A splicing mechanism of particular interest leads to an in-frame deletion of a 42 bp exon (exon 12 in the CRH-R1 genomic sequence) and loss of a 14 amino acid cassette from the C-terminal end of the putative TMD7 that allows generation of ‘‘TMD7-short’’ receptor variants. Splice variants arising from this exon deletion have been described for four members of the Secretin family, the CRH-R1 (R1d), CALC-R (CTRD13), PTH-R, and the type II receptor for vasoactive intestinal peptide (VIP)/PACAP (SD-VPAC2) [35–38], suggesting the presence of a highly conserved splicing ‘‘hot-spot’’. A closer look at the TMD7-short receptor variants Comparative sequence analysis uncovers some fascinating insights into the unique position of exon 12 within the genomic architecture of Secretin GPCRs. All 15 genes contain the 42 bp exon, which is present also in the genomic sequence of several members of the Adhesion family of GPCRs [39]. This raises the possibility that this exon has been conserved or ‘‘protected’’ throughout evolution of Secretin GPCRs to perform a function that is preserved in all members of the family. For example, these residues might be crucial for maintaining the proper three-dimensional configuration of the membrane-spanning portions of the receptor. The only other exon that appears to be conserved across the Secretin GPCR family is a 61 bp exon that is found four exons upstream of exon 12 (Figure 3a). 446
This exon, which is absent from the Adhesion family of GPCRs, encodes amino acid sequences of the TMD4 and Nterminal segment of EC2. Intriguingly, there is one striking difference between these two exons: the amino acid sequence encoded by exon 12, which follows the sequence pattern G-(L/F)-X-V-S/A-X-X-F/Y-C-F/Y-X-N-X-E, is highly conserved across all Secretin GPCRs. Frequency analysis of 14 individual amino acids in particular positions shows that 44% are identical in all receptors and 64% in at least 10 out of 15 Secretin GPCRs (Figure 3b). This exon contains the highest degree of conserved amino acids across the entire receptor sequence. By contrast, exon 8 (based on the CRH-R1 genomic sequence) encodes an unusually hypervariable amino acid sequence with only 10.5% of amino acids being conserved in ten or more Secretin GPCRs (Figure 4). Only the N and C-terminal segments exhibit a higher degree of amino acid variability, consistent with their well established roles in ligand recognition and signaling specificity. Key residues encoded by exon 8, such as a highly conserved Gly 4.52 (using the nomenclature of Ballesteros and Weinstein*) are found within the lipidexposed face of TMD4 of SCT-R and appear to form a functionally important interface for oligomerization [40]. This might provide some insights about the requirement for such sequence diversity. Extensive intra-family constitutive oligomerization has been reported for the Secretin GPCRs [41] and, thus, a hypervariable segment might ensure specificity of receptor–receptor interactions. * The most highly conserved residues in each transmembrane helix are given the number 50 in the Ballesteros and Weinstein nomenclature. In the prototypic rhodopsin receptor these are N1.50, D2.50, R3.50, W4.50, P5.50, P6.50 and P7.50.
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Figure 4. Frequency analysis of amino acids encoded by individual exons and their position in the structure of the GPCR. The analysis was based on amino acids conserved in at least 10 out of 15 Secretin GPCRs. Exon 12 (amino acids in the C terminus of TMD7) contains the highest degree of conserved amino acids across the entire receptor sequence. In contrast, exons 1, 2 and 13 (amino acids in the N and C terminus) encode hypervariable amino acid sequences with less than 10% of the amino acids being conserved in ten or more Secretin GPCRs.
Structural modifications and cellular expressionlocalization The loss of 14 amino acids from the C-terminal end of the putative TMD7 appears to have a major impact on receptor protein folding orientation and subcellular localization. In vitro studies utilizing CRH-R1d and CTRDe13 receptor ectopic over-expression in cellular models [42–44] suggest that plasma membrane TMD7short variants are hexahelical receptor proteins containing an extracellular C terminus probably consisting of EC3, the remainder of TMD7 and the C terminus. This is possibly due to reduced hydrophobicity of the residual sequence that follows EC3 and compromises the ability of the truncated TMD7 to anchor in the lipid bilayer. However, only a small fraction of TMD7-short receptor variants is localized to the plasma membrane and the majority of receptors are expressed as intracellular proteins, possibly in the endoplasmic reticulum (ER) [43,44], suggesting that the absence of TMD7 could affect receptor folding or transport to the cell surface. The biosynthetic path of GPCRs to the plasma membrane appears to involve interactions with various ER resident chaperones, including calnexin, calreticulin and the heat-shock protein (Hsp)70 family ER luminal protein BiP, which assist in the folding, processing, or cellular transport of GPCRs through the secretory pathway, and prevent export of premature cargo proteins [for detailed reviews see Refs 45 and 46]. In addition, GPCR oligomer-
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ization appears to be a pre-requisite to pass the ER quality control mechanism [46]. Plasma membrane expression of some Secretin GPCRs appears to be regulated by nonconventional escort proteins, including the family of receptor activity-modifying proteins (RAMPs) [47]. RAMP association with the CT receptor-like receptor (CRLR) traffics this receptor to the cell surface. The type of associated RAMP dictates the ligand-binding properties of the receptor and, thus, expression of distinct phenotypes. A similar function has been ascribed to RAMP interaction with the CALC-R. At least four other members of the Secretin family of GPCRs (VPAC1, PTH1-R, PTH2-R and GCG-R) are capable of interacting with RAMPs and, although their cell-surface expression is not affected by their association with RAMPs [48], they might have a more general role in modulating agonist-induced cell signaling. The receptor structural determinants allowing GPCRs to pass through the ER quality control mechanism and become available for export trafficking to the plasma membrane are not well characterized. Current evidence based on studies of TMD-containing proteins suggests that ER export is a selective process and is dictated by specific ER export motifs within the cargo proteins. Several such signals, including di-acidic motifs (Asp-X-Glu or similar), pairs of aromatic (Phe-Phe, Tyr-Tyr or Phe-Tyr) or bulky hydrophobic (Leu-Leu or Ile-Leu) amino acid residues have been identified in the cytoplasmic C terminus of TMDprotein cargos [49,50]. Similar putative ER-export motifs are present in the GPCR C terminus and, although experimental evidence is inconclusive, are likely to be involved in direct binding and interaction with specific protein partners [51]. For a number of Rhodopsin GPCRs (a2B, a1B and b2- adrenoceptor and angiotensin II type 1 receptor) the F(X)6LL motif in the membrane-proximal portion of the C terminus is essential for export from the ER, suggesting that this motif has a general role in GPCR export from the ER [52]. Recently, amino acids within the IC1 have been identified as important for GPCR export from the ER [53]. Disruption of specific ER-export motifs might also lead to inappropriate GPCR oligomer formation and GPCR retention in the ER, as demonstrated for the F(X)6LL motif of a(2B)-adrenoceptor [54]. Mutation studies of CRH-R1 [41] identified the cassette G-F-F within TMD7 as being crucial for successful CRH-R1 plasma membrane expression, because its absence leads to receptor intracellular retention. It is possible that in Secretin GPCRs, amino acid motifs present within the N terminus of the TMD7 segment encoded by exon 12 are crucial for appropriate protein folding and subsequent successful export trafficking; therefore, deletion of this sequence in TMD7-short receptor variants might render specific ER-export motifs inaccessible to exporting machinery and ultimately lead to defective export trafficking properties. Lisenbee and Miller [55] studied TMD7 of SCT-R and identified Gly 7.52, the first residue encoded by exon 12 and conserved across all Secretin GPCRs, as being crucial for efficient plasma membrane sorting. This Gly residue is part of a putative helix interaction motif (-GxxxG-) present in a subgroup of Secretin GPCRs, the SCT, VIP and GHRH receptors [55]. This transmembrane 447
Review segment appears to be central in the tertiary structure of Secretin GPCRs; in a helical packing model for Secretin GPCRs core domains, the GxxxG motif forms a portion of the helical face that is directed towards TMD2 and buried within the lipid-protected core [56]. These residues might facilitate intrahelical packing interactions that stabilize the receptor core domain and/or establish the conformation required for ligand binding and activation [55]. Signaling properties Available evidence suggests that the impact of TMD7 deletion on receptor binding characteristics differs among TMD7-short variants, and reflects the relative contribution of individual structural motifs that participate in the receptor binding pocket. For example, the binding affinity of CRH-R1d and SD-VPAC2 for their cognate agonists remains intact [35,38]; by contrast CALCRDe13 exhibits an impaired binding affinity and, unlike the wild type CALC-R, is unable to discriminate between different agonists, such as human and salmon calcitonin [37]. However, all TMD7-short receptor variants identified to date exhibit impaired signaling properties to downstream intracellular effectors. Studies on the Rhodopsin family of GPCRs that might be relevant for Secretin GPCRs, proposed that TMD7 has a crucial role in the mechanism of GPCRs activation that involves inward movement of extracellular segments, especially TMD6
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and TMD7, coupled to an outward movement of the intracellular segments of these helices (the proposed global toggle switch mechanism) [57]. The inability of the plasma membrane expressed proteins to relay signals across cells suggests that these variants might act as decoy receptors capable of competing with the wild type receptors for agonist binding and absorbing peptide bioactivity. Therefore, one can hypothesize that their intracellular retention is beneficial for the organisms because it would prevent tissue unresponsiveness to hormonal stimuli. However, TMD7-short variants might exhibit a ‘‘darker side’’ in hormonal signaling regulation because they appear to act as dominant negative (DN) regulators when co-expressed with the fully active receptors, to inhibit downstream signaling activity (Figure 5). Most studies suggest that this is achieved through hetero-oligomer formation, although the mechanism appears to be receptorspecific. The CALC-RDe13 and CRH-R1d variants associate with wild type receptors to form hetero-oligomers that are trapped intracellularly. This leads to a reduction in the signaling response through inhibition of wild type receptor cell surface expression [43,44]. Interestingly, the CRH-R1d interaction with the CRH-R2 receptor ‘‘rescues’’ plasma membrane expression of CRH-R1d [42]; enhancement of cell surface expression allows CRH-R1d to act as a DN regulator and heterologously attenuate CRH-R2b signaling. This suggests that under certain conditions and
Figure 5. A model for the potential regulation of hormonal signaling by TMD7-short GPCR variants. (a) Expression of variants containing exon 12 (A1, B) are fully active and able to elicit intracellular responses upon agonist activation. (b) Co-expression of TMD7-short variants (A2) might lead to inhibition of downstream signaling activity through hetero-oligomer formation (A1-A2) that traps active receptors within the cell and reduces expression of the cell surface A1 receptor. Alternatively, the plasma membrane expression of TMD7-short variants might be rescued through hetero-oligomer formation with suitable ‘‘partner’’ receptors (A2-B). Increased cell surface expression of TMD7-short variants might allow heterologous attenuation of receptor B downstream signaling through unknown mechanisms.
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Review with suitable ‘‘partner’’ proteins, biosynthetic trafficking pathways of TMD7-short variants can be modified to transport these proteins to the plasma membrane. As mentioned above, the relationship between GPCR dimerization and the transport of several GPCRs from the ER to the cell surface is well established [58]. Such interactions have implications in the pathogenic mechanisms of diseases associated with gain or loss-of-function mutations that result in GPCR misfolding in the ER [59,60] Pharmacological rescue of conformationally defective proteins using specific molecular chaperones [60], such as suitable receptor partners, might represent an attractive approach to overcoming such protein misfolding and misrouting defects. Putative mechanisms and regulators of splicing Little is known about the mechanisms or splicing factors regulating the generation of GPCR AS-variants. The process that is initiated by the assembly of spliceosomes onto each intron is not well understood and involves multiple small nuclear ribonucleoproteins (snRNPs) and numerous accessory proteins. Current knowledge suggests that splicing activator proteins can recognize and bind to specific cis-regulatory elements to promote (splicing enhancers) or repress (splicing silencers) inclusion or exclusion of an exon in the mature mRNA. These sequences can be located in the exons or introns [61]. In this complex network of interactions, four splicing activator factors, SF2/ASF, SC35, SRp40 and SRp55, are the best studied. As mentioned, little information is available about the splicing mechanisms controlling TMD7-short GPCR mRNA expression in mammalian cells. In the only available study investigating CRH-R1 variant expression in keratinocytes and melanocytes, Pisarchik et al. [33] reported that, in contrast to other melanoma cell lines, the SKMEL188 cell line exclusively expresses CRH-R1d, the TMD7-short variant of CRH-R1. This finding suggests
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an unusual predominance of splicing mechanisms promoting constitutive exon 12 skipping. Exon 12 retention by the mature mRNA transcript that encodes the fully active CRH-R1a can be rescued through activation of protein kinase C and protein kinase A-dependent pathways, implicating Ser/Thr kinases as important modulators of splicing mechanisms regulating TMD7-short variant expression (Figure 6). SR splicing factors are major targets of phosphorylation by specific SR kinases, including the SR protein kinases Clk and SRPK, as well as other kinases found downstream of cell membrane receptor-initiated pathways, such as PKB/Akt [62–64]. A biological example of TMD7-short GPCR variant regulation of potential physiological relevance is the observed up-regulation of CRHR1d mRNA in the human myometrium during the onset of labor [65]. This change reflects a widespread pattern of altered splicing events modulating myometrial gene regulation during pregnancy and labor, and orchestrated by changes in the expression profiles of several splicing factors [66]. It is possible that the labor-induced dramatic down–regulation of the splicing factors SRp40 and SC35, in particular, might modify the cellular environment in a way that favors exon 12 skipping and up-regulation of the TMD7-short CRHR1 mRNA variant. Understanding the basic molecular splicing mechanisms that govern TMD7-short Secretin GPCR variants expression will allow elucidation of their role in hormonal signaling regulation and mammalian pathophysiology. TMD7 splicing: implications for hormonal signallng in mammalian biology and pathophysiology Secretin GPCR ligands have important roles in a wide spectrum of physiological and pathophysiological processes ranging from adaptation to stressful environment to skeleton development, reproduction and control of energy balance and homeostasis; not surprisingly, these
Figure 6. Hypothetical regulation of CRHR1a and CRHR1d mRNA expression in SKMEL188 cells. Under basal conditions the splicing machinery promotes exon 12 skipping, possibly involving predominant activity of splicing repressors such as the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) or inactivation of splicing activators such as SF2/ASF, SC35, SRp40 and SRp55. Exon 12 retention in the mature mRNA transcript that encodes the fully active CRH-R1a can be enhanced through activation of protein kinase C- and A-dependent pathways that possibly reverse the balance of splicing activators/repressors.
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Review receptors represent targets for many currently used and novel drugs [67–72]. The true role of TMD7-short variants in modulating hormonal actions is largely unknown. All available evidence (albeit limited and circumstantial) suggests that in certain pathophysiological settings these variants might have a role in dampening hormonal bioactivity by attenuating receptor cell membrane expression and functional activity. Hetero-oligomer formation is a possible candidate mode of action, although this remains to be confirmed. Perhaps the most representative example of the potential of TMD7-short variants to influence hormonal bioactivity is the observation that SD-VPAC2 overexpression in Helper T Cells, Th2 cells, attenuates endogenous VIP-induced IL-4 production and prevents nuclear translocation of c-Maf and JunB proteins [73]. Concluding remarks and future perspectives Despite considerable diversity of GPCRs during evolution, necessary to ensure specificity of signal propagation in response to a diverse array of stimuli, these cell membrane proteins retain some conserved structural features with crucial roles for receptor function. An example of these conserved functional microdomains is the DRY motif, which is located at the boundary between TMD3 and IC2 of Rhodopsin GPCRs and has a pivotal role in regulating GPCR conformational states [74]. Similarly, the TMD7 of family Secretin GPCRs contains a highly conserved stretch of 14 amino acids encoded by a single exon across all family members. The presence of a highly conserved splice site can potentially remove this segment and generate receptor variants through alternative splicing, offering a novel regulatory mechanism to fine-tune the biological activity of Secretin GPCRs, and complement various post-transcriptional and post-translational modifications. Why is the TMD7 in this class of GPCRs designed to be targeted specifically by splicing and what is the biological and physiological significance of this splicing event? The precise functional significance of the TMD7 domain and the reasons for its unique conservation across Secretin GPCRs remain unknown, and further investigation is required (Box 1). Pre-mRNA splicing is emerging as a potent mechanism that allows generation of multiple receptor variants to form ‘‘a highly diversified receptor milieu’’ [14] that might be of potentially fundamental importance for cell biology and mammalian physiology and pathophysiology as well as future drug development. Although the mechanisms that govern alternative splicing decisions of Secretin GPCRs Box 1. Outstanding questions: – What is the structural role of the 14 amino acid segment encoded by exon 12 in Secretin GPCRs? – Does the expression of TMD7-short GPCR variants exhibit tissuespecific characteristics? – What is the true role of the TMD7-short variants in hormonal signaling and mammalian pathophysiology? – What are the splicing mechanisms controlling expression of these variants and do they exhibit conserved features? – What are the rules that govern hetero-oligomerization of TMD7short variants and partner receptor selection
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are virtually unknown, there is now strong evidence to suggest that the expression of crucial players such as various splicing factors is altered in various disease states (i.e. cancer). Thus, it is attractive to speculate that such modifications can result in aberrant patterns of GPCRs splicing with important consequences for hormonal signaling. There is now a wealth of data from global genomic and bioinformatics approaches available that can be used to test hypotheses and advance our understanding of the Secretin GPCR splicing regulation and understand the biological importance of this mechanism. Acknowledgements The authors thank Professor N. Europe-Finner for his helpful comments and suggestions.
References 1 Schellenberg, M.J. et al. (2008) Pre-mRNA splicing: a complex picture in higher definition. Trends Biochem. Sci. 33, 243–246 2 Maniatis, T. and Tasic, B. (2002) Alternative pre-mRNA splicing and proteome expansion in metazoans. Nature 418, 236–243 3 Sharp, P.A. (1994) Split genes and RNA splicing. Cell 77, 805–815 4 Wang, E.T. et al. (2008) Alternative isoform regulation in human tissue transcriptomes. Nature 456, 470–476 5 Xu, Q. et al. (2002) Genome-wide detection of tissue-specific alternative splicing in the human transcriptome. Nucleic Acids Res. 30, 3754–3766 6 Lewis, B.P. et al. (2003) Evidence for the widespread coupling of alternative splicing and nonsense-mediated mRNA decay in humans. Proc. Natl. Acad. Sci. U.S.A. 100, 189–192 7 Kreth, S. et al. (2008) Differential expression of 50 -UTR splice variants of the adenosine A2A receptor gene in human granulocytes: identification, characterization, and functional impact on activation. FASEB J. 22, 3276–3286 8 Monsma, F.J., Jr et al. (1989) Multiple D2 dopamine receptors produced by alternative RNA splicing. Nature 342, 926–929 9 Gentles, A.J. and Karlin, S. (1999) Why are human G-protein-coupled receptors predominantly intronless? Trends Genet. 15, 47–49 10 Valencia, P. et al. (2008) Splicing promotes rapid and efficient mRNA export in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 105, 3386– 3391 11 Wang, G.S. and Cooper, T.A. (2007) Splicing in disease: disruption of the splicing code and the decoding machinery. Nat. Rev. Genet. 8, 749– 761 12 Srebrow, A. and Kornblihtt, A.R. (2006) The connection between splicing and cancer. J. Cell Sci. 119, 2635–2641 13 Kishore, S. and Stamm, S. (2006) The snoRNA HBII-52 regulates alternative splicing of the serotonin receptor 2C. Science 311, 230–232 14 Einstein, R. et al. (2008) Alternative splicing of the G protein-coupled receptor superfamily in human airway smooth muscle diversifies the complement of receptors. Proc. Natl. Acad. Sci. U.S.A. 105, 5230– 5235 15 Laitinen, T. et al. (2004) Characterization of a common susceptibility locus for asthma-related traits. Science 304, 300–304 16 Bertolino, A. et al. (2009) Functional variants of the dopamine receptor D2 gene modulate prefronto-striatal phenotypes in schizophrenia. Brain 32, 417–425 17 Sarzani, R. et al. (2009) Altered pattern of cannabinoid type 1 receptor expression in adipose tissue of dysmetabolic and overweight patients. Metabolism 58, 361–367 18 Sztainberg, Y. et al. (2009) A novel corticotropin-releasing factor receptor splice variant exhibits dominant negative activity: a putative link to stress-induced heart disease. FASEB J. 23, 2186–2196 19 Fredriksson, R. et al. (2003) The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol. 63, 1256–1272 20 Kilpatrick, G.J. et al. (1999) 7TM receptors: the splicing on the cake. Trends Pharmacol. Sci. 20, 294–301 21 Bockaert, J. and Pin, J.P. (1999) Molecular tinkering of G proteincoupled receptors: an evolutionary success. EMBO J. 18, 1723–1729 22 Minneman, K.P. (2001) Splice variants of GPCRs. Molecular Interventions 1, 108–116
Review 23 Blondel, O. et al. (1998) Cloning, expression, and pharmacology of four human 5-hydroxytryptamine 4 receptor isoforms produced by alternative splicing in the carboxyl terminus. J. Neurochem. 70, 2252–2261 24 Spengler, D. et al. (1993) Differential signal transduction by five splice variants of the PACAP receptor. Nature 365, 170–175 25 Harmar, A.J. (2001) Family-B G-protein-coupled receptors. Genome Biol. 2, 3013.1–3013.10 26 Nordstro¨m, K.J. et al. (2009) The Secretin GPCRs descended from the family of Adhesion GPCRs. Mol. Biol. Evol. 26, 71–84 27 Xiong, Y. et al. (1995) Signaling properties of mouse and human corticotropin-releasing factor (CRF) receptors: decreased coupling efficiency of human type II CRF receptor. Endocrinology 136, 1828– 1834 28 Gorn, A.H. et al. (1995) Expression of two human skeletal calcitonin receptor isoforms cloned from a giant cell tumor of bone. The first intracellular domain modulates ligand binding and signal transduction. J. Clin. Invest. 95, 2680–2691 29 Markovic, D. et al. (2006) Differential responses of corticotropinreleasing hormone receptor type 1 variants to protein kinase C phosphorylation. J. Pharmacol. Exp. Ther. 319, 1032–1042 30 Nicot, A. and DiCicco-Bloom, E. (2001) Regulation of neuroblast mitosis is determined by PACAP receptor isoform expression. Proc. Natl. Acad. Sci. U.S.A. 98, 4758–4763 31 Harada, N. et al. (2008) A novel GIP receptor splice variant influences GIP sensitivity of pancreatic b-cells in obese mice. Am. J. Physiol. Endocrinol. Metab. 294, E61–E68 32 Nag, K. et al. (2007) Headless splice variant acting as dominant negative calcitonin receptor. Biochem. Biophys. Res. Commun. 362, 1037–1043 33 Pisarchik, A. and Slominski, A. (2004) Molecular and functional characterization of novel CRFR1 isoforms from the skin. Eur. J. Biochem. 271, 2821–2830 34 Chen, A.M. et al. (2005) A soluble mouse brain splice variant of type 2alpha corticotropin-releasing factor (CRF) receptor binds ligands and modulates their activity. Proc. Natl. Acad. Sci. U. S. A. 102, 2620–2625 35 Grammatopoulos, D.K. et al. (1999) A novel spliced variant of the type 1 corticotropin-releasing hormone receptor with a deletion in the seventh transmembrane domain present in the human pregnant term myometrium and fetal membranes. Mol. Endocrinol. 13, 2189–2202 36 Ding, C. et al. (1995) Identification of an alternative spliced form of PTH/PTHrP receptor mRNA in immrtalized renal tubular cells J. Bone Miner. Res. 10 (suppl. 1), S484 37 Shyu, J.F. et al. (1996) The deletion of 14 amino acids in the seventh transmembrane domain of a naturally occurring calcitonin receptor isoform alters ligand binding and selectively abolishes coupling to phospholipase C. J. Biol. Chem. 271, 31127–31134 38 Grinninger, C. et al. (2004) A natural variant type II G protein-coupled receptor for vasoactive intestinal peptide with altered function. J. Biol. Chem. 279, 40259–40262 39 Vanti, W.B. et al. (2003) Novel human G-protein-coupled receptors. Biochem. Biophys. Res. Commun. 305, 67–71 40 Harikumar, K.G. et al. (2007) Transmembrane segment IV contributes a functionally important interface for oligomerization of the class II G Protein-coupled Secretin receptor. J. Biol. Chem. 282, 30363–30372 41 Harikumar, K.G. et al. (2008) Pattern of intra-family heterooligomerization involving the G-protein-coupled Secretin receptor. J. Mol. Neurosci. 36, 279–285 42 Markovic, D. et al. (2008) Structural determinants critical for localization and signaling within the seventh transmembrane domain of the type 1 corticotropin releasing hormone receptor: lessons from the receptor variant R1d. Mol. Endocrinol. 22, 2505–2519 43 Zmijewski, M.A. and Slominski, A.T. (2008) CRF1 receptor splicing in epidermal keratinocytes: potential biological role and environmental regulations. J. Cell Physiol. 218, 593–602 44 Seck, T. et al. (2004) The De13 isoform of the calcitonin receptor forms a six transmembrane domain receptor with dominant negative effects on receptor surface expression and signaling. Mol. Endocrinol. 19, 2132–2144 45 Achour, L. et al. (2008) An escort for GPCRs: implications for regulation of receptor density at the cell surface. Trends Pharmacol. Sci. 29, 528–535
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46 Bulenger, S. et al. (2005) Emerging role of homo- and heterodimerization in G-protein-coupled receptor biosynthesis and maturation. Trends Pharmacol. Sci. 26, 131–137 47 Conner, A.C. et al. (2004) Heterodimers and family-B GPCRs: RAMPs, CGRP and adrenomedullin. Biochem. Soc. Trans. 32, 843–846 48 Christopoulos, A. et al. (2003) Novel receptor partners and function of receptor activity-modifying proteins. J. Biol. Chem. 278, 3293– 3297 49 Tan, C.M. et al. (2004) Membrane trafficking of G protein-coupled receptors. Annu. Rev. Pharmacol. Toxicol. 44, 559–609 50 Duvernay, M.T. et al. (2005) The regulatory mechanisms of export trafficking of G protein-coupled receptors. Cell. Signal. 17, 1457– 1465 51 Bermak, J.C. et al. (2001) Regulation of transport of the dopamine D1 receptor by a new membrane-associated ER protein. Nat. Cell Biol. 3, 492–498 52 Duvernay, M.T. et al. (2009) Anterograde trafficking of G proteincoupled receptors: function of the C-terminal F(X)6LL motif in export from the endoplasmic reticulum. Mol. Pharmacol. 75, 751–761 53 Duvernay, M.T. et al. (2009) A single conserved leucine residue on the first intracellular loop regulates ER export of G protein-coupled receptors. Traffic 10, 552–566 54 Zhou, F. et al. (2006) Cell-surface targeting of alpha2-adrenergic receptors – inhibition by a transport deficient mutant through dimerization. Cell. Signal. 18, 318–327 55 Lisenbee, C.S. and Miller, L.J. (2006) Secretin receptor oligomers form intracellularly during maturation through receptor core domains. Biochemistry 45, 8216–8226 56 Donnelly, D. (1997) The arrangement of the transmembrane helices in the Secretin receptor family of G-protein-coupled receptors. FEBS Lett. 409, 431–436 57 Schwartz, T.W. et al. (2006) Molecular mechanism of 7TM receptor activation–a global toggle switch model. Annu. Rev. Pharmacol. Toxicol. 46, 481–519 58 Salahpour, A. et al. (2004) Homodimerization of the b2-adrenergic receptor as a prerequisite for cell surface targeting. J. Biol. Chem. 279, 33390–33397 59 Ulloa-Aguirre, A. and Conn, P.M. (2009) Targeting of G protein-coupled receptors to the plasma membrane in health and disease. Front. Biosci. 14, 973–994 60 Conn, P.M. and Janovick, J.A. (2009) Drug development and the cellular quality control system. Trends Pharmacol. Sci. 30, 228–233 61 Blencowe, B.J. (2000) Exonic splicing enhancers: mechanism of action, diversity and role in human genetic diseases. Trends Biochem. Sci. 25, 106–110 62 Sanford, J.R. et al. (2005) Reversible phosphorylation differentially affects nuclear and cytoplasmic functions of splicing factor 2/ alternative splicing factor. Proc. Natl. Acad. Sci. U.S.A. 102, 15042– 15047 63 Patel, N.A. et al. (2001) Insulin regulates alternative splicing of protein kinase C beta II through a phosphatidylinositol 3-kinasedependent pathway involving the nuclear serine/arginine-rich splicing factor, SRp40, in skeletal muscle cells. J. Biol. Chem. 276, 22648–22654 64 Hagiwara, M. (2005) Alternative splicing: a new drug target of the postgenome era. Biochim. Biophys. Acta 1754, 324–331 65 Markovic, D. et al. (2007) The onset of labor alters corticotropinreleasing hormone type 1 receptor variant expression in human myometrium: putative role of interleukin-1beta. Endocrinology 148, 3205–3213 66 Tyson-Capper, A.J. (2007) Alternative splicing: an important mechanism for myometrial gene regulation that can be manipulated to target specific genes associated with preterm labour. BMC Pregnancy Childbirth. 7 (suppl. 1), S13 67 Kronenberg, H.M. (2006) PTHrP and skeletal development. Ann. NY. Acad. Sci. 1068, 1–13 68 Charmandari, E. et al. (2005) Endocrinology of the stress response. Annu. Rev. Physiol. 67, 259–284 69 Kuperman, Y. and Chen, A. (2008) Urocortins: emerging metabolic and energy homeostasis perspectives. Trends Endocrinol. Metab. 19, 122–129 70 Grammatopoulos, D.K. and Chrousos, G.P. (2002) Functional characteristics of CRH receptors and potential clinical applications
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Focus on the splicing of Secretin GPCRs transmembrane-domain 7 Danijela Markovic§ and Dimitris K. Grammatopoulos GPCR Pathophysiology Group, Division of Endocrinology and Metabolism, Clinical Sciences Research Institute, Warwick Medical School, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK
The family of G-protein coupled receptors (GPCRs) is one of the largest protein families in the mammalian genome with a fundamental role in cell biology. GPCR activity is finely tuned by various transcriptional, post-transcriptional and post-translational mechanisms. Alternative pre-mRNA splicing is now emerging as a crucial process regulating GPCR biological function. Intriguingly, this mechanism appears to extensively target the Secretin family of GPCRs, especially the exon that encodes a 14 amino acid sequence that forms the distal part of 7th transmembrane helix, and exhibits an unusually high level of sequence conservation among most Secretin GPCRs. Do the ‘‘TMD7-short’’ receptor variants have a role as novel regulators of GPCR signallng and, if so, what are the implications for hormonal actions and physiology? Alternative pre-mRNA splicing and GPCR superfamily diversity Mammalian evolution is tightly linked to highly conserved as well as divergent aspects of gene function. An important evolutionary mechanism generating considerable gene divergence and protein complexity is alternative pre-mRNA splicing [1,2]. This mechanism, discovered 30 years ago, allows production of multiple transcript isoforms from a single gene through exon skipping, alternative exon insertions, use of alternative 50 splice site and 30 splice site, and intron retention. These novel exonic combinations offer considerable plasticity toward changing environmental pressures, or stage of development. Although alternative splicing (AS) was originally considered to be an unusual event occurring in only 5% of human genes [3], this view has now been considerably revised. Indeed, computational analysis of AS has estimated that more than 90% of genes in humans are alternatively spliced [4]. Genome-wide analysis of expressed sequence tags (ESTs), datasets for discovering tissue-specific AS mechanisms, suggests that the brain contains 18% of all tissue-specific AS (TS-AS) events. Peripheral tissues with the highest number of TSAS forms are the retina, lung, liver, pancreas, placenta, ovary, uterus, testis, lymph, muscle and skin [5]. In addition, AS can provide a functional on/off switch for gene regulation because as many as one-third of AS events insert premature termination codons that usually result in mRNA degradation by nonsense-mediated decay [6]. Corresponding author: Grammatopoulos, D.K. ([email protected]) § Current address: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester LE1 9HN, UK..
A gene family extensively targeted by AS mechanisms is the superfamily of G-protein coupled receptors (GPCRs). These cell-surface proteins are the largest family of signaling molecules, with more than 1000 members, and fundamental roles in cell biology. AS appears to have a major role in the cellular machinery regulating GPCRs expression and activity through generation of functionally distinct receptor variants. AS of noncoding exons present in the 5’-untranslated region (UTR) of GPCR genes might be important for the efficient translation into protein [7]. The first GPCR splice variants were reported in 1989 for the rat D2 dopamine receptor [8], and subsequently a growing number of other GPCR genes have been identified exhibiting sequence diversity. An important prerequisite for generation of multiple GPCR splice variants is the presence of intronic sequences in their open reading frames (ORFs). Interestingly, it is believed that most mammalian GPCRs (>90%) lack introns in their protein-coding regions [9], a property found in less than 5% of human genes. At present, we do not understand why an intronless genomic architecture is preferred among mammalian GPCRs. It is possible that intronless GPCR genes are evolutionarily advantageous in complex eukaryotes [9], because of higher transcriptional fidelity and translational efficiency, although in a number of cases introns increase gene expression levels [10]. It is certainly intriguing that retaining introns allows some GPCRs to enrich their molecular diversity and potential functionality through encoded GPCR variants. Given the fundamental role of AS in mammalian biology, it is not surprising that disruption of this mechanism might contribute to the pathogenic mechanisms of disease or disease susceptibility. In fact, an unexpectedly large number of exonic mutations can cause disease by disrupting the splicing code. It is now well documented that certain types of cancer are associated with splicing abnormalities resulting in abnormal regulation of various proteins [11,12]. In this context, aberrant AS events affecting GPCRs function and hormonal responses might be a major determinant of disease development. The PraderWilli syndrome is the first example of a genetic disorder where a defect in pre-mRNA splicing and processing of the serotonin receptor 2C has been identified [13]. Furthermore, GPCR splicing might be important in the pathogenesis of various polygenic disorders, such as asthma, schizophrenia, obesity and stress-induced heart disease. [14–18]. A multitude of AS events modulating GPCR structural characteristics and function have been described that can
0968-0004/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2009.06.002
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generate distinct GPCR protein variants. Although the relationship between AS and protein structural modification appears to be unique for each GPCR, depending on its genomic architecture, recent evidence suggests that in some GPCR families, highly conserved structural motifs offer the potential for common patterns of specific AS events that might affect the structure of GPCRs and signaling potency of a plethora of hormonal signals. This previously unrecognised splicing potential highlights the multifaceted regulation of GPCRs in mammalian cells. Impact of alternative splicing on GPCR structure and function According to the GRAFS classification system [19], the superfamily of mammalian GPCRs can be broadly subdivided into five families according to their protein sequence homology, the ligand structure, and receptor function. These are: the Glutamate (G) family, Rhodopsin (R) family, Adhesion (A) family, Frizzled/Taste2 (F) family, and the Secretin (S) family [19]. AS variants have been described for more than 50 GPCRs [20–22]. Many GPCRs have multiple variants and some are species-specific [23] or exhibit tissue-specific distribution. A survey of GPCR splicing patterns suggests that AS-related structural variations can be divided into three main groups: (a) variations in the intracellular Cterminal tail length and sequence; (b) variations in the third intracellular (IC3) loop [24]; and (c) variations in the extracellular N terminus. By contrast, splice variants in the putative extracellular loops (EC) or transmembrane domains (TMD) are less frequent. Ultimately, the structural alterations resulting from the splice variations would determine the impact on receptor function [for detailed reviews see Refs 20 and 22].
The Secretin family of GPCRs: biology and splicing patterns The Secretin (or ‘‘brain-gut’’ neuropeptide) GPCRs (Table 1) have been identified throughout mammalian genomes, but not in plants, fungi or prokaryotes. All Secretin GPCRs have a complex genomic architecture comprising multiple exons and introns which appear to be extensively targeted by AS events. The Secretin GPCRs In humans, the Secretin receptors, encoded by 15 genes, and the Adhesion receptors, encoded by 33 genes, represent two distinct but related subfamilies of GPCR [25]. Evolution studies suggest that the Secretin GPCRs family could have descended from a subgroup of Adhesion GPCRs that contains receptors including GPR133 and GPR144 [26]. Secretin GPCRs bind large polypeptide hormones that share a high degree of amino acid identity and most often act in a paracrine or autocrine manner. Phylogenetic relationship analysis between the members of this family according to TMD1 to TMD7 sequence identities showed that the tree has four main subgroups: the corticotropinreleasing hormone (CRH) and Rs/calcitonin (CALC)-Rs, which are the two most ancient GPCR groups within the family [26], the parathyroid hormone (PTH)-Rs, glucagonlike peptide (GLP)-Rs/glucagon (GCG)-R/gastric inhibitory polypeptide (GIP)-R and the subgroup including Secretin and four other receptors [19] (Figure 1). Alternatively spliced variants of Secretin GPCRs Secretin GPCRs exhibit a diverse splicing pattern and a plethora of mRNA splice variants has been identified (Figure 2) in a wide range of tissues and cell types in
Table 1. Alternative splicing in secretin GPCRs. Receptor name
Gene name
Ligands
Calcitonin receptor
CALCR
Calcitonin receptor-like receptor
CALCRL
Corticotropin releasing hormone 1 receptor (CRH-R1) Corticotropin releasing hormone 2 receptor (CRH-R2) Gastric inhibitory polypeptide (GIP) receptor Glucagon receptor Glucagon-related peptide 1 (GLP-1) receptor Glucagon-related peptide 2 (GLP-2) receptor Growth hormone releasing hormone (GHRH) receptor PAC1 receptor
CRHR1
Calcitonin, amylin, calcitonin gene-related peptide Calcitonin gene-related peptide, adrenomedullin CRH, urocortin (UCN)
CRHR2
Parathyroid hormone 1 (PTH1) receptor Parathyroid hormone 2 (PTH2) receptor Secretin receptor VPAC1 receptor
PTHR1
VPAC2 receptor
VIPR2
444
Length of wild type receptor (amino acids) 474
Number of exons 12
461
12
415
14
UCN, UCN II, UCN III, CRH
411
12
GIPR
GIP
466
13
GCGR GLP1R
Glucagon GLP-1
477 463
13 13
GLP2R
GLP-2
553
13
GHRHR
GHRH
423
13
ADCYAP1R1
Pituitary adenylate cyclase-activating polypeptide (PACAP) Parathyroid hormone, PTH-related peptide Tuberoinfundibular peptide of 39 residues (TIP39) secretin Vasoactive intestinal peptide (VIP), PACAP VIP, PACAP
468
15
593
14
550
13
440 457
13 13
438
13
PTHR2 SCTR VIPR1
Review
Figure 1. Phylogenetic relationships of the Secretin family of GPCRs. These relationships are based on the sequence similarity of TMD1 and TMD7 according to Fredriksson et al. [19]. The tree consists of four main subgroups as indicated by the colored ovals.
human and other mammals. The resulting structural modifications might alter ligand binding and/or receptor signaling properties. For example, AS resulting in modifications of IC1 amino acid length of the CRH-R1 [27] and CALC-R receptors [28] appears to modulate G-protein coupling and post-translational modification [29]. Recent data have implicated some of these receptor variants in
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mammalian development and physiology as well as the pathophysiological mechanisms of disease [30,31]. Although the specific biological function of these receptor variants remains to be elucidated, it appears that some might act as dominant negative (DN) regulators of hormonal responses by interfering with wild type receptor cell membrane expression or activation by its cognate agonists [32–34]. The relevance of these receptor variants for mammalian physiology has been questioned, primarily because the lack of variant-specific antibodies prevents conclusive demonstration of protein expression in native tissues. Although aberrant or ‘‘random’’ splicing should always be considered as a potential source of receptor sequence variation, it is now evident that specific mechanisms have been developed or retained during mammalian evolution that allow the generation of discrete mRNAs and potentially encode functionally diverse protein isoforms. This idea is supported by the results of sequence comparison studies, which suggest that the overall complexity and splicing patterns are highly conserved across all members of Secretin GPCRs. A number of well conserved splice sites have been identified in sequences encoding amino acids present in EC1 and EC2, TMDs 4, 5, and 7 and within IC1 and IC3 (the latter is absent from the CALC-R/calcitonin-gene related peptide (CGRP)-R group of receptors) [26] (Figure 2). These conserved splice sites are present also in Adhesion GPCRs; in particular, the vertebrate orthologs of GPR144 and GPR133 have most (six) or all (seven) of the conserved splice sites found in Secretin GPCRs. This in silico prediction analysis is supported by in vitro studies that identified several predicted mRNA splice variants in mammalian tissues and cells; for example, mRNA variants at the
Figure 2. Examples of Secretin GPCR splice variants. A splicing map depicting examples of the different types of mRNA splice variants identified, involving members of the Secretin family of GPCRs, and their potential structural/functional consequences. Stars indicate the position of splice sites that are conserved in Secretin and Adhesion V GPCRs according to Nordstro¨m et al. [26].
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Figure 3. The conservation of exons in the Secretin family GPCRs. (a) Alignment of Secretin GPCRs according to individual exon nucleotide sequence length combinations demonstrating conservation of the -61 (pink) and 42 bp (red) exons across all members of Secretin GPCRs. (b) Amino acid sequence alignment as encoded by exon 12 (42 bp) in Secretin GPCRs. A pink background indicates residues conserved in all GPCRs; a blue background indicates residues conserved in 10 out of 15 GPCRs.
IC1 conserved splicing site, which permits insertion of variable length amino acid sequences and extension of IC1, have been described for the CRH-R1 and CALC-R [27,28]. A splicing mechanism of particular interest leads to an in-frame deletion of a 42 bp exon (exon 12 in the CRH-R1 genomic sequence) and loss of a 14 amino acid cassette from the C-terminal end of the putative TMD7 that allows generation of ‘‘TMD7-short’’ receptor variants. Splice variants arising from this exon deletion have been described for four members of the Secretin family, the CRH-R1 (R1d), CALC-R (CTRD13), PTH-R, and the type II receptor for vasoactive intestinal peptide (VIP)/PACAP (SD-VPAC2) [35–38], suggesting the presence of a highly conserved splicing ‘‘hot-spot’’. A closer look at the TMD7-short receptor variants Comparative sequence analysis uncovers some fascinating insights into the unique position of exon 12 within the genomic architecture of Secretin GPCRs. All 15 genes contain the 42 bp exon, which is present also in the genomic sequence of several members of the Adhesion family of GPCRs [39]. This raises the possibility that this exon has been conserved or ‘‘protected’’ throughout evolution of Secretin GPCRs to perform a function that is preserved in all members of the family. For example, these residues might be crucial for maintaining the proper three-dimensional configuration of the membrane-spanning portions of the receptor. The only other exon that appears to be conserved across the Secretin GPCR family is a 61 bp exon that is found four exons upstream of exon 12 (Figure 3a). 446
This exon, which is absent from the Adhesion family of GPCRs, encodes amino acid sequences of the TMD4 and Nterminal segment of EC2. Intriguingly, there is one striking difference between these two exons: the amino acid sequence encoded by exon 12, which follows the sequence pattern G-(L/F)-X-V-S/A-X-X-F/Y-C-F/Y-X-N-X-E, is highly conserved across all Secretin GPCRs. Frequency analysis of 14 individual amino acids in particular positions shows that 44% are identical in all receptors and 64% in at least 10 out of 15 Secretin GPCRs (Figure 3b). This exon contains the highest degree of conserved amino acids across the entire receptor sequence. By contrast, exon 8 (based on the CRH-R1 genomic sequence) encodes an unusually hypervariable amino acid sequence with only 10.5% of amino acids being conserved in ten or more Secretin GPCRs (Figure 4). Only the N and C-terminal segments exhibit a higher degree of amino acid variability, consistent with their well established roles in ligand recognition and signaling specificity. Key residues encoded by exon 8, such as a highly conserved Gly 4.52 (using the nomenclature of Ballesteros and Weinstein*) are found within the lipidexposed face of TMD4 of SCT-R and appear to form a functionally important interface for oligomerization [40]. This might provide some insights about the requirement for such sequence diversity. Extensive intra-family constitutive oligomerization has been reported for the Secretin GPCRs [41] and, thus, a hypervariable segment might ensure specificity of receptor–receptor interactions. * The most highly conserved residues in each transmembrane helix are given the number 50 in the Ballesteros and Weinstein nomenclature. In the prototypic rhodopsin receptor these are N1.50, D2.50, R3.50, W4.50, P5.50, P6.50 and P7.50.
Review
Figure 4. Frequency analysis of amino acids encoded by individual exons and their position in the structure of the GPCR. The analysis was based on amino acids conserved in at least 10 out of 15 Secretin GPCRs. Exon 12 (amino acids in the C terminus of TMD7) contains the highest degree of conserved amino acids across the entire receptor sequence. In contrast, exons 1, 2 and 13 (amino acids in the N and C terminus) encode hypervariable amino acid sequences with less than 10% of the amino acids being conserved in ten or more Secretin GPCRs.
Structural modifications and cellular expressionlocalization The loss of 14 amino acids from the C-terminal end of the putative TMD7 appears to have a major impact on receptor protein folding orientation and subcellular localization. In vitro studies utilizing CRH-R1d and CTRDe13 receptor ectopic over-expression in cellular models [42–44] suggest that plasma membrane TMD7short variants are hexahelical receptor proteins containing an extracellular C terminus probably consisting of EC3, the remainder of TMD7 and the C terminus. This is possibly due to reduced hydrophobicity of the residual sequence that follows EC3 and compromises the ability of the truncated TMD7 to anchor in the lipid bilayer. However, only a small fraction of TMD7-short receptor variants is localized to the plasma membrane and the majority of receptors are expressed as intracellular proteins, possibly in the endoplasmic reticulum (ER) [43,44], suggesting that the absence of TMD7 could affect receptor folding or transport to the cell surface. The biosynthetic path of GPCRs to the plasma membrane appears to involve interactions with various ER resident chaperones, including calnexin, calreticulin and the heat-shock protein (Hsp)70 family ER luminal protein BiP, which assist in the folding, processing, or cellular transport of GPCRs through the secretory pathway, and prevent export of premature cargo proteins [for detailed reviews see Refs 45 and 46]. In addition, GPCR oligomer-
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ization appears to be a pre-requisite to pass the ER quality control mechanism [46]. Plasma membrane expression of some Secretin GPCRs appears to be regulated by nonconventional escort proteins, including the family of receptor activity-modifying proteins (RAMPs) [47]. RAMP association with the CT receptor-like receptor (CRLR) traffics this receptor to the cell surface. The type of associated RAMP dictates the ligand-binding properties of the receptor and, thus, expression of distinct phenotypes. A similar function has been ascribed to RAMP interaction with the CALC-R. At least four other members of the Secretin family of GPCRs (VPAC1, PTH1-R, PTH2-R and GCG-R) are capable of interacting with RAMPs and, although their cell-surface expression is not affected by their association with RAMPs [48], they might have a more general role in modulating agonist-induced cell signaling. The receptor structural determinants allowing GPCRs to pass through the ER quality control mechanism and become available for export trafficking to the plasma membrane are not well characterized. Current evidence based on studies of TMD-containing proteins suggests that ER export is a selective process and is dictated by specific ER export motifs within the cargo proteins. Several such signals, including di-acidic motifs (Asp-X-Glu or similar), pairs of aromatic (Phe-Phe, Tyr-Tyr or Phe-Tyr) or bulky hydrophobic (Leu-Leu or Ile-Leu) amino acid residues have been identified in the cytoplasmic C terminus of TMDprotein cargos [49,50]. Similar putative ER-export motifs are present in the GPCR C terminus and, although experimental evidence is inconclusive, are likely to be involved in direct binding and interaction with specific protein partners [51]. For a number of Rhodopsin GPCRs (a2B, a1B and b2- adrenoceptor and angiotensin II type 1 receptor) the F(X)6LL motif in the membrane-proximal portion of the C terminus is essential for export from the ER, suggesting that this motif has a general role in GPCR export from the ER [52]. Recently, amino acids within the IC1 have been identified as important for GPCR export from the ER [53]. Disruption of specific ER-export motifs might also lead to inappropriate GPCR oligomer formation and GPCR retention in the ER, as demonstrated for the F(X)6LL motif of a(2B)-adrenoceptor [54]. Mutation studies of CRH-R1 [41] identified the cassette G-F-F within TMD7 as being crucial for successful CRH-R1 plasma membrane expression, because its absence leads to receptor intracellular retention. It is possible that in Secretin GPCRs, amino acid motifs present within the N terminus of the TMD7 segment encoded by exon 12 are crucial for appropriate protein folding and subsequent successful export trafficking; therefore, deletion of this sequence in TMD7-short receptor variants might render specific ER-export motifs inaccessible to exporting machinery and ultimately lead to defective export trafficking properties. Lisenbee and Miller [55] studied TMD7 of SCT-R and identified Gly 7.52, the first residue encoded by exon 12 and conserved across all Secretin GPCRs, as being crucial for efficient plasma membrane sorting. This Gly residue is part of a putative helix interaction motif (-GxxxG-) present in a subgroup of Secretin GPCRs, the SCT, VIP and GHRH receptors [55]. This transmembrane 447
Review segment appears to be central in the tertiary structure of Secretin GPCRs; in a helical packing model for Secretin GPCRs core domains, the GxxxG motif forms a portion of the helical face that is directed towards TMD2 and buried within the lipid-protected core [56]. These residues might facilitate intrahelical packing interactions that stabilize the receptor core domain and/or establish the conformation required for ligand binding and activation [55]. Signaling properties Available evidence suggests that the impact of TMD7 deletion on receptor binding characteristics differs among TMD7-short variants, and reflects the relative contribution of individual structural motifs that participate in the receptor binding pocket. For example, the binding affinity of CRH-R1d and SD-VPAC2 for their cognate agonists remains intact [35,38]; by contrast CALCRDe13 exhibits an impaired binding affinity and, unlike the wild type CALC-R, is unable to discriminate between different agonists, such as human and salmon calcitonin [37]. However, all TMD7-short receptor variants identified to date exhibit impaired signaling properties to downstream intracellular effectors. Studies on the Rhodopsin family of GPCRs that might be relevant for Secretin GPCRs, proposed that TMD7 has a crucial role in the mechanism of GPCRs activation that involves inward movement of extracellular segments, especially TMD6
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and TMD7, coupled to an outward movement of the intracellular segments of these helices (the proposed global toggle switch mechanism) [57]. The inability of the plasma membrane expressed proteins to relay signals across cells suggests that these variants might act as decoy receptors capable of competing with the wild type receptors for agonist binding and absorbing peptide bioactivity. Therefore, one can hypothesize that their intracellular retention is beneficial for the organisms because it would prevent tissue unresponsiveness to hormonal stimuli. However, TMD7-short variants might exhibit a ‘‘darker side’’ in hormonal signaling regulation because they appear to act as dominant negative (DN) regulators when co-expressed with the fully active receptors, to inhibit downstream signaling activity (Figure 5). Most studies suggest that this is achieved through hetero-oligomer formation, although the mechanism appears to be receptorspecific. The CALC-RDe13 and CRH-R1d variants associate with wild type receptors to form hetero-oligomers that are trapped intracellularly. This leads to a reduction in the signaling response through inhibition of wild type receptor cell surface expression [43,44]. Interestingly, the CRH-R1d interaction with the CRH-R2 receptor ‘‘rescues’’ plasma membrane expression of CRH-R1d [42]; enhancement of cell surface expression allows CRH-R1d to act as a DN regulator and heterologously attenuate CRH-R2b signaling. This suggests that under certain conditions and
Figure 5. A model for the potential regulation of hormonal signaling by TMD7-short GPCR variants. (a) Expression of variants containing exon 12 (A1, B) are fully active and able to elicit intracellular responses upon agonist activation. (b) Co-expression of TMD7-short variants (A2) might lead to inhibition of downstream signaling activity through hetero-oligomer formation (A1-A2) that traps active receptors within the cell and reduces expression of the cell surface A1 receptor. Alternatively, the plasma membrane expression of TMD7-short variants might be rescued through hetero-oligomer formation with suitable ‘‘partner’’ receptors (A2-B). Increased cell surface expression of TMD7-short variants might allow heterologous attenuation of receptor B downstream signaling through unknown mechanisms.
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Review with suitable ‘‘partner’’ proteins, biosynthetic trafficking pathways of TMD7-short variants can be modified to transport these proteins to the plasma membrane. As mentioned above, the relationship between GPCR dimerization and the transport of several GPCRs from the ER to the cell surface is well established [58]. Such interactions have implications in the pathogenic mechanisms of diseases associated with gain or loss-of-function mutations that result in GPCR misfolding in the ER [59,60] Pharmacological rescue of conformationally defective proteins using specific molecular chaperones [60], such as suitable receptor partners, might represent an attractive approach to overcoming such protein misfolding and misrouting defects. Putative mechanisms and regulators of splicing Little is known about the mechanisms or splicing factors regulating the generation of GPCR AS-variants. The process that is initiated by the assembly of spliceosomes onto each intron is not well understood and involves multiple small nuclear ribonucleoproteins (snRNPs) and numerous accessory proteins. Current knowledge suggests that splicing activator proteins can recognize and bind to specific cis-regulatory elements to promote (splicing enhancers) or repress (splicing silencers) inclusion or exclusion of an exon in the mature mRNA. These sequences can be located in the exons or introns [61]. In this complex network of interactions, four splicing activator factors, SF2/ASF, SC35, SRp40 and SRp55, are the best studied. As mentioned, little information is available about the splicing mechanisms controlling TMD7-short GPCR mRNA expression in mammalian cells. In the only available study investigating CRH-R1 variant expression in keratinocytes and melanocytes, Pisarchik et al. [33] reported that, in contrast to other melanoma cell lines, the SKMEL188 cell line exclusively expresses CRH-R1d, the TMD7-short variant of CRH-R1. This finding suggests
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an unusual predominance of splicing mechanisms promoting constitutive exon 12 skipping. Exon 12 retention by the mature mRNA transcript that encodes the fully active CRH-R1a can be rescued through activation of protein kinase C and protein kinase A-dependent pathways, implicating Ser/Thr kinases as important modulators of splicing mechanisms regulating TMD7-short variant expression (Figure 6). SR splicing factors are major targets of phosphorylation by specific SR kinases, including the SR protein kinases Clk and SRPK, as well as other kinases found downstream of cell membrane receptor-initiated pathways, such as PKB/Akt [62–64]. A biological example of TMD7-short GPCR variant regulation of potential physiological relevance is the observed up-regulation of CRHR1d mRNA in the human myometrium during the onset of labor [65]. This change reflects a widespread pattern of altered splicing events modulating myometrial gene regulation during pregnancy and labor, and orchestrated by changes in the expression profiles of several splicing factors [66]. It is possible that the labor-induced dramatic down–regulation of the splicing factors SRp40 and SC35, in particular, might modify the cellular environment in a way that favors exon 12 skipping and up-regulation of the TMD7-short CRHR1 mRNA variant. Understanding the basic molecular splicing mechanisms that govern TMD7-short Secretin GPCR variants expression will allow elucidation of their role in hormonal signaling regulation and mammalian pathophysiology. TMD7 splicing: implications for hormonal signallng in mammalian biology and pathophysiology Secretin GPCR ligands have important roles in a wide spectrum of physiological and pathophysiological processes ranging from adaptation to stressful environment to skeleton development, reproduction and control of energy balance and homeostasis; not surprisingly, these
Figure 6. Hypothetical regulation of CRHR1a and CRHR1d mRNA expression in SKMEL188 cells. Under basal conditions the splicing machinery promotes exon 12 skipping, possibly involving predominant activity of splicing repressors such as the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) or inactivation of splicing activators such as SF2/ASF, SC35, SRp40 and SRp55. Exon 12 retention in the mature mRNA transcript that encodes the fully active CRH-R1a can be enhanced through activation of protein kinase C- and A-dependent pathways that possibly reverse the balance of splicing activators/repressors.
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Review receptors represent targets for many currently used and novel drugs [67–72]. The true role of TMD7-short variants in modulating hormonal actions is largely unknown. All available evidence (albeit limited and circumstantial) suggests that in certain pathophysiological settings these variants might have a role in dampening hormonal bioactivity by attenuating receptor cell membrane expression and functional activity. Hetero-oligomer formation is a possible candidate mode of action, although this remains to be confirmed. Perhaps the most representative example of the potential of TMD7-short variants to influence hormonal bioactivity is the observation that SD-VPAC2 overexpression in Helper T Cells, Th2 cells, attenuates endogenous VIP-induced IL-4 production and prevents nuclear translocation of c-Maf and JunB proteins [73]. Concluding remarks and future perspectives Despite considerable diversity of GPCRs during evolution, necessary to ensure specificity of signal propagation in response to a diverse array of stimuli, these cell membrane proteins retain some conserved structural features with crucial roles for receptor function. An example of these conserved functional microdomains is the DRY motif, which is located at the boundary between TMD3 and IC2 of Rhodopsin GPCRs and has a pivotal role in regulating GPCR conformational states [74]. Similarly, the TMD7 of family Secretin GPCRs contains a highly conserved stretch of 14 amino acids encoded by a single exon across all family members. The presence of a highly conserved splice site can potentially remove this segment and generate receptor variants through alternative splicing, offering a novel regulatory mechanism to fine-tune the biological activity of Secretin GPCRs, and complement various post-transcriptional and post-translational modifications. Why is the TMD7 in this class of GPCRs designed to be targeted specifically by splicing and what is the biological and physiological significance of this splicing event? The precise functional significance of the TMD7 domain and the reasons for its unique conservation across Secretin GPCRs remain unknown, and further investigation is required (Box 1). Pre-mRNA splicing is emerging as a potent mechanism that allows generation of multiple receptor variants to form ‘‘a highly diversified receptor milieu’’ [14] that might be of potentially fundamental importance for cell biology and mammalian physiology and pathophysiology as well as future drug development. Although the mechanisms that govern alternative splicing decisions of Secretin GPCRs Box 1. Outstanding questions: – What is the structural role of the 14 amino acid segment encoded by exon 12 in Secretin GPCRs? – Does the expression of TMD7-short GPCR variants exhibit tissuespecific characteristics? – What is the true role of the TMD7-short variants in hormonal signaling and mammalian pathophysiology? – What are the splicing mechanisms controlling expression of these variants and do they exhibit conserved features? – What are the rules that govern hetero-oligomerization of TMD7short variants and partner receptor selection
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are virtually unknown, there is now strong evidence to suggest that the expression of crucial players such as various splicing factors is altered in various disease states (i.e. cancer). Thus, it is attractive to speculate that such modifications can result in aberrant patterns of GPCRs splicing with important consequences for hormonal signaling. There is now a wealth of data from global genomic and bioinformatics approaches available that can be used to test hypotheses and advance our understanding of the Secretin GPCR splicing regulation and understand the biological importance of this mechanism. Acknowledgements The authors thank Professor N. Europe-Finner for his helpful comments and suggestions.
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