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Billy K. C. Chow - One of the best experts on this subject based on the ideXlab platform.
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Functional Pairing of Class B1 Ligand-GPCR in Cephalochordate Provides Evidence of the Origin of PTH and PACAP/Glucagon Receptor Family
Molecular Biology and Evolution, 2015Co-Authors: Jason S. W. On, Cumming Duan, Billy K. C. ChowAbstract:: Several hypotheses have been proposed regarding the origin and evolution of the Secretin Family of peptides and receptors. However, identification of homologous ligand-receptor pairs in invertebrates and vertebrates is difficult because of the low levels of sequence identity between orthologs of distant species. In this study, five receptors structurally related to the vertebrate class B1 G protein-coupled receptor (GPCR) Family were characterized from amphioxus (Branchiostoma floridae). Phylogenetic analysis showed that they clustered with vertebrate parathyroid hormone receptors (PTHR) and pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagon receptors. These PTHR-like receptors shared synteny with several PTH and PACAP/glucagon receptors identified in spotted gar, Xenopus, and human, indicating that amphioxus preserves the ancestral chordate genomic organization of these receptor subfamilies. According to recent data by Mirabeau and Joly, amphioxus also expresses putative peptide ligands including homologs of PTH (bfPTH1 and 2) and PACAP/GLUC-like peptides (bfPACAP/GLUCs) that may interact with these receptors. Functional analyses showed that bfPTH1 and bfPTH2 activated one of the amphioxus receptors (bf98C) whereas bfPACAP/GLUCs strongly interacted with bf95. In summary, our data confirm the presence of PTH and PACAP/GLUC ligand-receptor pairs in amphioxus, demonstrating that functional homologs of vertebrate PTH and PACAP/glucagon GPCR subfamilies arose before the cephalochordate divergence from the ancestor of tunicates and vertebrates.
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functional pairing of class b1 ligand gpcr in cephalochordate provides evidence of the origin of pth and pacap glucagon receptor Family
Molecular Biology and Evolution, 2015Co-Authors: Jason S. W. On, Cumming Duan, Billy K. C. ChowAbstract:: Several hypotheses have been proposed regarding the origin and evolution of the Secretin Family of peptides and receptors. However, identification of homologous ligand-receptor pairs in invertebrates and vertebrates is difficult because of the low levels of sequence identity between orthologs of distant species. In this study, five receptors structurally related to the vertebrate class B1 G protein-coupled receptor (GPCR) Family were characterized from amphioxus (Branchiostoma floridae). Phylogenetic analysis showed that they clustered with vertebrate parathyroid hormone receptors (PTHR) and pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagon receptors. These PTHR-like receptors shared synteny with several PTH and PACAP/glucagon receptors identified in spotted gar, Xenopus, and human, indicating that amphioxus preserves the ancestral chordate genomic organization of these receptor subfamilies. According to recent data by Mirabeau and Joly, amphioxus also expresses putative peptide ligands including homologs of PTH (bfPTH1 and 2) and PACAP/GLUC-like peptides (bfPACAP/GLUCs) that may interact with these receptors. Functional analyses showed that bfPTH1 and bfPTH2 activated one of the amphioxus receptors (bf98C) whereas bfPACAP/GLUCs strongly interacted with bf95. In summary, our data confirm the presence of PTH and PACAP/GLUC ligand-receptor pairs in amphioxus, demonstrating that functional homologs of vertebrate PTH and PACAP/glucagon GPCR subfamilies arose before the cephalochordate divergence from the ancestor of tunicates and vertebrates.
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role of Secretin peptide Family and their receptors in the hypothalamic control of energy homeostasis
Hormone and Metabolic Research, 2013Co-Authors: Revathi Sekar, Billy K. C. ChowAbstract:Secretin Family of peptide hormones is a group of structurally related brain-gut peptides that exert their functions via interactions with the class B1 G protein-coupled receptors (GPCRs). Recent researches of these peptides and receptors in metabolism have been an area of intense focus for the development of promising drug targets as therapeutic potentials for metabolic disorders. The fact that agonists of GLP-1, a member in the Family, have already started being used as therapeutics clearly indicates the importance and relevance of further research on the clinical applications of these peptides. This review aims to provide an overview of the current understanding regarding the importance of this Family of peptides as well as their receptors in metabolism with special focus on their actions in the hypothalamus.
Laurence J Miller - One of the best experts on this subject based on the ideXlab platform.
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use of cysteine trapping to map spatial approximations between residues contributing to the helix n capping motif of Secretin and distinct residues within each of the extracellular loops of its receptor
Journal of Biological Chemistry, 2016Co-Authors: Maoqing Dong, Polo C H Lam, Andrew Orry, Patrick M Sexton, Arthur Christopoulos, Ruben Abagyan, Laurence J MillerAbstract:Amino-terminal regions of Secretin-Family peptides contain key determinants for biological activity and binding specificity, although the nature of interactions with receptors is unclear. A helix N-capping motif within this region has been postulated to directly contribute to agonist activity while also stabilizing formation of a helix extending toward the peptide carboxyl terminus and docking within the receptor amino terminus. We used cysteine trapping to systematically explore spatial approximations between cysteines replacing each residue in this motif of Secretin (sec), Phe(6), Thr(7), and Leu(10), and cysteines incorporated into the extracellular face of the receptor. Each peptide was a full agonist for cAMP, but had a lower binding affinity than natural hormone. These bound to COS cells expressing 61 receptor constructs incorporating cysteines in every position along each extracellular loop (ECL) and adjacent parts of transmembrane (TM) segments. Patterns of covalent labeling were distinct for each probe, with Cys(6)-sec labeling multiple residues in the carboxyl-terminal half of ECL2 and throughout ECL3, Cys(7)-sec predominantly labeling only single residues in the carboxyl-terminal end of ECL2 and the amino-terminal end of ECL3, and Cys(10)-sec not efficiently labeling any of these residues. These spatial constraints were used to refine our model of Secretin bound to its receptor, now bringing ECL3 above the amino terminus of the ligand and revealing possible charge-charge interactions between this part of Secretin and receptor residues in TM5, TM6, ECL2, and ECL3, which can orient and stabilize the peptide-receptor complex. This was validated by testing predicted approximations by mutagenesis and residue-residue complementation studies.
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Ligand binding and activation of the Secretin receptor, a prototypic Family B G protein-coupled receptor.
British Journal of Pharmacology, 2012Co-Authors: Laurence J Miller, Maoqing Dong, Kaleeckal G. HarikumarAbstract:The Secretin receptor is a prototypic member of Family B G protein-coupled receptors that binds and responds to a linear 27-residue peptide natural ligand. The carboxyl-terminal region of this peptide assumes a helical conformation that occupies the peptide-binding cleft within the structurally complex disulphide-bonded amino-terminal domain of this receptor. The amino terminus of Secretin is directed toward the core helical bundle domain of this receptor that seems to be structurally distinct from the analogous region of Family A G protein-coupled receptors. This amino-terminal region of Secretin is critical for its biological activity, to stimulate Gs coupling and the agonist-induced cAMP response. While the natural peptide ligand is known to span the two key receptor domains, with multiple residue-residue approximation constraints well established, the orientation of the receptor amino terminus relative to the receptor core helical bundle domain is still unclear. Fluorescence studies have established that the mid-region and carboxyl-terminal end of Secretin are protected by the receptor peptide-binding cleft and the amino terminus of Secretin is most exposed to the aqueous milieu as it is directed toward the receptor core, with the mid-region of the peptide becoming more exposed upon receptor activation. Like other Family B peptide hormone receptors, the Secretin receptor is constitutively present in a structurally specific homo-dimeric complex built around the lipid-exposed face of transmembrane segment four. This complex is important for facilitating G protein association and achieving the high affinity state of this receptor. LINKED ARTICLES This article is part of a themed section on Secretin Family (Class B) G Protein-Coupled Receptors. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.166.issue-1
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possible endogenous agonist mechanism for the activation of Secretin Family g protein coupled receptors
Molecular Pharmacology, 2006Co-Authors: Maoqing Dong, Delia I Pinon, Yan W Asmann, Laurence J MillerAbstract:The class B Family of G protein-coupled receptors contains several potentially important drug targets, yet our understanding of the molecular basis of ligand binding and receptor activation remains incomplete. Although a key role is recognized for the cysteine-rich, disulfide-bonded amino-terminal domain of these receptors, detailed insights into ligand docking and resultant conformational changes are not clear. We postulate that binding natural ligands to this domain results in a conformational change that exposes an endogenous ligand which interacts with the body of the receptor to activate it. In this work, we examined whether a synthetic peptide corresponding to a candidate region between the first and third conserved cysteines could act as an agonist. Indeed, this peptide was a weakly potent but fully efficacious agonist, stimulating a concentration-dependent cAMP response in Secretin receptor-bearing cells. This effect was maintained as the peptide length was reduced from 30 to 5, and ultimately, three residues focused on the conserved residue Asp49. The agonist potency was enhanced by cyclization through a diaminopropionic acid linker and by amino-terminal fatty acid acylation. Both ends of the cyclic peptide were shown to interact with the top of transmembrane segment 6 of the receptor, using probes with a photolabile benzoyl-phenylalanine on each end. Analogous observations were also made for two other members of this Family, the vasoactive intestinal polypeptide type 1 and calcitonin receptors. These data may provide a unique molecular mechanism and novel leads for the development of small-molecule agonists acting at potential drug targets within this physiologically important receptor Family.
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importance of the amino terminus in Secretin Family g protein coupled receptors intrinsic photoaffinity labeling establishes initial docking constraints for the calcitonin receptor
Journal of Biological Chemistry, 2004Co-Authors: Maoqing Dong, Delia I Pinon, Richard F Cox, Laurence J MillerAbstract:Abstract The calcitonin receptor is a member of the class B Family of G protein-coupled receptors, closely related to Secretin and parathyroid hormone receptors. Although mechanisms of ligand binding have been directly explored for those receptors, current knowledge of the molecular basis of calcitonin binding to its receptor is based only on receptor mutagenesis. In this work we have utilized the more direct approach of photoaffinity labeling to explore spatial approximations between distinct residues within calcitonin and its receptor. For this we have developed two human calcitonin analogues incorporating a photolabile p-benzoyl-l-phenylalanine residue in the mid-region and carboxyl-terminal half of the peptide in positions 16 and 26, respectively. Both probes specifically bound to the human calcitonin receptor with high affinity and were potent stimulants of cAMP accumulation in calcitonin receptor-bearing human embryonic kidney 293 cells. They covalently labeled the calcitonin receptor in a saturable and specific manner. Further purification, deglycosylation, specific chemical and enzymatic cleavage, and sequencing of labeled wild type and mutant calcitonin receptors identified the sites of labeling for the position 16 and 26 probes as receptor residues Phe137 and Thr30, respectively. Both were within the extracellular amino terminus of the calcitonin receptor, with the former adjacent to the first transmembrane segment and the latter within the distal amino-terminal tail of the receptor. These data are consistent with affinity labeling of other members of the class B G protein-coupled receptors using analogous probes and may suggest a common ligand binding mechanism for this Family.
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identification of two pairs of spatially approximated residues within the carboxyl terminus of Secretin and its receptor
Journal of Biological Chemistry, 2000Co-Authors: Maoqing Dong, Delia I Pinon, Yan W Asmann, Mengwei Zang, Laurence J MillerAbstract:The carboxyl-terminal domains of Secretin Family peptides have been shown to contain key determinants for high affinity binding to their receptors. In this work, we have examined the interaction between carboxyl-terminal residues within Secretin and the prototypic Secretin receptor. We previously utilized photoaffinity labeling to demonstrate spatial approximation between Secretin residue 22 and the receptor domain that includes the first 30 residues of the amino terminus (Dong, M., Wang, Y., Pinon, D. I., Hadac, E. M., and Miller, L. J. (1999) J. Biol. Chem. 274, 903-909). Here, we further refined the site of labeling with the p-benzoyl-phenylalanine (Bpa(22)) probe to receptor residue Leu(17) using progressive cleavage of wild type and mutant Secretin receptors (V13M and V16M) and sequence analysis. We also developed a new probe incorporating a photolabile Bpa at position 26 of Secretin, closer to its carboxyl terminus. This analogue was also a potent agonist (EC(50) = 72 +/- 6 pm) and bound to the Secretin receptor specifically and with high affinity (K(i) = 10.3 +/- 2.4 nm). It covalently labeled the Secretin receptor at a single site saturably and specifically. This was localized to the segment between residues Gly(34) and Ala(41) using chemical and enzymatic cleavage of labeled wild type and A41M mutant receptor constructs and immunoprecipitation of epitope-tagged receptor fragments. Radiochemical sequencing identified the site of covalent attachment as residue Leu(36). These new insights, along with our recent report of contact between residue 6 within the amino-terminal half of Secretin and this same amino-terminal region of this receptor (Dong, M., Wang, Y., Hadac, E. M., Pinon, D. I., Holicky, E. L., and Miller, L. J. (1999) J. Biol. Chem. 274, 19161-19167), support a key role for this region, making the molecular details of this interaction of major interest.
Debbie L. Hay - One of the best experts on this subject based on the ideXlab platform.
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the structure of Secretin Family gpcr peptide ligands implications for receptor pharmacology and drug development
Drug Discovery Today, 2012Co-Authors: Harriet A Watkins, Debbie L. HayAbstract:The Secretin Family G protein-coupled receptors, characterized by a large N-terminal extracellular domain and seven transmembrane helices, are drug targets in many diseases, including migraine, cardiovascular disease, diabetes, osteoporosis and inflammatory disorders. Their activating ligands are peptides with an average length of 30 amino acids. In this article we review the available structural data for these peptides and how this explains their activity. We emphasize how this information may be used to accelerate the development of new drugs against these receptors.
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the third extracellular loop of the human calcitonin receptor like receptor is crucial for the activation of adrenomedullin signalling
British Journal of Pharmacology, 2012Co-Authors: Kenji Kuwasako, Sayaka Nagata, Kazuo Kitamura, Debbie L. Hay, Tomomi Hikosaka, Johji KatoAbstract:BACKGROUND AND PURPOSE The extracellular loops (ECLs) in Family A GPCRs are important for ligand binding and receptor activation, but little is known about the function of Family B GPCR ECLs, especially ECL3. Calcitonin receptor-like receptor (CLR), a Family B GPCR, functions as a calcitonin gene-related peptide (CGRP) and an adrenomedullin (AM) receptor in association with three receptor activity-modifying proteins (RAMPs). Here, we examined the function of the ECL3 of human CLR within the CGRP and AM receptors. EXPERIMENTAL APPROACH A CLR ECL3 chimera, in which the ECL3 of CLR was substituted with that of VPAC2 (a Family B GPCR that is unable to interact with RAMPs), and CLR ECL3 point mutants were constructed and transiently transfected into HEK-293 cells along with each RAMP. Cell-surface expression of each receptor complex was then measured by flow cytometry; [125I]-CGRP and [125I]-AM binding and intracellular cAMP accumulation were also measured. KEY RESULTS Co-expression of the CLR ECL3 chimera with RAMP2 or RAMP3 led to significant reductions in the induction of cAMP signalling by AM, but CGRP signalling was barely affected, despite normal cell-surface expression of the receptors and normal [125I]-AM binding. The chimera had significantly decreased AM, but not CGRP, responses in the presence of RAMP1. Not all CLR ECL3 mutants supported these findings. CONCLUSIONS AND IMPLICATIONS The human CLR ECL3 is crucial for AM-induced cAMP responses via three CLR/RAMP heterodimers, and activation of these heterodimers probably relies on AM-induced conformational changes. This study provides a clue to the molecular basis of the activation of RAMP-based Family B GPCRs. LINKED ARTICLES This article is part of a themed section on Secretin Family (Class B) G Protein-Coupled Receptors. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.166.issue-1
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pharmacological characterization of rat amylin receptors implications for the identification of amylin receptor subtypes
British Journal of Pharmacology, 2012Co-Authors: Richard J. Bailey, Christopher S Walker, A H Ferner, Kerry M Loomes, Gordana Prijic, A Halim, Lynda Whiting, Anthony R J Phillips, Debbie L. HayAbstract:BACKGROUND AND PURPOSE Amylin (Amy) is an important glucoregulatory peptide and AMY receptors are clinical targets for diabetes and obesity. Human (h) AMY receptor subtypes are complexes of the calcitonin (CT) receptor with receptor activity-modifying proteins (RAMPs); their rodent counterparts have not been characterized. To allow identification of the most clinically relevant receptor subtype, the elucidation of rat (r) AMY receptor pharmacology is necessary. EXPERIMENTAL APPROACH Receptors were transiently transfected into COS-7 cells and cAMP responses measured in response to different agonists, with or without antagonists. Competition binding experiments were performed to determine rAmy affinity. KEY RESULTS rCT was the most potent agonist of rCT(a) receptors, whereas rAmy was most potent at rAMY1(a) and rAMY3(a) receptors. rAmy bound to these receptors with high affinity. Rat α-calcitonin gene-related peptide (CGRP) was equipotent to rAmy at both AMY receptors. Rat adrenomedullin (AM) and rAM2/intermedin activated all three receptors but were most effective at rAMY3(a). AC187, AC413 and sCT8-32 were potent antagonists at all three receptors. rαCGRP8-37 displayed selectivity for rAMY receptors over rCT(a) receptors. rAMY8-37 was a weak antagonist but was more effective at rAMY1(a) than rAMY3(a). CONCLUSIONS AND IMPLICATIONS AMY receptors were generated by co-expression of rCT(a) with rRAMP1 or 3, forming rAMY1(a) and rAMY3(a) receptors, respectively. CGRP was more potent at rAMY than at hAMY receptors. No antagonist tested was able to differentiate the rAMY receptor subtypes. The data emphasize the need for and provide a useful resource for developing new CT or AMY receptor ligands as pharmacological tools or potential clinical candidates. LINKED ARTICLES This article is part of a themed section on Secretin Family (Class B) G Protein-Coupled Receptors. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.166.issue-1
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Secretin Family class b g protein coupled receptors from molecular to clinical perspectives
British Journal of Pharmacology, 2012Co-Authors: David R Poyner, Debbie L. HayAbstract:Family B G protein-coupled receptors represent an important but under-researched group of receptors. This edition of the British Journal of Pharmacology considers the roles and pharmacology of a number of these receptors. Whilst common themes emerge, it is clear that more work is needed to understand the details of each receptor in order to properly exploit them therapeutically.
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structural insights into ramp modification of Secretin Family g protein coupled receptors implications for drug development
Trends in Pharmacological Sciences, 2011Co-Authors: Jack U Flanagan, Julia K Archbold, Harriet A Watkins, Joseph J Gingell, Debbie L. HayAbstract:Secretin Family G protein-coupled receptors (GPCRs) are important therapeutic targets for migraine, diabetes, bone disorders, inflammatory disorders and cardiovascular disease. They possess a large N-terminal extracellular domain (ECD) known to be the primary ligand-binding determinant. Structural determination of several Secretin Family GPCR ECDs in complex with peptide ligands has been achieved recently, providing insight into the molecular determinants of hormone binding. Some Secretin Family GPCRs associate with receptor activity-modifying proteins (RAMPs), resulting in changes to receptor pharmacology. Recently, the first crystal structure of a RAMP ECD in complex with a Secretin Family GPCR was solved, revealing the elegant mechanism governing receptor selectivity of small molecule antagonists of the calcitonin gene-related peptide (CGRP) receptor. Here we review the structural basis of ligand binding to Secretin Family GPCRs, concentrating on recent progress made on the structural basis of RAMP-modified GPCR pharmacology and its implications for rational drug design.
Deborah M Power - One of the best experts on this subject based on the ideXlab platform.
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peptide Family members
2013Co-Authors: Joao C R Cardoso, Florbela A Vieira, Ana S Gomes, Deborah M PowerAbstract:Background: The Secretin Family is a pleotropic group of brain-gut peptides with affinity for class 2 G-protein coupled receptors (Secretin Family GPCRs) proposed to have emerged early in the metazoan radiation via gene or genome duplications. In human, 10 members exist and sequence and functional homologues and ligand-receptor pairs have been characterised in representatives of most vertebrate classes. Secretin-like Family GPCR homologues have also been isolated in non-vertebrate genomes however their corresponding ligands have not been convincingly identified and their evolution remains enigmatic. Results: In silico sequence comparisons failed to retrieve a non-vertebrate (porifera, cnidaria, protostome and early deuterostome) Secretin Family homologue. In contrast, Secretin Family members were identified in lamprey, several teleosts and tetrapods and comparative studies revealed that sequence and structure is in general maintained. Sequence comparisons and phylogenetic analysis revealed that PACAP, VIP and GCG are the most highly conserved members and two major peptide subfamilies exist; i) PACAP-like which includes PACAP, PRP, VIP, PH, GHRH, SCT and ii) GCG-like which includes GCG, GLP1, GLP2 and GIP. Conserved regions flanking Secretin Family members were established by comparative analysis of the Takifugu, Xenopus, chicken and human genomes and gene homologues were identified in nematode, Drosophila and Ciona genomes but no gene linkage occurred. However, in Drosophila an
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the serendipitous origin of chordate Secretin peptide Family members
BMC Evolutionary Biology, 2010Co-Authors: Joao C R Cardoso, Florbela A Vieira, Ana Gomes, Deborah M PowerAbstract:The Secretin Family is a pleotropic group of brain-gut peptides with affinity for class 2 G-protein coupled receptors (Secretin Family GPCRs) proposed to have emerged early in the metazoan radiation via gene or genome duplications. In human, 10 members exist and sequence and functional homologues and ligand-receptor pairs have been characterised in representatives of most vertebrate classes. Secretin-like Family GPCR homologues have also been isolated in non-vertebrate genomes however their corresponding ligands have not been convincingly identified and their evolution remains enigmatic. In silico sequence comparisons failed to retrieve a non-vertebrate (porifera, cnidaria, protostome and early deuterostome) Secretin Family homologue. In contrast, Secretin Family members were identified in lamprey, several teleosts and tetrapods and comparative studies revealed that sequence and structure is in general maintained. Sequence comparisons and phylogenetic analysis revealed that PACAP, VIP and GCG are the most highly conserved members and two major peptide subfamilies exist; i) PACAP-like which includes PACAP, PRP, VIP, PH, GHRH, SCT and ii) GCG-like which includes GCG, GLP1, GLP2 and GIP. Conserved regions flanking Secretin Family members were established by comparative analysis of the Takifugu, Xenopus, chicken and human genomes and gene homologues were identified in nematode, Drosophila and Ciona genomes but no gene linkage occurred. However, in Drosophila and nematode genes which flank vertebrate Secretin Family members were identified in the same chromosome. Receptors of the Secretin-like Family GPCRs are present in protostomes but no sequence homologues of the vertebrate cognate ligands have been identified. It has not been possible to determine when the ligands evolved but it seems likely that it was after the protostome-deuterostome divergence from an exon that was part of an existing gene or gene fragment by rounds of gene/genome duplication. The duplicate exon under different evolutionary pressures originated the chordate PACAP-like and GCG-like subFamily groups. This event occurred after the emergence of the metazoan Secretin GPCRs and led to the establishment of novel peptide-receptor interactions that contributed to the generation of novel physiological functions in the chordate lineage.
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evolution of Secretin Family gpcr members in the metazoa
BMC Evolutionary Biology, 2006Co-Authors: Joao C R Cardoso, Melody S Clark, Florbela A Vieira, Vanda C Pinto, Deborah M PowerAbstract:Background Comparative approaches using protostome and deuterostome data have greatly contributed to understanding gene function and organismal complexity. The Family 2 G-protein coupled receptors (GPCRs) are one of the largest and best studied hormone and neuropeptide receptor families. They are suggested to have arisen from a single ancestral gene via duplication events. Despite the recent identification of receptor members in protostome and early deuterostome genomes, relatively little is known about their function or origin during metazoan divergence. In this study a comprehensive description of Family 2 GPCR evolution is given based on in silico and expression analyses of the invertebrate receptor genes.
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evolution of Secretin Family gpcr members in the metazoa
BMC Evolutionary Biology, 2006Co-Authors: Joao C R Cardoso, Melody S Clark, Florbela A Vieira, Vanda C Pinto, Deborah M PowerAbstract:Comparative approaches using protostome and deuterostome data have greatly contributed to understanding gene function and organismal complexity. The Family 2 G-protein coupled receptors (GPCRs) are one of the largest and best studied hormone and neuropeptide receptor families. They are suggested to have arisen from a single ancestral gene via duplication events. Despite the recent identification of receptor members in protostome and early deuterostome genomes, relatively little is known about their function or origin during metazoan divergence. In this study a comprehensive description of Family 2 GPCR evolution is given based on in silico and expression analyses of the invertebrate receptor genes. Family 2 GPCR members were identified in the invertebrate genomes of the nematodes C. elegans and C. briggsae, the arthropods D. melanogaster and A. gambiae (mosquito) and in the tunicate C. intestinalis. This suggests that they are of ancient origin and have evolved through gene/genome duplication events. Sequence comparisons and phylogenetic analyses have demonstrated that the immediate gene environment, with regard to gene content, is conserved between the protostome and deuterostome receptor genomic regions. Also that the protostome genes are more like the deuterostome Corticotrophin Releasing Factor (CRF) and Calcitonin/Calcitonin Gene-Related Peptide (CAL/CGRP) receptors members than the other Family 2 GPCR members. The evolution of Family 2 GPCRs in deuterostomes is characterised by acquisition of new Family members, with SCT (Secretin) receptors only present in tetrapods. Gene structure is characterised by an increase in intron number with organismal complexity with the exception of the vertebrate CAL/CGRP receptors. The Family 2 GPCR members provide a good example of gene duplication events occurring in tandem with increasing organismal complexity during metazoan evolution. The putative ancestral receptors are proposed to be more like the deuterostome CAL/CGRP and CRF receptors and this may be associated with their fundamental role in calcium regulation and the stress response, both of which are essential for survival.
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the Secretin g protein coupled receptor Family teleost receptors
Journal of Molecular Endocrinology, 2005Co-Authors: Joao C R Cardoso, Melody S Clark, F A Viera, Paul D Bridge, A Gilles, Deborah M PowerAbstract:Twenty-one members of the Secretin Family (Family 2) of G-protein-coupled receptors (GPCRs) were identified via directed cloning and data-mining of the Fugu Genome Consortium database, representing the most comprehensive description of Secretin GPCRs in a teleost fish to date. Duplicated genes were identified for many of the Family members, namely the receptors for pituitary adenylate cyclase-activating polypeptide (PACAP)/vasoactive intestinal peptide (VIP), calcitonin, calcitonin gene-related peptide (CGRP), growth hormone releasing hormone (GHRH), glucagon receptor/glucagon-like peptide (GLP) and parathyroid hormone-related peptide (PTHrP)/PTH. Mining of other teleost genomes (zebrafish and Tetraodon) revealed that the duplicated genes identified in the Takifugu genome were also present in these fish. Additional database searching of the Escherichia coli, yeast, Drosophila, Caenorhabditis elegans and Ciona genomes revealed that the Family 2 of GPCRs were only present in the multicellular organisms. Orthologues of all the human Secretin receptors were identified with the exception of Secretin itself. Additional database searches in the Fugu Genome Consortium database also failed to reveal a Secretin ligand and so it is hypothesised that both the receptor and the ligand evolved after the divergence of teleost/tetrapod lineages. Phylogenetic analysis at both the protein and the DNA level provided strong support for each of the individual receptor Family groupings, but weak support between groups, making evolutionary inferences difficult. A more critical analysis of the PACAP/VIP receptor Family confirmed previous hypotheses that the vasoactive intestinal peptide receptor (VPAC1R) gene is the ancestral form of the receptor.
Maoqing Dong - One of the best experts on this subject based on the ideXlab platform.
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use of cysteine trapping to map spatial approximations between residues contributing to the helix n capping motif of Secretin and distinct residues within each of the extracellular loops of its receptor
Journal of Biological Chemistry, 2016Co-Authors: Maoqing Dong, Polo C H Lam, Andrew Orry, Patrick M Sexton, Arthur Christopoulos, Ruben Abagyan, Laurence J MillerAbstract:Amino-terminal regions of Secretin-Family peptides contain key determinants for biological activity and binding specificity, although the nature of interactions with receptors is unclear. A helix N-capping motif within this region has been postulated to directly contribute to agonist activity while also stabilizing formation of a helix extending toward the peptide carboxyl terminus and docking within the receptor amino terminus. We used cysteine trapping to systematically explore spatial approximations between cysteines replacing each residue in this motif of Secretin (sec), Phe(6), Thr(7), and Leu(10), and cysteines incorporated into the extracellular face of the receptor. Each peptide was a full agonist for cAMP, but had a lower binding affinity than natural hormone. These bound to COS cells expressing 61 receptor constructs incorporating cysteines in every position along each extracellular loop (ECL) and adjacent parts of transmembrane (TM) segments. Patterns of covalent labeling were distinct for each probe, with Cys(6)-sec labeling multiple residues in the carboxyl-terminal half of ECL2 and throughout ECL3, Cys(7)-sec predominantly labeling only single residues in the carboxyl-terminal end of ECL2 and the amino-terminal end of ECL3, and Cys(10)-sec not efficiently labeling any of these residues. These spatial constraints were used to refine our model of Secretin bound to its receptor, now bringing ECL3 above the amino terminus of the ligand and revealing possible charge-charge interactions between this part of Secretin and receptor residues in TM5, TM6, ECL2, and ECL3, which can orient and stabilize the peptide-receptor complex. This was validated by testing predicted approximations by mutagenesis and residue-residue complementation studies.
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Ligand binding and activation of the Secretin receptor, a prototypic Family B G protein-coupled receptor.
British Journal of Pharmacology, 2012Co-Authors: Laurence J Miller, Maoqing Dong, Kaleeckal G. HarikumarAbstract:The Secretin receptor is a prototypic member of Family B G protein-coupled receptors that binds and responds to a linear 27-residue peptide natural ligand. The carboxyl-terminal region of this peptide assumes a helical conformation that occupies the peptide-binding cleft within the structurally complex disulphide-bonded amino-terminal domain of this receptor. The amino terminus of Secretin is directed toward the core helical bundle domain of this receptor that seems to be structurally distinct from the analogous region of Family A G protein-coupled receptors. This amino-terminal region of Secretin is critical for its biological activity, to stimulate Gs coupling and the agonist-induced cAMP response. While the natural peptide ligand is known to span the two key receptor domains, with multiple residue-residue approximation constraints well established, the orientation of the receptor amino terminus relative to the receptor core helical bundle domain is still unclear. Fluorescence studies have established that the mid-region and carboxyl-terminal end of Secretin are protected by the receptor peptide-binding cleft and the amino terminus of Secretin is most exposed to the aqueous milieu as it is directed toward the receptor core, with the mid-region of the peptide becoming more exposed upon receptor activation. Like other Family B peptide hormone receptors, the Secretin receptor is constitutively present in a structurally specific homo-dimeric complex built around the lipid-exposed face of transmembrane segment four. This complex is important for facilitating G protein association and achieving the high affinity state of this receptor. LINKED ARTICLES This article is part of a themed section on Secretin Family (Class B) G Protein-Coupled Receptors. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.166.issue-1
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possible endogenous agonist mechanism for the activation of Secretin Family g protein coupled receptors
Molecular Pharmacology, 2006Co-Authors: Maoqing Dong, Delia I Pinon, Yan W Asmann, Laurence J MillerAbstract:The class B Family of G protein-coupled receptors contains several potentially important drug targets, yet our understanding of the molecular basis of ligand binding and receptor activation remains incomplete. Although a key role is recognized for the cysteine-rich, disulfide-bonded amino-terminal domain of these receptors, detailed insights into ligand docking and resultant conformational changes are not clear. We postulate that binding natural ligands to this domain results in a conformational change that exposes an endogenous ligand which interacts with the body of the receptor to activate it. In this work, we examined whether a synthetic peptide corresponding to a candidate region between the first and third conserved cysteines could act as an agonist. Indeed, this peptide was a weakly potent but fully efficacious agonist, stimulating a concentration-dependent cAMP response in Secretin receptor-bearing cells. This effect was maintained as the peptide length was reduced from 30 to 5, and ultimately, three residues focused on the conserved residue Asp49. The agonist potency was enhanced by cyclization through a diaminopropionic acid linker and by amino-terminal fatty acid acylation. Both ends of the cyclic peptide were shown to interact with the top of transmembrane segment 6 of the receptor, using probes with a photolabile benzoyl-phenylalanine on each end. Analogous observations were also made for two other members of this Family, the vasoactive intestinal polypeptide type 1 and calcitonin receptors. These data may provide a unique molecular mechanism and novel leads for the development of small-molecule agonists acting at potential drug targets within this physiologically important receptor Family.
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importance of the amino terminus in Secretin Family g protein coupled receptors intrinsic photoaffinity labeling establishes initial docking constraints for the calcitonin receptor
Journal of Biological Chemistry, 2004Co-Authors: Maoqing Dong, Delia I Pinon, Richard F Cox, Laurence J MillerAbstract:Abstract The calcitonin receptor is a member of the class B Family of G protein-coupled receptors, closely related to Secretin and parathyroid hormone receptors. Although mechanisms of ligand binding have been directly explored for those receptors, current knowledge of the molecular basis of calcitonin binding to its receptor is based only on receptor mutagenesis. In this work we have utilized the more direct approach of photoaffinity labeling to explore spatial approximations between distinct residues within calcitonin and its receptor. For this we have developed two human calcitonin analogues incorporating a photolabile p-benzoyl-l-phenylalanine residue in the mid-region and carboxyl-terminal half of the peptide in positions 16 and 26, respectively. Both probes specifically bound to the human calcitonin receptor with high affinity and were potent stimulants of cAMP accumulation in calcitonin receptor-bearing human embryonic kidney 293 cells. They covalently labeled the calcitonin receptor in a saturable and specific manner. Further purification, deglycosylation, specific chemical and enzymatic cleavage, and sequencing of labeled wild type and mutant calcitonin receptors identified the sites of labeling for the position 16 and 26 probes as receptor residues Phe137 and Thr30, respectively. Both were within the extracellular amino terminus of the calcitonin receptor, with the former adjacent to the first transmembrane segment and the latter within the distal amino-terminal tail of the receptor. These data are consistent with affinity labeling of other members of the class B G protein-coupled receptors using analogous probes and may suggest a common ligand binding mechanism for this Family.
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identification of two pairs of spatially approximated residues within the carboxyl terminus of Secretin and its receptor
Journal of Biological Chemistry, 2000Co-Authors: Maoqing Dong, Delia I Pinon, Yan W Asmann, Mengwei Zang, Laurence J MillerAbstract:The carboxyl-terminal domains of Secretin Family peptides have been shown to contain key determinants for high affinity binding to their receptors. In this work, we have examined the interaction between carboxyl-terminal residues within Secretin and the prototypic Secretin receptor. We previously utilized photoaffinity labeling to demonstrate spatial approximation between Secretin residue 22 and the receptor domain that includes the first 30 residues of the amino terminus (Dong, M., Wang, Y., Pinon, D. I., Hadac, E. M., and Miller, L. J. (1999) J. Biol. Chem. 274, 903-909). Here, we further refined the site of labeling with the p-benzoyl-phenylalanine (Bpa(22)) probe to receptor residue Leu(17) using progressive cleavage of wild type and mutant Secretin receptors (V13M and V16M) and sequence analysis. We also developed a new probe incorporating a photolabile Bpa at position 26 of Secretin, closer to its carboxyl terminus. This analogue was also a potent agonist (EC(50) = 72 +/- 6 pm) and bound to the Secretin receptor specifically and with high affinity (K(i) = 10.3 +/- 2.4 nm). It covalently labeled the Secretin receptor at a single site saturably and specifically. This was localized to the segment between residues Gly(34) and Ala(41) using chemical and enzymatic cleavage of labeled wild type and A41M mutant receptor constructs and immunoprecipitation of epitope-tagged receptor fragments. Radiochemical sequencing identified the site of covalent attachment as residue Leu(36). These new insights, along with our recent report of contact between residue 6 within the amino-terminal half of Secretin and this same amino-terminal region of this receptor (Dong, M., Wang, Y., Hadac, E. M., Pinon, D. I., Holicky, E. L., and Miller, L. J. (1999) J. Biol. Chem. 274, 19161-19167), support a key role for this region, making the molecular details of this interaction of major interest.