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Debbie L. Hay - One of the best experts on this subject based on the ideXlab platform.
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probing the mechanism of receptor activity modifying protein modulation of gpcr ligand selectivity through rational design of potent adrenomedullin and calcitonin gene related peptide antagonists
Molecular Pharmacology, 2018Co-Authors: Jason M. Booe, Debbie L. Hay, Margaret L. Warner, Amanda M Roehrkasse, Augen A. PioszakAbstract:Binding of the vasodilator peptides adrenomedullin (AM) and calcitonin gene–related peptide (CGRP) to the class B G protein–coupled receptor calcitonin receptor–like receptor (CLR) is modulated by receptor activity–modifying proteins (RAMPs). RAMP1 favors CGRP, whereas RAMP2 and RAMP3 favor AM. Crystal structures of peptide-bound RAMP1/2-CLR extracellular domain (ECD) heterodimers suggested RAMPs alter ligand preference through direct peptide contacts and allosteric modulation of CLR. Here, we probed this dual mechanism through rational structure-guided design of AM and CGRP antagonist variants. Variants were characterized for binding to purified RAMP1/2-CLR ECD and for antagonism of the full-length CGRP (RAMP1:CLR), AM1 (RAMP2:CLR), and AM2 (RAMP3:CLR) receptors. Short nanomolar affinity AM(37–52) and CGRP(27–37) variants were obtained through substitutions including AM S45W/Q50W and CGRP K35W/A36S designed to stabilize their β-turn. K46L and Y52F substitutions designed to exploit RAMP allosteric effects and direct peptide contacts, respectively, yielded AM variants with selectivity for the CGRP receptor over the AM1 receptor. AM(37–52) S45W/K46L/Q50W/Y52F exhibited nanomolar potency at the CGRP receptor and micromolar potency at AM1. A 2.8-A resolution crystal structure of this variant bound to the RAMP1-CLR ECD confirmed that it bound as designed. CGRP(27–37) N31D/S34P/K35W/A36S exhibited potency and selectivity comparable to the traditional antagonist CGRP(8–37). Giving this variant the ability to contact RAMP2 through the F37Y substitution increased affinity for AM1, but it still preferred the CGRP receptor. These potent peptide antagonists with altered selectivity inform the development of AM/CGRP-based pharmacological tools and support the hypothesis that RAMPs alter CLR ligand selectivity through allosteric effects and direct peptide contacts.
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update on the pharmacology of calcitonin cgrp family of peptides iuphar review 25
British Journal of Pharmacology, 2018Co-Authors: Debbie L. Hay, David R Poyner, Michael L Garelja, Christopher S. WalkerAbstract:The calcitonin/calcitonin gene-related peptide (CGRP) family of peptides includes calcitonin, α and β CGRP, amylin, adrenomedullin (AM) and adrenomedullin 2/intermedin (AM2/IMD). Their receptors consist of one of two G protein-coupled receptors (GPCRs), the calcitonin receptor (CTR) or the calcitonin receptor-like receptor (CLR). Further diversity arises from heterodimerisation of these GPCRs with one of three receptor activity-modifying proteins (RAMPs). This gives the CGRP receptor (CLR/RAMP1), the AM1 and AM2 receptors (CLR/RAMP2 or RAMP3) and the AMY1, AMY2 and AMY3 receptors (CTR/RAMPs1-3 complexes, respectively). Apart from the CGRP receptor, there are only peptide antagonists widely available for these receptors and these have limited selectivity, thus defining the function of each receptor in vivo remains challenging. Further challenges arise from the probable co-expression of CTR with the CTR/RAMP complexes and species-dependent splice variants of the CTR (CT(a) and CT(b)). Furthermore, the AMY1(a) receptor is activated equally well by both amylin and CGRP and the preferred receptor for AM2/IMD has been unclear. However, there are clear therapeutic rationales for developing agents against the various receptors for these peptides. For example many agents targeting the CGRP system are in clinical trials and pramlintide, an amylin analogue, is an approved therapy for insulin-requiring diabetes. This review provides an update on the pharmacology of the calcitonin family of peptides by members of the corresponding subcommittee of the International Union of Basic and Clinical Pharmacology and colleagues.
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Receptor activity-modifying proteins; multifunctional G protein-coupled receptor accessory proteins.
Biochemical Society transactions, 2016Co-Authors: Debbie L. Hay, Joseph J Gingell, Christopher S. Walker, Graham Ladds, Christopher A. Reynolds, David R PoynerAbstract:Receptor activity-modifying proteins (RAMPs) are single pass membrane proteins initially identified by their ability to determine the pharmacology of the calcitonin receptor-like receptor (CLR), a family B G protein-coupled receptor (GPCR). It is now known that RAMPs can interact with a much wider range of GPCRs. This review considers recent developments on the structure of the complexes formed between the extracellular domains (ECDs) of CLR and RAMP1 or RAMP2 as these provide insights as to how the RAMPs direct ligand binding. The range of RAMP interactions is also considered; RAMPs can interact with numerous family B GPCRs as well as examples of family A and family C GPCRs. They influence receptor expression at the cell surface, trafficking, ligand binding and G protein coupling. The GPCR-RAMP interface offers opportunities for drug targeting, illustrated by examples of drugs developed for migraine.
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receptor activity modifying protein dependent effects of mutations in the calcitonin receptor like receptor implications for adrenomedullin and calcitonin gene related peptide pharmacology
British Journal of Pharmacology, 2014Co-Authors: Harriet A Watkins, David R Poyner, Richard J. Bailey, Christopher S. Walker, James Barwell, Debbie L. HayAbstract:Background and Purpose Receptor activity-modifying proteins (RAMPs) define the pharmacology of the calcitonin receptor-like receptor (CLR). The interactions of the different RAMPs with this class B GPCR yield high-affinity calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) receptors. However, the mechanism for this is unclear. Experimental Approach Guided by receptor models, we mutated residues in the N-terminal helix of CLR, RAMP2 and RAMP3 hypothesized to be involved in peptide interactions. These were assayed for cAMP production with AM, AM2 and CGRP together with their cell surface expression. Binding studies were also conducted for selected mutants. Key Results An important domain for peptide interactions on CLR from I32 to I52 was defined. Although I41 was universally important for binding and receptor function, the role of other residues depended on both ligand and RAMP. Peptide binding to CLR/RAMP3 involved a more restricted range of residues than that to CLR/RAMP1 or CLR/RAMP2. E101 of RAMP2 had a major role in AM interactions, and F111/W84 of RAMP2/3 was important with each peptide. Conclusions and Implications RAMP-dependent effects of CLR mutations suggest that the different RAMPs control accessibility of peptides to binding residues situated on the CLR N-terminus. RAMP3 appears to alter the role of specific residues at the CLR-RAMP interface compared with RAMP1 and RAMP2.
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Receptor activity-modifying protein-dependent impairment of calcitonin receptor splice variant Δ(1–47)hCT(a) function
British journal of pharmacology, 2013Co-Authors: Maoqing Dong, Laurence J Miller, Harriet A Watkins, Denise Wootten, Debbie L. HayAbstract:Background and Purpose Alternative splicing expands proteome diversity to GPCRs. Distinct receptor variants have been identified for a secretin family GPCR, the calcitonin receptor (CTR). The possible functional contributions of these receptor variants are further altered by their potential interactions with receptor activity-modifying proteins (RAMPs). One variant of the human CTR lacks the first 47 residues at its N terminus [Δ(1–47)hCT(a)]. However, very little is known about the pharmacology of this variant or its ability to interact with RAMPs to form amylin receptors. Experimental Approach Δ(1–47)hCT(a) was characterized both with and without RAMPs in Cos7 and/or HEK293S cells. The receptor expression (ELISA assays) and function (cAMP and pERK1/2 assays) for up to six agonists and two antagonists were determined. Key Results Despite lacking 47 residues at the N terminus, Δ(1–47)hCT(a) was still able to express at the cell surface, but displayed a generalized reduction in peptide potency. Δ(1–47)hCT(a) retained its ability to interact with RAMP1 and formed a functional amylin receptor; this also appeared to be the case with RAMP3. On the other hand, its interaction with RAMP2 and resultant amylin receptor was reduced to a greater extent. Conclusions and Implications Δ(1–47)hCT(a) acts as a functional receptor at the cell surface. It exhibits altered receptor function, depending on whether it associates with a RAMP and which RAMP it interacts with. Therefore, the presence of this variant in tissues will potentially contribute to altered peptide binding and signalling, depending on the RAMP distribution in tissues.
David R Poyner - One of the best experts on this subject based on the ideXlab platform.
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update on the pharmacology of calcitonin cgrp family of peptides iuphar review 25
British Journal of Pharmacology, 2018Co-Authors: Debbie L. Hay, David R Poyner, Michael L Garelja, Christopher S. WalkerAbstract:The calcitonin/calcitonin gene-related peptide (CGRP) family of peptides includes calcitonin, α and β CGRP, amylin, adrenomedullin (AM) and adrenomedullin 2/intermedin (AM2/IMD). Their receptors consist of one of two G protein-coupled receptors (GPCRs), the calcitonin receptor (CTR) or the calcitonin receptor-like receptor (CLR). Further diversity arises from heterodimerisation of these GPCRs with one of three receptor activity-modifying proteins (RAMPs). This gives the CGRP receptor (CLR/RAMP1), the AM1 and AM2 receptors (CLR/RAMP2 or RAMP3) and the AMY1, AMY2 and AMY3 receptors (CTR/RAMPs1-3 complexes, respectively). Apart from the CGRP receptor, there are only peptide antagonists widely available for these receptors and these have limited selectivity, thus defining the function of each receptor in vivo remains challenging. Further challenges arise from the probable co-expression of CTR with the CTR/RAMP complexes and species-dependent splice variants of the CTR (CT(a) and CT(b)). Furthermore, the AMY1(a) receptor is activated equally well by both amylin and CGRP and the preferred receptor for AM2/IMD has been unclear. However, there are clear therapeutic rationales for developing agents against the various receptors for these peptides. For example many agents targeting the CGRP system are in clinical trials and pramlintide, an amylin analogue, is an approved therapy for insulin-requiring diabetes. This review provides an update on the pharmacology of the calcitonin family of peptides by members of the corresponding subcommittee of the International Union of Basic and Clinical Pharmacology and colleagues.
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Receptor activity-modifying proteins; multifunctional G protein-coupled receptor accessory proteins.
Biochemical Society transactions, 2016Co-Authors: Debbie L. Hay, Joseph J Gingell, Christopher S. Walker, Graham Ladds, Christopher A. Reynolds, David R PoynerAbstract:Receptor activity-modifying proteins (RAMPs) are single pass membrane proteins initially identified by their ability to determine the pharmacology of the calcitonin receptor-like receptor (CLR), a family B G protein-coupled receptor (GPCR). It is now known that RAMPs can interact with a much wider range of GPCRs. This review considers recent developments on the structure of the complexes formed between the extracellular domains (ECDs) of CLR and RAMP1 or RAMP2 as these provide insights as to how the RAMPs direct ligand binding. The range of RAMP interactions is also considered; RAMPs can interact with numerous family B GPCRs as well as examples of family A and family C GPCRs. They influence receptor expression at the cell surface, trafficking, ligand binding and G protein coupling. The GPCR-RAMP interface offers opportunities for drug targeting, illustrated by examples of drugs developed for migraine.
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receptor activity modifying protein dependent effects of mutations in the calcitonin receptor like receptor implications for adrenomedullin and calcitonin gene related peptide pharmacology
British Journal of Pharmacology, 2014Co-Authors: Harriet A Watkins, David R Poyner, Richard J. Bailey, Christopher S. Walker, James Barwell, Debbie L. HayAbstract:Background and Purpose Receptor activity-modifying proteins (RAMPs) define the pharmacology of the calcitonin receptor-like receptor (CLR). The interactions of the different RAMPs with this class B GPCR yield high-affinity calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) receptors. However, the mechanism for this is unclear. Experimental Approach Guided by receptor models, we mutated residues in the N-terminal helix of CLR, RAMP2 and RAMP3 hypothesized to be involved in peptide interactions. These were assayed for cAMP production with AM, AM2 and CGRP together with their cell surface expression. Binding studies were also conducted for selected mutants. Key Results An important domain for peptide interactions on CLR from I32 to I52 was defined. Although I41 was universally important for binding and receptor function, the role of other residues depended on both ligand and RAMP. Peptide binding to CLR/RAMP3 involved a more restricted range of residues than that to CLR/RAMP1 or CLR/RAMP2. E101 of RAMP2 had a major role in AM interactions, and F111/W84 of RAMP2/3 was important with each peptide. Conclusions and Implications RAMP-dependent effects of CLR mutations suggest that the different RAMPs control accessibility of peptides to binding residues situated on the CLR N-terminus. RAMP3 appears to alter the role of specific residues at the CLR-RAMP interface compared with RAMP1 and RAMP2.
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Receptor activity modifying proteins (RAMPs) interact with the VPAC2 receptor and CRF1 receptors and modulate their function
British journal of pharmacology, 2013Co-Authors: Denise Wootten, James Barwell, Kathleen M. Caron, Helena Lindmark, Mahita Kadmiel, Helen H. Willcockson, Tomas Drmota, David R PoynerAbstract:Background and Purpose Although it is established that the receptor activity modifying proteins (RAMPs) can interact with a number of GPCRs, little is known about the consequences of these interactions. Here the interaction of RAMPs with the glucagon-like peptide 1 receptor (GLP-1 receptor), the human vasoactive intestinal polypeptide/pituitary AC-Activating peptide 2 receptor (VPAC) and the type 1 corticotrophin releasing factor receptor (CRF) has been examined. Experimental Approach GPCRs were co-transfected with RAMPs in HEK 293S and CHO-K1 cells. Cell surface expression of RAMPs and GPCRs was examined by elisa. Where there was evidence for interactions, agonist-stimulated cAMP production, Ca mobilization and GTPγS binding to G, G, G and G were examined. The ability of CRF to stimulate adrenal corticotrophic hormone release in Ramp2 mice was assessed. Key Results The GLP-1 receptor failed to enhance the cell surface expression of any RAMP. VPAC enhanced the cell surface expression of all three RAMPs. CRF enhanced the cell surface expression of RAMP2; the cell surface expression of CRF was also increased. There was no effect on agonist-stimulated cAMP production. However, there was enhanced G-protein coupling in a receptor and agonist-dependent manner. The CRF: RAMP2 complex resulted in enhanced elevation of intracellular calcium to CRF and urocortin 1 but not sauvagine. In Ramp2 mice, there was a loss of responsiveness to CRF. Conclusions and Implications The VPAC and CRF receptors interact with RAMPs. This modulates G-protein coupling in an agonist-specific manner. For CRF, coupling to RAMP2 may be of physiological significance. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.
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Calcitonin Gene-Related Peptide and Adrenomedullin Receptors
Encyclopedia of Biological Chemistry, 2013Co-Authors: Debbie L. Hay, Alex C. Conner, David R PoynerAbstract:Calcitonin gene-related peptide (CGRP) and adrenomedullin are related peptides, which are potent vasodilators. The CGRP and adrenomedullin receptors are unusual receptors, comprising a G-protein-coupled receptor (GPCR) with receptor activity-modifying proteins (RAMPs). The GPCR, the calcitonin receptor-like receptor (CLR), forms a CGRP receptor with RAMP1, whereas CLR with RAMP2 or RAMP3 forms two subtypes of adrenomedullin receptor. This article covers the pharmacology of these receptors, their distribution, and potential involvement in disease.
Patrick M. Sexton - One of the best experts on this subject based on the ideXlab platform.
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structure function analysis of amino acid 74 of human RAMP1 and ramp3 and its role in peptide interactions with adrenomedullin and calcitonin gene related peptide receptors
Peptides, 2011Co-Authors: David R Poyner, Patrick M. Sexton, George Christopoulos, Debbie L. HayAbstract:The receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are complexes of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying proteins (RAMP). The CGRP receptor is a CLR/RAMP1 pairing whereas CLR/RAMP2 and CLR/RAMP3 constitute two subtypes of AM receptor: AM(1) and AM(2), respectively. Previous studies identified Glu74 in RAMP3 to be important for AM binding and potency. To further understand the importance of this residue and its equivalent in RAMP1 (Trp74) we substituted the native amino acids with several others. In RAMP3, these were Trp, Phe, Tyr, Ala, Ser, Thr, Arg and Asn; in RAMP1, Glu, Phe, Tyr, Ala and Asn substitutions were made. The mutant RAMPs were co-expressed with CLR in Cos7 cells; receptor function in response to AM, AM(2)/intermedin and CGRP was measured in a cAMP assay and cell surface expression was determined by ELISA. Phe reduced AM potency in RAMP3 but had no effect in RAMP1. In contrast, Tyr had no effect in RAMP3 but enhanced AM potency in RAMP1. Most other substitutions had a small effect on AM potency in both receptors whereas there was little impact on CGRP or AM(2) potency. Overall, these data suggest that the geometry and charge of the residue at position 74 contribute to how AM interacts with the AM(2) and CGRP receptors and confirms the role of this position in dictating differential AM pharmacology at the AM(2) and CGRP receptors.
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Structure–function analysis of amino acid 74 of human RAMP1 and RAMP3 and its role in peptide interactions with adrenomedullin and calcitonin gene-related peptide receptors
Peptides, 2011Co-Authors: David R Poyner, Patrick M. Sexton, George Christopoulos, Debbie L. HayAbstract:The receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are complexes of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying proteins (RAMP). The CGRP receptor is a CLR/RAMP1 pairing whereas CLR/RAMP2 and CLR/RAMP3 constitute two subtypes of AM receptor: AM(1) and AM(2), respectively. Previous studies identified Glu74 in RAMP3 to be important for AM binding and potency. To further understand the importance of this residue and its equivalent in RAMP1 (Trp74) we substituted the native amino acids with several others. In RAMP3, these were Trp, Phe, Tyr, Ala, Ser, Thr, Arg and Asn; in RAMP1, Glu, Phe, Tyr, Ala and Asn substitutions were made. The mutant RAMPs were co-expressed with CLR in Cos7 cells; receptor function in response to AM, AM(2)/intermedin and CGRP was measured in a cAMP assay and cell surface expression was determined by ELISA. Phe reduced AM potency in RAMP3 but had no effect in RAMP1. In contrast, Tyr had no effect in RAMP3 but enhanced AM potency in RAMP1. Most other substitutions had a small effect on AM potency in both receptors whereas there was little impact on CGRP or AM(2) potency. Overall, these data suggest that the geometry and charge of the residue at position 74 contribute to how AM interacts with the AM(2) and CGRP receptors and confirms the role of this position in dictating differential AM pharmacology at the AM(2) and CGRP receptors.
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Molecular basis of association of receptor activity-modifying protein 3 with the family B G protein-coupled secretin receptor.
Biochemistry, 2009Co-Authors: Kaleeckal G. Harikumar, George Christopoulos, Patrick M. Sexton, John Simms, Laurence J MillerAbstract:The three receptor activity-modifying proteins (RAMPs) have been recognized as being important for the trafficking and function of a subset of family B G protein-coupled receptors, although the structural basis for this has not been well established. In the current work, we use morphological fluorescence techniques, bioluminescence resonance energy transfer, and bimolecular fluorescence complementation to demonstrate that the secretin receptor associates specifically with RAMP3, but not with RAMP1 or RAMP2. We use truncation constructs, peptide competition experiments, and chimeric secretin−GLP1 receptor constructs to establish that this association is structurally specific, dependent on the intramembranous region of the RAMP and TM6 and TM7 of this receptor. There were no observed changes in secretin-stimulated cAMP, intracellular calcium, ERK1/2 phosphorylation, or receptor internalization in receptor-bearing COS or CHO-K1 cells in the presence or absence of exogenous RAMP transfection, although the sec...
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identification of n terminal receptor activity modifying protein residues important for calcitonin gene related peptide adrenomedullin and amylin receptor function
Molecular Pharmacology, 2008Co-Authors: George Christopoulos, Patrick M. Sexton, Arthur Christopoulos, Richard J. Bailey, Debbie L. HayAbstract:Calcitonin-family receptors comprise calcitonin receptor-like receptor (CL) or calcitonin receptor and receptor activity-modifying protein (RAMP) pairings. Calcitonin gene-related peptide (CGRP) receptors are CL/RAMP1, whereas adrenomedullin (AM) receptors are CL/RAMP2 (AM1 receptor) or CL/RAMP3 (AM2 receptor). Amylin (Amy) receptors are RAMP hetero-oligomers with the calcitonin receptor (AMY1, AMY2, and AMY3, respectively). How RAMPs change G protein-coupled receptor pharmacology is not fully understood. We exploited sequence differences between RAMP1 and RAMP3 to identify individual residues capable of altering receptor pharmacology. Alignment of human RAMPs revealed eight residues that are conserved in RAMP2 and RAMP3 but are different in RAMP1. We hypothesized that residues in RAMP2 and RAMP3, but not RAMP1, are responsible for making CL/RAMP2 and CL/RAMP3 AM receptors. Using site-directed mutagenesis, we introduced individual RAMP3 residues into RAMP1 and vice versa in these eight positions. Mutant or wild-type RAMPs were transfected into Cos7 cells with CL or the insert-negative form of the calcitonin receptor [CT(a)]. Agonist-stimulated cAMP production and cell-surface expression of constructs were measured. Position 74 in RAMP1 and RAMP3 was critical for determining AM potency and affinity, and Phe93 in RAMP1 was an important contributor to αCGRP potency at CGRP receptors. Mutant RAMP/CT(a) receptor complexes displayed different phenotypes. It is noteworthy that RAMP1 S103N and W74E mutations led to enhanced rAmy potency, probably related to increased cell-surface expression of these complexes. This differs from the effect on CL-based receptors where expression was unchanged. Targeted substitution has emphasized the importance of position 74 in RAMP1/RAMP3 as a key determinant of AM pharmacology.
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Receptor activity-modifying proteins differentially modulate the G protein-coupling efficiency of amylin receptors.
Endocrinology, 2008Co-Authors: Maria Morfis, George Christopoulos, Arthur Christopoulos, Nandasena Tilakaratne, Sebastian G.b. Furness, Timothy David Werry, Patrick M. SextonAbstract:Receptor activity-modifying proteins (RAMPs) 1, 2, and 3 are prototypic G protein-coupled receptor accessory proteins that can alter not only receptor trafficking but also receptor phenotype. Specific RAMP interaction with the calcitonin receptor (CTR) generates novel and distinct receptors for the peptide amylin; however, the role of RAMPs in receptor signaling is not understood. The current study demonstrates that RAMP interaction with the CTRa in COS-7 or HEK-293 cells leads to selective modulation of signaling pathways activated by the receptor complex. There was a 20- to 30-fold induction in amylin potency at CTR/RAMP1 (AMY1) and CTR/RAMP3 (AMY3) receptors, compared with CTR alone, for formation of the second-messenger cAMP that parallels an increase in amylin binding affinity. In contrast, only 2- to 5-fold induction of amylin potency was seen for mobilization of intracellular Ca++ or activation of ERK1/2. In addition, in COS-7 cells, the increase in amylin potency for Ca++ mobilization was 2-fold g...
George Christopoulos - One of the best experts on this subject based on the ideXlab platform.
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structure function analysis of amino acid 74 of human RAMP1 and ramp3 and its role in peptide interactions with adrenomedullin and calcitonin gene related peptide receptors
Peptides, 2011Co-Authors: David R Poyner, Patrick M. Sexton, George Christopoulos, Debbie L. HayAbstract:The receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are complexes of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying proteins (RAMP). The CGRP receptor is a CLR/RAMP1 pairing whereas CLR/RAMP2 and CLR/RAMP3 constitute two subtypes of AM receptor: AM(1) and AM(2), respectively. Previous studies identified Glu74 in RAMP3 to be important for AM binding and potency. To further understand the importance of this residue and its equivalent in RAMP1 (Trp74) we substituted the native amino acids with several others. In RAMP3, these were Trp, Phe, Tyr, Ala, Ser, Thr, Arg and Asn; in RAMP1, Glu, Phe, Tyr, Ala and Asn substitutions were made. The mutant RAMPs were co-expressed with CLR in Cos7 cells; receptor function in response to AM, AM(2)/intermedin and CGRP was measured in a cAMP assay and cell surface expression was determined by ELISA. Phe reduced AM potency in RAMP3 but had no effect in RAMP1. In contrast, Tyr had no effect in RAMP3 but enhanced AM potency in RAMP1. Most other substitutions had a small effect on AM potency in both receptors whereas there was little impact on CGRP or AM(2) potency. Overall, these data suggest that the geometry and charge of the residue at position 74 contribute to how AM interacts with the AM(2) and CGRP receptors and confirms the role of this position in dictating differential AM pharmacology at the AM(2) and CGRP receptors.
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Structure–function analysis of amino acid 74 of human RAMP1 and RAMP3 and its role in peptide interactions with adrenomedullin and calcitonin gene-related peptide receptors
Peptides, 2011Co-Authors: David R Poyner, Patrick M. Sexton, George Christopoulos, Debbie L. HayAbstract:The receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are complexes of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying proteins (RAMP). The CGRP receptor is a CLR/RAMP1 pairing whereas CLR/RAMP2 and CLR/RAMP3 constitute two subtypes of AM receptor: AM(1) and AM(2), respectively. Previous studies identified Glu74 in RAMP3 to be important for AM binding and potency. To further understand the importance of this residue and its equivalent in RAMP1 (Trp74) we substituted the native amino acids with several others. In RAMP3, these were Trp, Phe, Tyr, Ala, Ser, Thr, Arg and Asn; in RAMP1, Glu, Phe, Tyr, Ala and Asn substitutions were made. The mutant RAMPs were co-expressed with CLR in Cos7 cells; receptor function in response to AM, AM(2)/intermedin and CGRP was measured in a cAMP assay and cell surface expression was determined by ELISA. Phe reduced AM potency in RAMP3 but had no effect in RAMP1. In contrast, Tyr had no effect in RAMP3 but enhanced AM potency in RAMP1. Most other substitutions had a small effect on AM potency in both receptors whereas there was little impact on CGRP or AM(2) potency. Overall, these data suggest that the geometry and charge of the residue at position 74 contribute to how AM interacts with the AM(2) and CGRP receptors and confirms the role of this position in dictating differential AM pharmacology at the AM(2) and CGRP receptors.
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Molecular basis of association of receptor activity-modifying protein 3 with the family B G protein-coupled secretin receptor.
Biochemistry, 2009Co-Authors: Kaleeckal G. Harikumar, George Christopoulos, Patrick M. Sexton, John Simms, Laurence J MillerAbstract:The three receptor activity-modifying proteins (RAMPs) have been recognized as being important for the trafficking and function of a subset of family B G protein-coupled receptors, although the structural basis for this has not been well established. In the current work, we use morphological fluorescence techniques, bioluminescence resonance energy transfer, and bimolecular fluorescence complementation to demonstrate that the secretin receptor associates specifically with RAMP3, but not with RAMP1 or RAMP2. We use truncation constructs, peptide competition experiments, and chimeric secretin−GLP1 receptor constructs to establish that this association is structurally specific, dependent on the intramembranous region of the RAMP and TM6 and TM7 of this receptor. There were no observed changes in secretin-stimulated cAMP, intracellular calcium, ERK1/2 phosphorylation, or receptor internalization in receptor-bearing COS or CHO-K1 cells in the presence or absence of exogenous RAMP transfection, although the sec...
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identification of n terminal receptor activity modifying protein residues important for calcitonin gene related peptide adrenomedullin and amylin receptor function
Molecular Pharmacology, 2008Co-Authors: George Christopoulos, Patrick M. Sexton, Arthur Christopoulos, Richard J. Bailey, Debbie L. HayAbstract:Calcitonin-family receptors comprise calcitonin receptor-like receptor (CL) or calcitonin receptor and receptor activity-modifying protein (RAMP) pairings. Calcitonin gene-related peptide (CGRP) receptors are CL/RAMP1, whereas adrenomedullin (AM) receptors are CL/RAMP2 (AM1 receptor) or CL/RAMP3 (AM2 receptor). Amylin (Amy) receptors are RAMP hetero-oligomers with the calcitonin receptor (AMY1, AMY2, and AMY3, respectively). How RAMPs change G protein-coupled receptor pharmacology is not fully understood. We exploited sequence differences between RAMP1 and RAMP3 to identify individual residues capable of altering receptor pharmacology. Alignment of human RAMPs revealed eight residues that are conserved in RAMP2 and RAMP3 but are different in RAMP1. We hypothesized that residues in RAMP2 and RAMP3, but not RAMP1, are responsible for making CL/RAMP2 and CL/RAMP3 AM receptors. Using site-directed mutagenesis, we introduced individual RAMP3 residues into RAMP1 and vice versa in these eight positions. Mutant or wild-type RAMPs were transfected into Cos7 cells with CL or the insert-negative form of the calcitonin receptor [CT(a)]. Agonist-stimulated cAMP production and cell-surface expression of constructs were measured. Position 74 in RAMP1 and RAMP3 was critical for determining AM potency and affinity, and Phe93 in RAMP1 was an important contributor to αCGRP potency at CGRP receptors. Mutant RAMP/CT(a) receptor complexes displayed different phenotypes. It is noteworthy that RAMP1 S103N and W74E mutations led to enhanced rAmy potency, probably related to increased cell-surface expression of these complexes. This differs from the effect on CL-based receptors where expression was unchanged. Targeted substitution has emphasized the importance of position 74 in RAMP1/RAMP3 as a key determinant of AM pharmacology.
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Receptor activity-modifying proteins differentially modulate the G protein-coupling efficiency of amylin receptors.
Endocrinology, 2008Co-Authors: Maria Morfis, George Christopoulos, Arthur Christopoulos, Nandasena Tilakaratne, Sebastian G.b. Furness, Timothy David Werry, Patrick M. SextonAbstract:Receptor activity-modifying proteins (RAMPs) 1, 2, and 3 are prototypic G protein-coupled receptor accessory proteins that can alter not only receptor trafficking but also receptor phenotype. Specific RAMP interaction with the calcitonin receptor (CTR) generates novel and distinct receptors for the peptide amylin; however, the role of RAMPs in receptor signaling is not understood. The current study demonstrates that RAMP interaction with the CTRa in COS-7 or HEK-293 cells leads to selective modulation of signaling pathways activated by the receptor complex. There was a 20- to 30-fold induction in amylin potency at CTR/RAMP1 (AMY1) and CTR/RAMP3 (AMY3) receptors, compared with CTR alone, for formation of the second-messenger cAMP that parallels an increase in amylin binding affinity. In contrast, only 2- to 5-fold induction of amylin potency was seen for mobilization of intracellular Ca++ or activation of ERK1/2. In addition, in COS-7 cells, the increase in amylin potency for Ca++ mobilization was 2-fold g...
Tanenao Eto - One of the best experts on this subject based on the ideXlab platform.
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functions of the cytoplasmic tails of the human receptor activity modifying protein components of calcitonin gene related peptide and adrenomedullin receptors
Journal of Biological Chemistry, 2006Co-Authors: Yuan-ning Cao, Tanenao Eto, Chunping Chu, Shuji Iwatsubo, Kazuo KitamuraAbstract:Next Section Abstract Receptor activity-modifying proteins (RAMPs) enable calcitonin receptor-like receptor (CRLR) to function as a calcitonin gene-related peptide receptor (CRLR/RAMP1) or an adrenomedullin (AM) receptor (CRLR/RAMP2 or -3). Here we investigated the functions of the cytoplasmic C-terminal tails (C-tails) of human RAMP1, -2, and -3 (hRAMP1, -2, and -3) by cotransfecting their C-terminal deletion or progressive truncation mutants into HEK-293 cells stably expressing hCRLR. Deletion of the C-tail from hRAMP1 had little effect on the surface expression, function, or intracellular trafficking of the mutant heterodimers. By contrast, deletion of the C-tail from hRAMP2 disrupted transport of hCRLR to the cell surface, resulting in significant reductions in 125I-hAM binding and evoked cAMP accumulation. The transfection efficiency for the hRAMP2 mutant was comparable with that for wild-type hRAMP2; moreover, immunocytochemical analysis showed that the mutant hRAMP2 remained within the endoplasmic reticulum. FACS analysis revealed that deleting the C-tail from hRAMP3 markedly enhances AM-evoked internalization of the mutant heterodimers, although there was no change in agonist affinity. Truncating the C-tails by removing the six C-terminal amino acids of hRAMP2 and -3 or exchanging their C-tails with one another had no effect on surface expression, agonist affinity, or internalization of hCRLR, which suggests that the highly conserved Ser-Lys sequence within hRAMP C-tails is involved in cellular trafficking of the two AM receptors. Notably, deleting the respective C-tails from hRAMPs had no effect on lysosomal sorting of hCRLR. Thus, the respective C-tails of hRAMP2 and -3 differentially affect hCRLR surface delivery and internalization.
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Characterization of the human calcitonin gene-related peptide receptor subtypes associated with receptor activity-modifying proteins
Molecular pharmacology, 2004Co-Authors: Kenji Kuwasako, Kazuo Kitamura, Yasuko Nagoshi, Yuan-ning Cao, Toshihiro Tsuruda, Tanenao EtoAbstract:Coexpression of receptor activity-modifying proteins (RAMPs) with calcitonin receptor 2 (CTR2) or calcitonin receptor-like receptor (CRLR) leads to the formation of four functional heterodimeric receptors for human calcitonin gene-related peptide (hCGRP). In this study, we transfected hCGRP receptors into human embryonic kidney 293 cells and examined their pharmacological profiles using three dominant-negative (DN) RAMP mutants and various hCGRPα analogs. Fluorescence-activated cell-sorting analysis revealed that their cotransfection with CTR2 induced cell surface expression of all three RAMPs, and the three CTR2/RAMP heterodimers mediated equivalent levels of cAMP production in response to hCGRPα that were approximately 50-fold greater than were seen with CTR2 alone. By contrast, [Tyr0]hCGRPα binding and signaling were markedly weaker with CTR2/RAMP2 or -3 than with CTR2/RAMP1 or CRLR/RAMP1; likewise, 125I-[His10]hCGRPα bound most potently to CTR2/RAMP1. When CTR2 was coexpressed with DN RAMP1 or -2, hCGRPα-evoked responses were similar to those seen with CTR2 alone, despite the expression of both CTR2 and DN RAMP at the cell surface. But coexpression of DN RAMP3 with CTR2 significantly diminished hCGRPα signaling compared with that seen with CTR2 alone, indicating that DN RAMP3 is able to function as a negative regulator of CTR2 function. Competition experiments showed the relative agonist sensitivity of the four receptors to be hCGRPα > [Tyr0]hCGRPα > [Cys(Et)2,7]hCGRPα > [Cys(ACM)2,7]hCGRPα. Of the linear analogs, [Cys(ACM)2,7]hCGRPα (ACM, acetylmethoxy) enhanced cAMP formation only via CTR2/RAMP1, whereas [Cys(Et2,7)]hCGRPα acted via CRLR/RAMP1 and somewhat less potently via CTR2/RAMP1. Thus, among the three CGRP8–37-insensitive receptors, CTR2/RAMP1 is most sensitive to the two linear analogs, suggesting that it could be classified as a CGRP2 receptor. Moreover, the combined use of iodinated CGRPα analogs may be useful for defining the CGRP1 receptor.
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Adrenomedullin receptors: pharmacological features and possible pathophysiological roles
Peptides, 2004Co-Authors: Kenji Kuwasako, Kazuo Kitamura, Yasuko Nagoshi, Yuan-ning Cao, Tanenao EtoAbstract:Abstract Three receptor activity modifying proteins (RAMPs) chaperone calcitonin-like receptor (CLR) to the cell surface. RAMP2 enables CLR to form an adrenomedullin (AM)-specific receptor that is sensitive to AM-(22–52) (AM 1 receptor). RAMP3 enables CLR to form an AM receptor sensitive to both calcitonin gene-related peptide (CGRP)-(8–37) and AM-(22–52) (AM 2 receptor), though rat and mouse AM 2 receptors show a clear preference for CGRPα-(8–37) over AM-(22–52). RAMP1 enables CRL to form the CGRP-(8–37)-sensitive CGRP 1 receptor, which can also be activated by higher concentrations of AM. Here we review the available information on the pharmacological features and possible pathophysiological roles of the aforementioned AM receptors.
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Angiotensin II modulates gene expression of adrenomedullin receptor components in rat cardiomyocytes.
Life sciences, 2003Co-Authors: Kazuya Mishima, Johji Kato, Kazuo Kitamura, Takuroh Imamura, Tanenao EtoAbstract:Both adrenomedullin (AM) and angiotensin II (Ang II) are locally-acting hormones in the cardiac ventricles. Previously we reported that AM inhibits Ang II-induced hypertrophy of cultured rat neonatal cardiomyocytes. In this study, we examined whether Ang II affects the gene expression of the AM receptor components of calcitonin-receptor-like receptor (CRLR) and receptor-activity-modifying protein (RAMP) in rat cardiomyocytes. The mRNA levels of RAMP1 and RAMP3 were significantly elevated following 24-h treatment with Ang II without a change of those of RAMP2 and CRLR. AM increased the intracellular cAMP level and the cAMP accumulation by AM was significantly amplified by the 24-h preincubation with Ang II. The effects of Ang II on RAMP1 and RAMP3 expression were abolished by an Ang II type 1 (AT1) receptor antagonist, but not by an AT2 receptor antagonist. Thus, Ang II modulates gene expression of the AM receptor components via AT1 receptor, suggesting alteration of AM actions by Ang II in cultured rat cardiomyocytes.
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Identification of the human receptor activity : modifying protein 1 domains responsible for agonist binding specificity
The Journal of biological chemistry, 2003Co-Authors: Kazuo Kitamura, Yasuko Nagoshi, Yuan-ning Cao, Tanenao EtoAbstract:Abstract When co-expressed with receptor activity-modifying protein (RAMP) 1, calcitonin receptor-like receptor (CRLR) can function as a receptor for both calcitonin gene-related peptide (CGRP) and adrenomedullin (AM). To investigate the structural determinants of ligand binding specificity, we examined the extracellular domain of human (h) RAMP1 using various deletion mutants. Co-expression of the hRAMP1 mutants with hCRLR in HEK-293 cells revealed that deletion of residues 91–94, 96–100, or 101–103 blocked [125I]CGRP binding and completely abolished intracellular cAMP accumulation normally elicited by CGRP or AM. On the other hand, the deletion of residues 78–80 or 88–90 significantly attenuated only AM-evoked responses. In all of these cases, the receptor heterodimers were fully expressed at the cell surface. Substituting alanine for residues 91–103 one at a time had little effect on CGRP-induced responses, indicating that although this segment is essential for high affinity agonist binding to the receptors, none of the residues directly interacts with either CGRP or AM. This finding suggests that RAMPs probably determine ligand specificity by contributing to the structure of the ligand-binding pocket or by allosteric modulation of the conformation of the receptor. Interestingly, the L94A mutant up-regulated surface expression of the receptor heterodimer to a greater degree than wild-type hRAMP1, thereby increasing CGRP binding and signaling. L94A also significantly increased cell surface expression of the hRAMP1 deletion mutant D101–103 when co-transfected with hCRLR, and expression of a L94A/D101–103 double mutant markedly attenuated the activity of endogenous RAMP1 in HEK-293T cells.
