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Debbie L. Hay - One of the best experts on this subject based on the ideXlab platform.
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deconvoluting the molecular control of binding and signaling at the amylin 3 Receptor ramp3 alters signal propagation through extracellular loops of the Calcitonin Receptor
ACS Pharmacology & Translational Science, 2019Co-Authors: Vi Pham, Yue Zhu, Emma Dal Maso, Christopher A Reynolds, Giuseppe Deganutti, Silvia Atanasio, Caroline A Hick, Dehua Yang, Arthur Christopoulos, Debbie L. HayAbstract:Amylin is coexpressed with insulin in pancreatic islet β-cells and has potent effects on gastric emptying and food intake. The effect of amylin on satiation has been postulated to involve AMY3 Receptors (AMY3R) that are heteromers of the Calcitonin Receptor (CTR) and Receptor activity-modifying protein 3 (RAMP3). Understanding the molecular control of signaling through the AMY3R is thus important for peptide drug targeting of this Receptor. We have previously used alanine scanning mutagenesis to study the contribution of the extracellular surface of the CTR to binding and signaling initiated by Calcitonin (CT) and related peptides (Dal Maso, E., et al. (2019) The molecular control of Calcitonin Receptor signaling. ACS Pharmacol. Transl. Sci. 2, 31–51). That work revealed ligand- and pathway-specific effects of mutation, with extracellular loops (ECLs) 2 and 3 particularly important in the distinct propagation of signaling mediated by individual peptides. In the current study, we have used equivalent alani...
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Deconvoluting the Molecular Control of Binding and Signaling at the Amylin 3 Receptor: RAMP3 Alters Signal Propagation through Extracellular Loops of the Calcitonin Receptor
2019Co-Authors: Vi Pham, Yue Zhu, Emma Dal Maso, Christopher A Reynolds, Giuseppe Deganutti, Silvia Atanasio, Caroline A Hick, Dehua Yang, Arthur Christopoulos, Debbie L. HayAbstract:Amylin is coexpressed with insulin in pancreatic islet β-cells and has potent effects on gastric emptying and food intake. The effect of amylin on satiation has been postulated to involve AMY3 Receptors (AMY3R) that are heteromers of the Calcitonin Receptor (CTR) and Receptor activity-modifying protein 3 (RAMP3). Understanding the molecular control of signaling through the AMY3R is thus important for peptide drug targeting of this Receptor. We have previously used alanine scanning mutagenesis to study the contribution of the extracellular surface of the CTR to binding and signaling initiated by Calcitonin (CT) and related peptides (Dal Maso, E., et al. (2019) The molecular control of Calcitonin Receptor signaling. ACS Pharmacol. Transl. Sci. 2, 31–51). That work revealed ligand- and pathway-specific effects of mutation, with extracellular loops (ECLs) 2 and 3 particularly important in the distinct propagation of signaling mediated by individual peptides. In the current study, we have used equivalent alanine scanning of ECL2 and ECL3 of the CTR in the context of coexpression with RAMP3 to form AMY3Rs, to examine functional affinity and efficacy of peptides in cAMP accumulation and extracellular signal-regulated kinase (ERK) phosphorylation (pERK). The effect of mutation was determined on representatives of the three major distinct classes of CT peptide, salmon CT (sCT), human CT (hCT), and porcine CT (pCT), as well as rat amylin (rAmy) or human α-CGRP (Calcitonin gene-related peptide, hCGRP) whose potency is enhanced by RAMP interaction. We demonstrate that the dynamic nature of CTR ECL2 and ECL3 in propagation of signaling is fundamentally altered when complexed with RAMP3 to form the AMY3R, despite only having predicted direct interactions with ECL2. Moreover, the work shows that the role of these loops in Receptor signaling is highly peptide dependent, illustrating that even subtle changes to peptide sequence may change signaling output downstream of the Receptor
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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 Have Limited Effects on the Class B G Protein-Coupled Receptor Calcitonin Receptor-Like Receptor Stalk
2018Co-Authors: Michael L Garelja, Harriet A Watkins, Joseph J. Gingell, Christina A. Walker, Andrew Siow, Sung H. Yang, Paul W.r. Harris, Margaret A. Brimble, Debbie L. HayAbstract:The Calcitonin Receptor-like Receptor (CLR) is a class B G protein-coupled Receptor (GPCR) that forms the basis of three pharmacologically distinct Receptors, the Calcitonin gene-related peptide (CGRP) Receptor, and two adrenomedullin (AM) Receptors. These three Receptors are created by CLR interacting with three Receptor activity-modifying proteins (RAMPs). Class B GPCRs have an N-terminal extracellular domain (ECD) and transmembrane bundle that are both important for binding endogenous ligands. These two domains are joined together by a stretch of amino acids that is referred to as the “stalk”. Studies of other class B GPCRs suggest that the stalk may act as hinge, allowing the ECD to adopt multiple conformations. It is unclear what the role of the stalk is within CLR and whether RAMPs can influence its function. Therefore, this study investigated the role of this region using an alanine scan. Effects of mutations were measured with all three RAMPs through cell surface expression, cAMP production and, in select cases, radioligand binding and total cell expression assays. Most mutants did not affect expression or cAMP signaling. CLR C127A, N140A, F142A, and L144A impaired cell surface expression with all three RAMPs. T125A decreased the potency of all peptides at all Receptors. N128A, V135A, and L139A showed ligand-dependent effects. While the stalk appears to play a role in CLR function, the effect of RAMPs on this region seems limited, in contrast to their effects on the structure of CLR in other Receptor regions
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Calcitonin and amylin Receptor peptide interaction mechanisms insights into peptide binding modes and allosteric modulation of the Calcitonin Receptor by Receptor activity modifying proteins
Journal of Biological Chemistry, 2016Co-Authors: Sangmin Lee, Debbie L. Hay, Augen A PioszakAbstract:Receptor activity-modifying proteins (RAMP1-3) determine the selectivity of the class B G protein-coupled Calcitonin Receptor (CTR) and the CTR-like Receptor (CLR) for Calcitonin (CT), amylin (Amy), Calcitonin gene-related peptide (CGRP), and adrenomedullin (AM) peptides. RAMP1/2 alter CLR selectivity for CGRP/AM in part by RAMP1 Trp-84 or RAMP2 Glu-101 contacting the distinct CGRP/AM C-terminal residues. It is unclear whether RAMPs use a similar mechanism to modulate CTR affinity for CT and Amy, analogs of which are therapeutics for bone disorders and diabetes, respectively. Here, we reproduced the peptide selectivity of intact CTR, AMY1 (CTR·RAMP1), and AMY2 (CTR·RAMP2) Receptors using purified CTR extracellular domain (ECD) and tethered RAMP1- and RAMP2-CTR ECD fusion proteins and antagonist peptides. All three proteins bound salmon Calcitonin (sCT). Tethering RAMPs to CTR enhanced binding of rAmy, CGRP, and the AMY antagonist AC413. Peptide alanine-scanning mutagenesis and modeling of Receptor-bound sCT and AC413 supported a shared non-helical CGRP-like conformation for their TN(T/V)G motif prior to the C terminus. After this motif, the peptides diverged; the sCT C-terminal Pro was crucial for Receptor binding, whereas the AC413/rAmy C-terminal Tyr had little or no influence on binding. Accordingly, mutant RAMP1 W84A- and RAMP2 E101A-CTR ECD retained AC413/rAmy binding. ECD binding and cell-based signaling assays with antagonist sCT/AC413/rAmy variants with C-terminal residue swaps indicated that the C-terminal sCT/rAmy residue identity affects affinity more than selectivity. rAmy(8-37) Y37P exhibited enhanced antagonism of AMY1 while retaining selectivity. These results reveal unexpected differences in how RAMPs determine CTR and CLR peptide selectivity and support the hypothesis that RAMPs allosterically modulate CTR peptide affinity.
Nigel W Bunnett - One of the best experts on this subject based on the ideXlab platform.
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Calcitonin Receptor like Receptor clr Receptor activity modifying protein 1 ramp1 and Calcitonin gene related peptide cgrp immunoreactivity in the rat trigeminovascular system differences between peripheral and central cgrp Receptor distribution
The Journal of Comparative Neurology, 2008Co-Authors: Jochen K Lennerz, Victor Ruhle, Eugene P Ceppa, Eileen F Grady, Nigel W Bunnett, Winfried Neuhuber, Karl MesslingerAbstract:Calcitonin gene-related peptide (CGRP) is a key mediator in primary headaches including migraine. Animal models of meningeal nociception demonstrate both peripheral and central CGRP effects; however, the target structures remain unclear. To study the distribution of CGRP Receptors in the rat trigeminovascular system we used antibodies recognizing two components of the CGRP Receptor, the Calcitonin Receptor-like Receptor (CLR) and the Receptor activity-modifying protein 1 (RAMP1). In the cranial dura mater, CLR and RAMP1 immunoreactivity (-ir) was found within arterial blood vessels, mononuclear cells, and Schwann cells, but not sensory axons. In the trigeminal ganglion, besides Schwann and satellite cells, CLR- and RAMP1-ir was found in subpopulations of CGRP-ir neurons where colocalization of CGRP- and RAMP1-ir was very rare (≈0.6%). CLR- and RAMP1-ir was present on central, but not peripheral, axons. In the spinal trigeminal nucleus, CLR- and RAMP1-ir was localized to “glomerular structures,” partly colocalized with CGRP-ir. However, CLR- and RAMP1-ir was lacking in central glia and neuronal cell bodies. We conclude that CGRP Receptors are associated with structural targets of known CGRP effects (vasodilation, mast cell degranulation) and targets of unknown function (Schwann cells). In the spinal trigeminal nucleus, CGRP Receptors are probably located on neuronal processes, including primary afferent endings, suggesting involvement in presynaptic regulation of nociceptive transmission. Thus, in the trigeminovascular system CGRP Receptor localization suggests multiple targets for CGRP in the pathogenesis of primary headaches. J. Comp. Neurol. 507:1277–1299, 2008. © 2008 Wiley-Liss, Inc.
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Calcitonin Receptor like Receptor clr Receptor activity modifying protein 1 ramp1 and Calcitonin gene related peptide cgrp immunoreactivity in the rat trigeminovascular system differences between peripheral and central cgrp Receptor distribution
The Journal of Comparative Neurology, 2008Co-Authors: Jochen K Lennerz, Victor Ruhle, Eugene P Ceppa, Eileen F Grady, Nigel W Bunnett, Winfried Neuhuber, Karl MesslingerAbstract:Calcitonin gene-related peptide (CGRP) is a key mediator in primary headaches including migraine. Animal models of meningeal nociception demonstrate both peripheral and central CGRP effects; however, the target structures remain unclear. To study the distribution of CGRP Receptors in the rat trigeminovascular system we used antibodies recognizing two components of the CGRP Receptor, the Calcitonin Receptor-like Receptor (CLR) and the Receptor activity-modifying protein 1 (RAMP1). In the cranial dura mater, CLR and RAMP1 immunoreactivity (-ir) was found within arterial blood vessels, mononuclear cells, and Schwann cells, but not sensory axons. In the trigeminal ganglion, besides Schwann and satellite cells, CLR- and RAMP1-ir was found in subpopulations of CGRP-ir neurons where colocalization of CGRP- and RAMP1-ir was very rare ( approximately 0.6%). CLR- and RAMP1-ir was present on central, but not peripheral, axons. In the spinal trigeminal nucleus, CLR- and RAMP1-ir was localized to "glomerular structures," partly colocalized with CGRP-ir. However, CLR- and RAMP1-ir was lacking in central glia and neuronal cell bodies. We conclude that CGRP Receptors are associated with structural targets of known CGRP effects (vasodilation, mast cell degranulation) and targets of unknown function (Schwann cells). In the spinal trigeminal nucleus, CGRP Receptors are probably located on neuronal processes, including primary afferent endings, suggesting involvement in presynaptic regulation of nociceptive transmission. Thus, in the trigeminovascular system CGRP Receptor localization suggests multiple targets for CGRP in the pathogenesis of primary headaches.
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endothelin converting enzyme 1 regulates endosomal sorting of Calcitonin Receptor like Receptor and β arrestins
Journal of Cell Biology, 2007Co-Authors: Benjamin E. Padilla, Dirk Roosterman, Graeme S. Cottrell, Laurent Muller, Martin Steinhoff, Stella Pikios, Nigel W BunnettAbstract:Although cell surface metalloendopeptidases degrade neuropeptides in the extracellular fluid to terminate signaling, the function of peptidases in endosomes is unclear. We report that isoforms of endothelin-converting enzyme-1 (ECE-1a–d) are present in early endosomes, where they degrade neuropeptides and regulate post-endocytic sorting of Receptors. Calcitonin gene-related peptide (CGRP) co-internalizes with Calcitonin Receptor-like Receptor (CLR), Receptor activity-modifying protein 1 (RAMP1), β-arrestin2, and ECE-1 to early endosomes, where ECE-1 degrades CGRP. CGRP degradation promotes CLR/RAMP1 recycling and β-arrestin2 redistribution to the cytosol. ECE-1 inhibition or knockdown traps CLR/RAMP1 and β-arrestin2 in endosomes and inhibits CLR/RAMP1 recycling and resensitization, whereas ECE-1 overexpression has the opposite effect. ECE-1 does not regulate either the resensitization of Receptors for peptides that are not ECE-1 substrates (e.g., angiotensin II), or the recycling of the bradykinin B2 Receptor, which transiently interacts with β-arrestins. We propose a mechanism by which endosomal ECE-1 degrades neuropeptides in endosomes to disrupt the peptide/Receptor/β-arrestin complex, freeing internalized Receptors from β-arrestins and promoting recycling and resensitization.
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endothelin converting enzyme 1 regulates endosomal sorting of Calcitonin Receptor like Receptor and beta arrestins
Journal of Cell Biology, 2007Co-Authors: Benjamin E. Padilla, Dirk Roosterman, Graeme S. Cottrell, Laurent Muller, Martin Steinhoff, Stella Pikios, Nigel W BunnettAbstract:Although cell surface metalloendopeptidases degrade neuropeptides in the extracellular fluid to terminate signaling, the function of peptidases in endosomes is unclear. We report that isoforms of endothelin-converting enzyme-1 (ECE-1a-d) are present in early endosomes, where they degrade neuropeptides and regulate post-endocytic sorting of Receptors. Calcitonin gene-related peptide (CGRP) co-internalizes with Calcitonin Receptor-like Receptor (CLR), Receptor activity-modifying protein 1 (RAMP1), beta-arrestin2, and ECE-1 to early endosomes, where ECE-1 degrades CGRP. CGRP degradation promotes CLR/RAMP1 recycling and beta-arrestin2 redistribution to the cytosol. ECE-1 inhibition or knockdown traps CLR/RAMP1 and beta-arrestin2 in endosomes and inhibits CLR/RAMP1 recycling and resensitization, whereas ECE-1 overexpression has the opposite effect. ECE-1 does not regulate either the resensitization of Receptors for peptides that are not ECE-1 substrates (e.g., angiotensin II), or the recycling of the bradykinin B(2) Receptor, which transiently interacts with beta-arrestins. We propose a mechanism by which endosomal ECE-1 degrades neuropeptides in endosomes to disrupt the peptide/Receptor/beta-arrestin complex, freeing internalized Receptors from beta-arrestins and promoting recycling and resensitization.
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Calcitonin Receptor-like Receptor and Receptor activity modifying protein 1 in the rat dorsal horn: Localization in glutamatergic presynaptic terminals containing opioids and adrenergic α2C Receptors
Neuroscience, 2007Co-Authors: Juan-carlos Marvizon, O. A. Pérez, E. F. Grady, Nigel W Bunnett, B Song, W. Chen, A. J. ToddAbstract:Calcitonin gene-related peptide (CGRP) is abundant in the central terminals of primary afferents. However, the function of CGRP Receptors in the spinal cord remains unclear. CGRP Receptors are heterodimers of Calcitonin Receptor-like Receptor (CRLR) and Receptor activity modifying protein 1 (RAMP1). We studied the localization of CRLR and RAMP1 in the rat dorsal horn using well-characterized antibodies against them, which labeled numerous puncta in laminae I-II. In addition, RAMP1 was found in cell bodies, forming patches at the cell surface. The CRLR- and RAMP1-immunoreactive puncta were further characterized using double and triple labeling. Colocalization was quantified in confocal stacks using Imaris software. CRLR did not colocalize with primary afferent markers, indicating that these puncta were not primary afferent terminals. CRLR- and RAMP1-immunoreactive puncta contained synaptophysin and vesicular glutamate transporter-2 (VGLUT2), showing that they were glutamatergic presynaptic terminals. Electron microscopic immunohistochemistry confirmed that CRLR immunoreactivity was present in axonal boutons that were not in synaptic glomeruli. Using tyramide signal amplification for double labeling with the CRLR and RAMP1 antibodies, we found some clear instances of colocalization of CRLR with RAMP1 in puncta, but their overall colocalization was low. In particular, CRLR was absent from RAMP1-containing cells. Many of the puncta stained for CRLR and RAMP1 were labeled by anti-opioid and anti-enkephalin antibodies. CRLR and, to a lesser extent, RAMP1 also colocalized with adrenergic α2C Receptors. Triple label studies demonstrated three-way colocalization of CRLR-VGLUT2-synaptophysin, CRLR-VGLUT2-opioids, and CRLR-opioids-α2C Receptors. In conclusion, CRLR is located in glutamatergic presynaptic terminals in the dorsal horn that contain α2C adrenergic Receptors and opioids. Some of these terminals contain RAMP1, which may form CGRP Receptors with CRLR, but in others CRLR may form other Receptors, possibly by dimerizing with RAMP2 or RAMP3. These findings suggest that CGRP or adrenomedullin Receptors modulate opioid release in the dorsal horn. © 2007 IBRO.
Jochen K Lennerz - One of the best experts on this subject based on the ideXlab platform.
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Calcitonin Receptor like Receptor clr Receptor activity modifying protein 1 ramp1 and Calcitonin gene related peptide cgrp immunoreactivity in the rat trigeminovascular system differences between peripheral and central cgrp Receptor distribution
The Journal of Comparative Neurology, 2008Co-Authors: Jochen K Lennerz, Victor Ruhle, Eugene P Ceppa, Eileen F Grady, Nigel W Bunnett, Winfried Neuhuber, Karl MesslingerAbstract:Calcitonin gene-related peptide (CGRP) is a key mediator in primary headaches including migraine. Animal models of meningeal nociception demonstrate both peripheral and central CGRP effects; however, the target structures remain unclear. To study the distribution of CGRP Receptors in the rat trigeminovascular system we used antibodies recognizing two components of the CGRP Receptor, the Calcitonin Receptor-like Receptor (CLR) and the Receptor activity-modifying protein 1 (RAMP1). In the cranial dura mater, CLR and RAMP1 immunoreactivity (-ir) was found within arterial blood vessels, mononuclear cells, and Schwann cells, but not sensory axons. In the trigeminal ganglion, besides Schwann and satellite cells, CLR- and RAMP1-ir was found in subpopulations of CGRP-ir neurons where colocalization of CGRP- and RAMP1-ir was very rare (≈0.6%). CLR- and RAMP1-ir was present on central, but not peripheral, axons. In the spinal trigeminal nucleus, CLR- and RAMP1-ir was localized to “glomerular structures,” partly colocalized with CGRP-ir. However, CLR- and RAMP1-ir was lacking in central glia and neuronal cell bodies. We conclude that CGRP Receptors are associated with structural targets of known CGRP effects (vasodilation, mast cell degranulation) and targets of unknown function (Schwann cells). In the spinal trigeminal nucleus, CGRP Receptors are probably located on neuronal processes, including primary afferent endings, suggesting involvement in presynaptic regulation of nociceptive transmission. Thus, in the trigeminovascular system CGRP Receptor localization suggests multiple targets for CGRP in the pathogenesis of primary headaches. J. Comp. Neurol. 507:1277–1299, 2008. © 2008 Wiley-Liss, Inc.
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Calcitonin Receptor like Receptor clr Receptor activity modifying protein 1 ramp1 and Calcitonin gene related peptide cgrp immunoreactivity in the rat trigeminovascular system differences between peripheral and central cgrp Receptor distribution
The Journal of Comparative Neurology, 2008Co-Authors: Jochen K Lennerz, Victor Ruhle, Eugene P Ceppa, Eileen F Grady, Nigel W Bunnett, Winfried Neuhuber, Karl MesslingerAbstract:Calcitonin gene-related peptide (CGRP) is a key mediator in primary headaches including migraine. Animal models of meningeal nociception demonstrate both peripheral and central CGRP effects; however, the target structures remain unclear. To study the distribution of CGRP Receptors in the rat trigeminovascular system we used antibodies recognizing two components of the CGRP Receptor, the Calcitonin Receptor-like Receptor (CLR) and the Receptor activity-modifying protein 1 (RAMP1). In the cranial dura mater, CLR and RAMP1 immunoreactivity (-ir) was found within arterial blood vessels, mononuclear cells, and Schwann cells, but not sensory axons. In the trigeminal ganglion, besides Schwann and satellite cells, CLR- and RAMP1-ir was found in subpopulations of CGRP-ir neurons where colocalization of CGRP- and RAMP1-ir was very rare ( approximately 0.6%). CLR- and RAMP1-ir was present on central, but not peripheral, axons. In the spinal trigeminal nucleus, CLR- and RAMP1-ir was localized to "glomerular structures," partly colocalized with CGRP-ir. However, CLR- and RAMP1-ir was lacking in central glia and neuronal cell bodies. We conclude that CGRP Receptors are associated with structural targets of known CGRP effects (vasodilation, mast cell degranulation) and targets of unknown function (Schwann cells). In the spinal trigeminal nucleus, CGRP Receptors are probably located on neuronal processes, including primary afferent endings, suggesting involvement in presynaptic regulation of nociceptive transmission. Thus, in the trigeminovascular system CGRP Receptor localization suggests multiple targets for CGRP in the pathogenesis of primary headaches.
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 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, Christopher S. Walker, David R Poyner, Richard J. Bailey, 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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Structure-function analysis of RAMP1-RAMP3 chimeras.
Biochemistry, 2010Co-Authors: John Simms, Debbie L. Hay, Richard J. Bailey, Mark Wheatley, Daniel L. Rathbone, David R PoynerAbstract:The role of Receptor activity modifying protein 1 (RAMP1) in forming Receptors with the Calcitonin Receptor-like Receptor (CLR) and the Calcitonin Receptor (CTR) was examined by producing chimeras between RAMP1 and RAMP3. RAMPs have three extracellular helices. Exchange of helix 1 of the RAMPs or residues 62-69 in helix 2 greatly reduced CLR trafficking (a marker for CLR association). Modeling suggests that these exchanges alter the CLR recognition site on RAMP1, which is more exposed than on RAMP3. Exchange of residues 86-89 of RAMP1 had no effect on the trafficking of CLR but reduced the potency of human (h) alphaCGRP and adrenomedullin. However, these alterations to RAMP1 had no effect on the potency of hbetaCGRP. These residues of RAMP1 lie at the junction of helix 3 and its connecting loop with helix 2. Modeling suggests that the loop is more exposed in RAMP1 than RAMP3; it may play an important role in peptide binding, either directly or indirectly. Exchange of residues 90-94 of RAMP1 caused a modest reduction in CLR expression and a 15-fold decrease in CGRP potency. It is unlikely that the decrease in expression is enough to explain the reduction in potency, and so these may have dual roles in recognizing CLR and CGRP. For CTR, only 6 out of 26 chimeras covering the extracellular part of RAMP1 did not reduce agonist potency. Thus the association of CTR with RAMP1 seems more sensitive to changes in RAMP1 structure induced by the chimeras than is CLR.
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pharmacological discrimination of Calcitonin Receptor Receptor activity modifying protein complexes
Molecular Pharmacology, 2005Co-Authors: George Christopoulos, David R PoynerAbstract:Calcitonin (CT) Receptors dimerize with Receptor activity-modifying proteins (RAMPs) to create high-affinity amylin (AMY) Receptors, but there is no reliable means of pharmacologically distinguishing these Receptors. We used agonists and antagonists to define their pharmacology, expressing the CT (a) Receptor alone or with RAMPs in COS-7 cells and measuring cAMP accumulation. Intermedin short, otherwise known as adrenomedullin 2, mirrored the action of αCGRP, being a weak agonist at CT(a), AMY 2(a), and AMY3(a) Receptors but considerably more potent at AMY1(a) Receptors. Likewise, the linear Calcitonin gene-related peptide (CGRP) analogs (Cys(ACM)2,7)hαCGRP and (Cys(Et) 2,7)haCGRP were only effective at AMY1(a) Receptors, but they were partial agonists. As previously observed in COS-7 cells, there was little induction of the AMY2(a) Receptor phenotype; thus, AMY 2(a) was not examined further in this study. The antagonist peptide salmon Calcitonin8-32 (sCT8-32) did not discriminate strongly between CT and AMY Receptors; however, AC187 was a more effective antagonist of AMY responses at AMY Receptors, and AC413 additionally showed modest selectivity for AMY1(a) over AMY3(a) Receptors. CGRP8-37 also demonstrated Receptor-dependent effects. CGRP 8-37 more effectively antagonized AMY at AMY1(a) than AMY3(a) Receptors, although it was only a weak antagonist of both, but it did not inhibit responses at the CT(a) Receptor. Low CGRP 8-37 affinity and agonism by linear CGRP analogs at AMY 1(a) are the classic signature of a CGRP2 Receptor. Our data indicate that careful use of combinations of agonists and antagonists may allow pharmacological discrimination of CT(a), AMY1(a), and AMY3(a) Receptors, providing a means to delineate the physiological significance of these Receptors. Copyright © 2005 The American Society for Pharmacology and Experimental Therapeutics.
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international union of pharmacology xxxii the mammalian Calcitonin gene related peptides adrenomedullin amylin and Calcitonin Receptors
Pharmacological Reviews, 2002Co-Authors: David R Poyner, Jan A. Fischer, Walter Born, Ian W Marshall, Remi Quirion, Roman Muff, Steven Michael FoordAbstract:The Calcitonin family of peptides comprises Calcitonin, amylin two Calcitonin gene-related peptides (CGRPs), and adrenomedullin. The first Calcitonin Receptor was cloned in 1991. Its pharmacology is complicated by the existence of several splice variants. The Receptors for the other members the family are made up of subunits. The Calcitonin-like Receptor (CL Receptor) requires a single transmembrane domain protein, termed Receptor activity modifying protein, RAMP1, to function as a CGRP Receptor. RAMP2 and -3 enable the same CL Receptor to behave as an adrenomedullin Receptor. Although the Calcitonin Receptor does not require RAMP to bind and respond to Calcitonin, it can associate with the RAMPs, resulting in a series of Receptors that typically have high affinity for amylin and varied affinity for CGRP. This review aims to reconcile what is observed when the Receptors are reconstituted in vitro with the properties they show in native cells and tissues. Experimental conditions must be rigorously controlled because different degrees of protein expression may markedly modify pharmacology in such a complex situation. Recommendations, which follow International Union of Pharmacology guidelines, are made for the nomenclature of these multimeric Receptors.
Karl Messlinger - One of the best experts on this subject based on the ideXlab platform.
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Calcitonin Receptor like Receptor clr Receptor activity modifying protein 1 ramp1 and Calcitonin gene related peptide cgrp immunoreactivity in the rat trigeminovascular system differences between peripheral and central cgrp Receptor distribution
The Journal of Comparative Neurology, 2008Co-Authors: Jochen K Lennerz, Victor Ruhle, Eugene P Ceppa, Eileen F Grady, Nigel W Bunnett, Winfried Neuhuber, Karl MesslingerAbstract:Calcitonin gene-related peptide (CGRP) is a key mediator in primary headaches including migraine. Animal models of meningeal nociception demonstrate both peripheral and central CGRP effects; however, the target structures remain unclear. To study the distribution of CGRP Receptors in the rat trigeminovascular system we used antibodies recognizing two components of the CGRP Receptor, the Calcitonin Receptor-like Receptor (CLR) and the Receptor activity-modifying protein 1 (RAMP1). In the cranial dura mater, CLR and RAMP1 immunoreactivity (-ir) was found within arterial blood vessels, mononuclear cells, and Schwann cells, but not sensory axons. In the trigeminal ganglion, besides Schwann and satellite cells, CLR- and RAMP1-ir was found in subpopulations of CGRP-ir neurons where colocalization of CGRP- and RAMP1-ir was very rare (≈0.6%). CLR- and RAMP1-ir was present on central, but not peripheral, axons. In the spinal trigeminal nucleus, CLR- and RAMP1-ir was localized to “glomerular structures,” partly colocalized with CGRP-ir. However, CLR- and RAMP1-ir was lacking in central glia and neuronal cell bodies. We conclude that CGRP Receptors are associated with structural targets of known CGRP effects (vasodilation, mast cell degranulation) and targets of unknown function (Schwann cells). In the spinal trigeminal nucleus, CGRP Receptors are probably located on neuronal processes, including primary afferent endings, suggesting involvement in presynaptic regulation of nociceptive transmission. Thus, in the trigeminovascular system CGRP Receptor localization suggests multiple targets for CGRP in the pathogenesis of primary headaches. J. Comp. Neurol. 507:1277–1299, 2008. © 2008 Wiley-Liss, Inc.
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Calcitonin Receptor like Receptor clr Receptor activity modifying protein 1 ramp1 and Calcitonin gene related peptide cgrp immunoreactivity in the rat trigeminovascular system differences between peripheral and central cgrp Receptor distribution
The Journal of Comparative Neurology, 2008Co-Authors: Jochen K Lennerz, Victor Ruhle, Eugene P Ceppa, Eileen F Grady, Nigel W Bunnett, Winfried Neuhuber, Karl MesslingerAbstract:Calcitonin gene-related peptide (CGRP) is a key mediator in primary headaches including migraine. Animal models of meningeal nociception demonstrate both peripheral and central CGRP effects; however, the target structures remain unclear. To study the distribution of CGRP Receptors in the rat trigeminovascular system we used antibodies recognizing two components of the CGRP Receptor, the Calcitonin Receptor-like Receptor (CLR) and the Receptor activity-modifying protein 1 (RAMP1). In the cranial dura mater, CLR and RAMP1 immunoreactivity (-ir) was found within arterial blood vessels, mononuclear cells, and Schwann cells, but not sensory axons. In the trigeminal ganglion, besides Schwann and satellite cells, CLR- and RAMP1-ir was found in subpopulations of CGRP-ir neurons where colocalization of CGRP- and RAMP1-ir was very rare ( approximately 0.6%). CLR- and RAMP1-ir was present on central, but not peripheral, axons. In the spinal trigeminal nucleus, CLR- and RAMP1-ir was localized to "glomerular structures," partly colocalized with CGRP-ir. However, CLR- and RAMP1-ir was lacking in central glia and neuronal cell bodies. We conclude that CGRP Receptors are associated with structural targets of known CGRP effects (vasodilation, mast cell degranulation) and targets of unknown function (Schwann cells). In the spinal trigeminal nucleus, CGRP Receptors are probably located on neuronal processes, including primary afferent endings, suggesting involvement in presynaptic regulation of nociceptive transmission. Thus, in the trigeminovascular system CGRP Receptor localization suggests multiple targets for CGRP in the pathogenesis of primary headaches.