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Walter Born - One of the best experts on this subject based on the ideXlab platform.
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Protection of Angiotensin II–Induced Vascular Hypertrophy in Vascular Smooth Muscle–Targeted Receptor Activity-Modifying Protein 2 Transgenic Mice
2016Co-Authors: Lihuan Liang, Jan A. Fischer, Walter Born, Christina W. Tam, Gabor Pozsgai, Richard Siow, Natalie Clark, Julie Keeble, Knut Husmann, Robin PostonAbstract:Abstract—The vasodilator and vascular regulatory peptide adrenomedullin (AM), a member of the calcitonin gene-related peptide family of peptides, is predicted to play a pivotal protective role in cardiovascular dysfunction. The principle AM (AM1) Receptor is composed of a G Protein–linked calcitonin Receptor-like Receptor and a Receptor Activity-Modifying Protein (Receptor Activity-Modifying Protein 2). There is little knowledge of the Receptors via which AM acts in diseases. Using smooth muscle-targeted Receptor activity–modifying Protein 2 transgenic mice with increased vascular density of functional AM1 Receptors, we demonstrate that Receptor Activity-Modifying Protein 2 transgenic mice are not protected against angiotensin II–induced hypertension or cardiac hypertrophy. However, vascular hypertrophy, together with vascular cell adhesion molecule 1 and monocyte chemotactic Protein 1 expression, is significantly reduced in the aortic walls of transgenic mice, as determined by histological techniques. This indicates that the AM1 vascular smooth muscle Receptor can mediate local protection in vivo. This is supported by proliferation studies in cultured smooth muscle cells. By comparison, levels of hypotension and inflammation in a shock model were similar to those in wild-type mice. Thus, a role of the AM1 Receptor in the vasoactive component could not be detected, and evidence is provided to show that the hypotensive response to AM is subject to desensitization in vivo. The finding that the vascular smooth muscle AM1 Receptor acts at a local level to protect against hypertension-induced vascular hypertrophy and inflammation provides evidence that targeting this Receptor may be a beneficial therapeutic approach. (Hypertension. 2009;54:1254-1261.
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Protection of Angiotensin II-Induced Vascular Hypertrophy in Vascular Smooth Muscle-Targeted Receptor Activity-Modifying Protein 2 Transgenic Mice
Hypertension (Dallas Tex. : 1979), 2009Co-Authors: Lihuan Liang, Jan A. Fischer, Walter Born, Christina W. Tam, Gabor Pozsgai, Natalie Clark, Julie Keeble, Knut Husmann, Richard C.m. Siow, Robin PostonAbstract:The vasodilator and vascular regulatory peptide adrenomedullin (AM), a member of the calcitonin gene-related peptide family of peptides, is predicted to play a pivotal protective role in cardiovascular dysfunction. The principle AM (AM1) Receptor is composed of a G Protein–linked calcitonin Receptor-like Receptor and a Receptor Activity-Modifying Protein (Receptor Activity-Modifying Protein 2). There is little knowledge of the Receptors via which AM acts in diseases. Using smooth muscle-targeted Receptor activity–modifying Protein 2 transgenic mice with increased vascular density of functional AM1 Receptors, we demonstrate that Receptor Activity-Modifying Protein 2 transgenic mice are not protected against angiotensin II–induced hypertension or cardiac hypertrophy. However, vascular hypertrophy, together with vascular cell adhesion molecule 1 and monocyte chemotactic Protein 1 expression, is significantly reduced in the aortic walls of transgenic mice, as determined by histological techniques. This indicates that the AM1 vascular smooth muscle Receptor can mediate local protection in vivo. This is supported by proliferation studies in cultured smooth muscle cells. By comparison, levels of hypotension and inflammation in a shock model were similar to those in wild-type mice. Thus, a role of the AM1 Receptor in the vasoactive component could not be detected, and evidence is provided to show that the hypotensive response to AM is subject to desensitization in vivo. The finding that the vascular smooth muscle AM1 Receptor acts at a local level to protect against hypertension-induced vascular hypertrophy and inflammation provides evidence that targeting this Receptor may be a beneficial therapeutic approach.
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The function of conserved cysteine residues in the extracellular domain of human Receptor-Activity-Modifying Protein 1
FEBS letters, 2003Co-Authors: Sarah Steiner, Jan A. Fischer, Walter Born, Roman MuffAbstract:The Receptor-Activity-Modifying Protein (RAMP) 1 is a single-transmembrane-domain Protein associated with the calcitonin-like Receptor (CLR) to reveal a calcitonin gene-related peptide (CGRP) Receptor. The extracellular region of RAMP1 contains six conserved cysteines. Here, Cys27 in myc-tagged human (h) RAMP1 was deleted (hRAMP1Δ1), and Cys40, Cys57, Cys72, Cys82 and Cys104 were each replaced by Ala. In COS-7 cells expressing hCLR/myc-hRAMP1Δ1 or -C82A, cell surface expression, [125I]hαCGRP binding and cAMP formation in response to hαCGRP were similar to those of hCLR/myc-hRAMP1. Cell surface expression of myc-hRAMP1-C72A was reduced to 24±7% of myc-hRAMP1, and that of -C40A, -C57A and -C104A was below 10%. [125I]hαCGRP binding of hCLR/myc-hRAMP1-C72A was 13±3% of hCLR/myc-hRAMP1 and it was undetectable in hCLR/myc-hRAMP1-C40A-, -C57A- and -C104A-expressing cells. Maximal cAMP stimulation by hαCGRP in hCLR/myc-hRAMP1-C40A- and -C72A-expressing cells was 14±1% and 33±2% of that of the hCLR/myc-hRAMP1 with comparable EC50. But cAMP stimulation was abolished in cells expressing hCLR/myc-hRAMP1-C57A and -C104A. In conclusion, CGRP Receptor function was not affected by the deletion of Cys27 or the substitution of Cys82 by Ala in hRAMP1, but it was impaired by the substitution of Cys40, Cys57, Cys72 and Cys104 by Ala. These four cysteines are required for the transport of hRAMP1 together with the CLR to the cell surface.
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Receptor-Activity-Modifying Protein 1 forms heterodimers with two G-Protein-coupled Receptors to define ligand recognition
Biochemical Journal, 2000Co-Authors: Kerstin Leuthauser, Jan A. Fischer, Roman Muff, Remo Gujer, Amaya Aldecoa, R. Anne Mckinney, Walter BornAbstract:Receptor-Activity-Modifying Proteins (RAMPs) with single transmembrane domains define the function of two G-Protein-coupled Receptors of the B family. Cell-surface complexes of human RAMP1 (hRAMP1) and human calcitonin (CT) Receptor isotype 2 (hCTR2) or rat CT-Receptor-like Receptor (rCRLR) have now been identified through Protein cross-linking, co-immunoprecipitation and confocal microscopy. They are two distinct CT-gene-related peptide (CGRP) Receptors coupled to cAMP production and pharmacologically distinguished by the CT and CGRP antagonists salmon CT(8-32) and human or rat CGRP(8-37). Thus direct molecular interactions of hRAMP1 with hCTR2 or rCRLR are required for CGRP recognition. hCTR2, moreover, adopts non-traditional functions through its association with hRAMP1.
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mammalian calcitonin Receptor like Receptor Receptor activity modifying Protein complexes define calcitonin gene related peptide and adrenomedullin Receptors in drosophila schneider 2 cells
FEBS Letters, 2000Co-Authors: Amaya Aldecoa, Jan A. Fischer, Remo Gujer, Walter BornAbstract:Differential glycosylation of human and rat (r) calcitonin (CT) Receptor-like Receptors (CRLR) as a result of interactions with accessory Receptor Activity-Modifying Proteins (RAMP)1 or -2 was considered to define CT gene-related peptide (CGRP) or adrenomedullin (ADM) Receptors in mammalian cells. Here, Drosophila Schneider (S2) cells stably co-expressed rCRLR and RAMP1 or -2 as functional CGRP or ADM Receptors. Different from mammalian cells, rCRLR expressed in S2 cells are uniformly glycosylated Proteins independent of RAMP1 or RAMP2. Bis(sulfosuccinimidyl)suberate cross-linking revealed Receptor components with the size of rCRLR, increased by the molecular weights of the corresponding RAMPs and [125I]CGRP or [125I]ADM. In conclusion, [125I]CGRP/rCRLR/RAMP1 and [125I]ADM/rCRLR/RAMP2 complexes have been recognized in Drosophila S2 cells.
Kenji Kuwasako - One of the best experts on this subject based on the ideXlab platform.
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characterization of the single transmembrane domain of human Receptor activity modifying Protein 3 in adrenomedullin Receptor internalization
Biochemical and Biophysical Research Communications, 2012Co-Authors: Kenji Kuwasako, Sayaka Nagata, Kazuo Kitamura, Naomi Nozaki, Johji KatoAbstract:Abstract Two Receptor Activity-Modifying Proteins (RAMP2 and RAMP3) enable calcitonin Receptor-like Receptor (CLR) to function as two heterodimeric Receptors (CLR/RAMP2 and CLR/RAMP3) for adrenomedullin (AM), a potent cardiovascular protective peptide. Following AM stimulation, both Receptors undergo rapid internalization through a clathrin-dependent pathway, after which CLR/RAMP3, but not CLR/RAMP2, can be recycled to the cell surface for resensitization. However, human (h)RAMP3 mediates CLR internalization much less efficiently than does hRAMP2. Therefore, the molecular basis of the single transmembrane domain (TMD) and the intracellular domain of hRAMP3 during AM Receptor internalization was investigated by transiently transfecting various RAMP chimeras and mutants into HEK-293 cells stably expressing hCLR. Flow cytometric analysis revealed that substituting the RAMP3 TMD with that of RAMP2 markedly enhanced AM-induced internalization of CLR. However, this replacement did not enhance the cell surface expression of CLR, [125I]AM binding affinity or AM-induced cAMP response. More detailed analyses showed that substituting the Thr130–Val131 sequence in the RAMP3 TMD with the corresponding sequence (Ile157–Pro158) from RAMP2 significantly enhanced AM-mediated CLR internalization. In contrast, substituting the RAMP3 target sequence with Ala130–Ala131 did not significantly affect CLR internalization. Thus, the RAMP3 TMD participates in the negative regulation of CLR/RAMP3 internalization, and the aforementioned introduction of the Ile–Pro sequence into the RAMP3 TMD may be a strategy for promoting Receptor internalization/resensitization.
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Function of the cytoplasmic tail of human calcitonin Receptor-like Receptor in complex with Receptor Activity-Modifying Protein 2.
Biochemical and biophysical research communications, 2010Co-Authors: Kenji Kuwasako, Sayaka Nagata, Kazuo Kitamura, Tomomi Hikosaka, Johji KatoAbstract:Abstract Receptor Activity-Modifying Protein 2 (RAMP2) enables calcitonin Receptor-like Receptor (CRLR) to form an adrenomedullin (AM)-specific Receptor. Here we investigated the function of the cytoplasmic C-terminal tail (C-tail) of human (h)CRLR by co-transfecting its C-terminal mutants into HEK-293 cells stably expressing hRAMP2. Deleting the C-tail from CRLR disrupted AM-evoked cAMP production or Receptor internalization, but did not affect [125I]AM binding. We found that CRLR residues 428–439 are required for AM-evoked cAMP production, though deleting this region had little effect on Receptor internalization. Moreover, pretreatment with pertussis toxin (100 ng/mL) led to significant increases in AM-induced cAMP production via wild-type CRLR/RAMP2 complexes. This effect was canceled by deleting CRLR residues 454–457, suggesting Gi couples to this region. Flow cytometric analysis revealed that CRLR truncation mutants lacking residues in the Ser/Thr-rich region extending from Ser449 to Ser467 were unable to undergo AM-induced Receptor internalization and, in contrast to the effect on wild-type CRLR, overexpression of GPCR kinases-2, -3 and -4 failed to promote internalization of CRLR mutants lacking residues 449–467. Thus, the hCRLR C-tail is crucial for AM-evoked cAMP production and internalization of the CRLR/RAMP2, while the Receptor internalization is dependent on the aforementioned GPCR kinases, but not Gs coupling.
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functions of the extracellular histidine residues of Receptor activity modifying Proteins vary within adrenomedullin Receptors
Biochemical and Biophysical Research Communications, 2008Co-Authors: Kenji Kuwasako, Sayaka Nagata, Kazuo Kitamura, Johji KatoAbstract:Abstract Receptor Activity-Modifying Protein (RAMP)-2 and -3 chaperone calcitonin Receptor-like Receptor (CRLR) to the plasma membrane, where together they form heterodimeric adrenomedullin (AM) Receptors. We investigated the contributions made by His residues situated in the RAMP extracellular domain to AM Receptor trafficking and Receptor signaling by co-expressing hCRLR and V5-tagged-hRAMP2 or -3 mutants in which a His residue was substituted with Ala in HEK-293 cells. Flow cytometric analysis revealed that hRAMP2-H71A mediated normal hCRLR surface delivery, but the resultant heterodimers showed significantly diminished [125I]AM binding and AM-evoked cAMP production. Expression of hRAMP2-H124A and -H127A impaired surface delivery of hCRLR, which impaired or abolishing AM binding and Receptor signaling. Although hRAMP3-H97A mediated full surface delivery of hCRLR, the resultant heterodimers showed impaired AM binding and signaling. Other His residues appeared uninvolved in hCRLR-related functions. Thus, the His residues of hRAMP2 and -3 differentially govern AM Receptor function.
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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: Kenji Kuwasako, Tanenao Eto, Yuan-ning Cao, 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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Angiotensin II modulates gene expression of adrenomedullin Receptor components in rat cardiomyocytes.
Life sciences, 2003Co-Authors: Kazuya Mishima, Kenji Kuwasako, 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.
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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Receptor activity modifying Protein dependent and independent activation mechanisms in the coupling of calcitonin gene related peptide and adrenomedullin Receptors to gs
Biochemical Pharmacology, 2017Co-Authors: Michael J Woolley, David R Poyner, John Simms, Christopher A. Reynolds, Christopher S. Walker, Juan Carlos Mobarec, Michael L Garelja, Alex C Conner, Debbie L HayAbstract:Calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) Receptors are heteromers of the calcitonin Receptor-like Receptor (CLR), a class B G Protein-coupled Receptor, and one of three Receptor Activity-Modifying Proteins (RAMPs). How CGRP and AM activate CLR and how this process is modulated by RAMPs is unclear. We have defined how CGRP and AM induce Gs-coupling in CLR-RAMP heteromers by measuring the effect of targeted mutagenesis in the CLR transmembrane domain on cAMP production, modeling the active state conformations of CGRP and AM Receptors in complex with the Gs C-terminus and conducting molecular dynamics simulations in an explicitly hydrated lipidic bilayer. The largest effects on Receptor signaling were seen with H295A5.40b, I298A5.43b, L302A5.47b, N305A5.50b, L345A6.49b and E348A6.52b, F349A6.53b and H374A7.47b (class B numbering in superscript). Many of these residues are likely to form part of a group in close proximity to the peptide binding site and link to a network of hydrophilic and hydrophobic residues, which undergo rearrangements to facilitate Gs binding. Residues closer to the extracellular loops displayed more pronounced RAMP or ligand-dependent effects. Mutation of H3747.47b to alanine increased AM potency 100-fold in the CGRP Receptor. The molecular dynamics simulation showed that TM5 and TM6 pivoted around TM3. The data suggest that hydrophobic interactions are more important for CLR activation than other class B GPCRs, providing new insights into the mechanisms of activation of this class of Receptor. Furthermore the data may aid in the understanding of how RAMPs modulate the signaling of other class B GPCRs.
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An allosteric role for Receptor Activity-Modifying Proteins in defining GPCR pharmacology
Cell discovery, 2016Co-Authors: Joseph J. Gingell, Patrick M. Sexton, David R Poyner, John Simms, Harriet A. Watkins, James Barwell, Augen A. Pioszak, Debbie L HayAbstract:G Protein-coupled Receptors are allosteric Proteins that control transmission of external signals to regulate cellular response. Although agonist binding promotes canonical G Protein signalling transmitted through conformational changes, G Protein-coupled Receptors also interact with other Proteins. These include other G Protein-coupled Receptors, other Receptors and channels, regulatory Proteins and Receptor-modifying Proteins, notably Receptor Activity-Modifying Proteins (RAMPs). RAMPs have at least 11 G Protein-coupled Receptor partners, including many class B G Protein-coupled Receptors. Prototypic is the calcitonin Receptor, with altered ligand specificity when co-expressed with RAMPs. To gain molecular insight into the consequences of this Protein–Protein interaction, we combined molecular modelling with mutagenesis of the calcitonin Receptor extracellular domain, assessed in ligand binding and functional assays. Although some calcitonin Receptor residues are universally important for peptide interactions (calcitonin, amylin and calcitonin gene-related peptide) in calcitonin Receptor alone or with Receptor Activity-Modifying Protein, others have RAMP-dependent effects, whereby mutations decreased amylin/calcitonin gene-related peptide potency substantially only when RAMP was present. Remarkably, the key residues were completely conserved between calcitonin Receptor and AMY Receptors, and between subtypes of AMY Receptor that have different ligand preferences. Mutations at the interface between calcitonin Receptor and RAMP affected ligand pharmacology in a RAMP-dependent manner, suggesting that RAMP may allosterically influence the calcitonin Receptor conformation. Supporting this, molecular dynamics simulations suggested that the calcitonin Receptor extracellular N-terminal domain is more flexible in the presence of Receptor Activity-Modifying Protein 1. Thus, RAMPs may act in an allosteric manner to generate a spectrum of unique calcitonin Receptor conformational states, explaining the pharmacological preferences of calcitonin Receptor-RAMP complexes. This provides novel insight into our understanding of G Protein-coupled Receptor-Protein interaction that is likely broadly applicable for this Receptor class.
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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.
Jan A. Fischer - One of the best experts on this subject based on the ideXlab platform.
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Protection of Angiotensin II–Induced Vascular Hypertrophy in Vascular Smooth Muscle–Targeted Receptor Activity-Modifying Protein 2 Transgenic Mice
2016Co-Authors: Lihuan Liang, Jan A. Fischer, Walter Born, Christina W. Tam, Gabor Pozsgai, Richard Siow, Natalie Clark, Julie Keeble, Knut Husmann, Robin PostonAbstract:Abstract—The vasodilator and vascular regulatory peptide adrenomedullin (AM), a member of the calcitonin gene-related peptide family of peptides, is predicted to play a pivotal protective role in cardiovascular dysfunction. The principle AM (AM1) Receptor is composed of a G Protein–linked calcitonin Receptor-like Receptor and a Receptor Activity-Modifying Protein (Receptor Activity-Modifying Protein 2). There is little knowledge of the Receptors via which AM acts in diseases. Using smooth muscle-targeted Receptor activity–modifying Protein 2 transgenic mice with increased vascular density of functional AM1 Receptors, we demonstrate that Receptor Activity-Modifying Protein 2 transgenic mice are not protected against angiotensin II–induced hypertension or cardiac hypertrophy. However, vascular hypertrophy, together with vascular cell adhesion molecule 1 and monocyte chemotactic Protein 1 expression, is significantly reduced in the aortic walls of transgenic mice, as determined by histological techniques. This indicates that the AM1 vascular smooth muscle Receptor can mediate local protection in vivo. This is supported by proliferation studies in cultured smooth muscle cells. By comparison, levels of hypotension and inflammation in a shock model were similar to those in wild-type mice. Thus, a role of the AM1 Receptor in the vasoactive component could not be detected, and evidence is provided to show that the hypotensive response to AM is subject to desensitization in vivo. The finding that the vascular smooth muscle AM1 Receptor acts at a local level to protect against hypertension-induced vascular hypertrophy and inflammation provides evidence that targeting this Receptor may be a beneficial therapeutic approach. (Hypertension. 2009;54:1254-1261.
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Protection of Angiotensin II-Induced Vascular Hypertrophy in Vascular Smooth Muscle-Targeted Receptor Activity-Modifying Protein 2 Transgenic Mice
Hypertension (Dallas Tex. : 1979), 2009Co-Authors: Lihuan Liang, Jan A. Fischer, Walter Born, Christina W. Tam, Gabor Pozsgai, Natalie Clark, Julie Keeble, Knut Husmann, Richard C.m. Siow, Robin PostonAbstract:The vasodilator and vascular regulatory peptide adrenomedullin (AM), a member of the calcitonin gene-related peptide family of peptides, is predicted to play a pivotal protective role in cardiovascular dysfunction. The principle AM (AM1) Receptor is composed of a G Protein–linked calcitonin Receptor-like Receptor and a Receptor Activity-Modifying Protein (Receptor Activity-Modifying Protein 2). There is little knowledge of the Receptors via which AM acts in diseases. Using smooth muscle-targeted Receptor activity–modifying Protein 2 transgenic mice with increased vascular density of functional AM1 Receptors, we demonstrate that Receptor Activity-Modifying Protein 2 transgenic mice are not protected against angiotensin II–induced hypertension or cardiac hypertrophy. However, vascular hypertrophy, together with vascular cell adhesion molecule 1 and monocyte chemotactic Protein 1 expression, is significantly reduced in the aortic walls of transgenic mice, as determined by histological techniques. This indicates that the AM1 vascular smooth muscle Receptor can mediate local protection in vivo. This is supported by proliferation studies in cultured smooth muscle cells. By comparison, levels of hypotension and inflammation in a shock model were similar to those in wild-type mice. Thus, a role of the AM1 Receptor in the vasoactive component could not be detected, and evidence is provided to show that the hypotensive response to AM is subject to desensitization in vivo. The finding that the vascular smooth muscle AM1 Receptor acts at a local level to protect against hypertension-induced vascular hypertrophy and inflammation provides evidence that targeting this Receptor may be a beneficial therapeutic approach.
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The function of conserved cysteine residues in the extracellular domain of human Receptor-Activity-Modifying Protein 1
FEBS letters, 2003Co-Authors: Sarah Steiner, Jan A. Fischer, Walter Born, Roman MuffAbstract:The Receptor-Activity-Modifying Protein (RAMP) 1 is a single-transmembrane-domain Protein associated with the calcitonin-like Receptor (CLR) to reveal a calcitonin gene-related peptide (CGRP) Receptor. The extracellular region of RAMP1 contains six conserved cysteines. Here, Cys27 in myc-tagged human (h) RAMP1 was deleted (hRAMP1Δ1), and Cys40, Cys57, Cys72, Cys82 and Cys104 were each replaced by Ala. In COS-7 cells expressing hCLR/myc-hRAMP1Δ1 or -C82A, cell surface expression, [125I]hαCGRP binding and cAMP formation in response to hαCGRP were similar to those of hCLR/myc-hRAMP1. Cell surface expression of myc-hRAMP1-C72A was reduced to 24±7% of myc-hRAMP1, and that of -C40A, -C57A and -C104A was below 10%. [125I]hαCGRP binding of hCLR/myc-hRAMP1-C72A was 13±3% of hCLR/myc-hRAMP1 and it was undetectable in hCLR/myc-hRAMP1-C40A-, -C57A- and -C104A-expressing cells. Maximal cAMP stimulation by hαCGRP in hCLR/myc-hRAMP1-C40A- and -C72A-expressing cells was 14±1% and 33±2% of that of the hCLR/myc-hRAMP1 with comparable EC50. But cAMP stimulation was abolished in cells expressing hCLR/myc-hRAMP1-C57A and -C104A. In conclusion, CGRP Receptor function was not affected by the deletion of Cys27 or the substitution of Cys82 by Ala in hRAMP1, but it was impaired by the substitution of Cys40, Cys57, Cys72 and Cys104 by Ala. These four cysteines are required for the transport of hRAMP1 together with the CLR to the cell surface.
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Receptor-Activity-Modifying Protein 1 forms heterodimers with two G-Protein-coupled Receptors to define ligand recognition
Biochemical Journal, 2000Co-Authors: Kerstin Leuthauser, Jan A. Fischer, Roman Muff, Remo Gujer, Amaya Aldecoa, R. Anne Mckinney, Walter BornAbstract:Receptor-Activity-Modifying Proteins (RAMPs) with single transmembrane domains define the function of two G-Protein-coupled Receptors of the B family. Cell-surface complexes of human RAMP1 (hRAMP1) and human calcitonin (CT) Receptor isotype 2 (hCTR2) or rat CT-Receptor-like Receptor (rCRLR) have now been identified through Protein cross-linking, co-immunoprecipitation and confocal microscopy. They are two distinct CT-gene-related peptide (CGRP) Receptors coupled to cAMP production and pharmacologically distinguished by the CT and CGRP antagonists salmon CT(8-32) and human or rat CGRP(8-37). Thus direct molecular interactions of hRAMP1 with hCTR2 or rCRLR are required for CGRP recognition. hCTR2, moreover, adopts non-traditional functions through its association with hRAMP1.
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mammalian calcitonin Receptor like Receptor Receptor activity modifying Protein complexes define calcitonin gene related peptide and adrenomedullin Receptors in drosophila schneider 2 cells
FEBS Letters, 2000Co-Authors: Amaya Aldecoa, Jan A. Fischer, Remo Gujer, Walter BornAbstract:Differential glycosylation of human and rat (r) calcitonin (CT) Receptor-like Receptors (CRLR) as a result of interactions with accessory Receptor Activity-Modifying Proteins (RAMP)1 or -2 was considered to define CT gene-related peptide (CGRP) or adrenomedullin (ADM) Receptors in mammalian cells. Here, Drosophila Schneider (S2) cells stably co-expressed rCRLR and RAMP1 or -2 as functional CGRP or ADM Receptors. Different from mammalian cells, rCRLR expressed in S2 cells are uniformly glycosylated Proteins independent of RAMP1 or RAMP2. Bis(sulfosuccinimidyl)suberate cross-linking revealed Receptor components with the size of rCRLR, increased by the molecular weights of the corresponding RAMPs and [125I]CGRP or [125I]ADM. In conclusion, [125I]CGRP/rCRLR/RAMP1 and [125I]ADM/rCRLR/RAMP2 complexes have been recognized in Drosophila S2 cells.
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: Kenji Kuwasako, Tanenao Eto, Yuan-ning Cao, 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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Angiotensin II modulates gene expression of adrenomedullin Receptor components in rat cardiomyocytes.
Life sciences, 2003Co-Authors: Kazuya Mishima, Kenji Kuwasako, 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: Kenji Kuwasako, 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.
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The calcitonin Receptor-like Receptor/Receptor Activity-Modifying Protein 1 heterodimer can function as a calcitonin gene-related peptide-(8-37)-sensitive adrenomedullin Receptor
European journal of pharmacology, 2002Co-Authors: Yasuko Nagoshi, Kenji Kuwasako, Johji Kato, Kazuo Kitamura, Kaoru Ito, Tomohiko Uemura, Tanenao EtoAbstract:Abstract The Receptor Activity-Modifying Protein (RAMP)/calcitonin Receptor-like (CRL) Receptor heterodimer is thought to function as a Receptor for either a calcitonin gene-related peptide (CGRP) (CRL Receptor/RAMP1) or adrenomedullin (CRL Receptor/RAMP2 or -3), depending on the RAMP isoform present. We examined the Receptor specificity of adrenomedullin-induced increases in cAMP in human embryonic kidney (HEK)293 cells coexpressing human CRL Receptor and human RAMP1 or RAMP2. In cells expressing CRL Receptor/RAMP1, adrenomedulin-induced increases in cAMP were comparable to those induced by α-CGRP, and the CGRP Receptor antagonist α-CGRP-(8–37), but not the adrenomedullin Receptor antagonist adrenomedullin-(22–52), blocked the adrenomedullin-evoked responses. Cells expressing CRL Receptor/RAMP2 responded more selectively to adrenomedullin; in this case, the effect was blocked by adrenomedullin-(22–52) but not by α-CGRP-(8–37). Real-time quantitative polymerase chain reaction confirmed that cotransfection of CRL Receptor and RAMP1 had no effect on the endogenous expression of RAMP2. Thus, CRL Receptor/RAMP1 likely functions as an adrenomedullin Receptor as well as a CGRP Receptor, which may explain why many of the actions of adrenomedullin are potently antagonized by α-CGRP-(8–37).
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the calcitonin Receptor like Receptor Receptor activity modifying Protein 1 heterodimer can function as a calcitonin gene related peptide 8 37 sensitive adrenomedullin Receptor
European Journal of Pharmacology, 2002Co-Authors: Yasuko Nagoshi, Kenji Kuwasako, Johji Kato, Kazuo Kitamura, Kaoru Ito, Tomohiko Uemura, Tanenao EtoAbstract:Abstract The Receptor Activity-Modifying Protein (RAMP)/calcitonin Receptor-like (CRL) Receptor heterodimer is thought to function as a Receptor for either a calcitonin gene-related peptide (CGRP) (CRL Receptor/RAMP1) or adrenomedullin (CRL Receptor/RAMP2 or -3), depending on the RAMP isoform present. We examined the Receptor specificity of adrenomedullin-induced increases in cAMP in human embryonic kidney (HEK)293 cells coexpressing human CRL Receptor and human RAMP1 or RAMP2. In cells expressing CRL Receptor/RAMP1, adrenomedulin-induced increases in cAMP were comparable to those induced by α-CGRP, and the CGRP Receptor antagonist α-CGRP-(8–37), but not the adrenomedullin Receptor antagonist adrenomedullin-(22–52), blocked the adrenomedullin-evoked responses. Cells expressing CRL Receptor/RAMP2 responded more selectively to adrenomedullin; in this case, the effect was blocked by adrenomedullin-(22–52) but not by α-CGRP-(8–37). Real-time quantitative polymerase chain reaction confirmed that cotransfection of CRL Receptor and RAMP1 had no effect on the endogenous expression of RAMP2. Thus, CRL Receptor/RAMP1 likely functions as an adrenomedullin Receptor as well as a CGRP Receptor, which may explain why many of the actions of adrenomedullin are potently antagonized by α-CGRP-(8–37).
