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1.
Udawela M Christopoulos G Morfis M Tilakaratne N Christopoulos A Sexton PM 《Regulatory peptides》2008,145(1-3):65-71
Receptor activity modifying proteins (RAMPs) interact with calcitonin receptors to produce novel amylin receptor phenotypes. We have recently demonstrated that the short intracellular C-terminus of RAMPs plays a key role in the function of amylin receptors derived from the CTa calcitonin receptor through the use of chimeric RAMPs and RAMPs that are truncated at the C-terminus [15, Udawela M, Christopoulos G, Morfis M, Christopoulos A, Ye S, Tilakaratne N, Sexton PM. A critical role for the short intracellular C terminus in receptor activity modifying protein function. Mol Pharmacol 2006;70:1750-60., 18, Udawela M, Christopoulos G, Tilakaratne N, Christopoulos A, Albiston A, Sexton PM. Distinct receptor activity-modifying protein domains differentially modulate interaction with calcitonin receptors. Mol Pharmacol 2006;69:1984-89.]. The calcitonin receptor in humans is expressed as two major alternatively spliced isoforms termed CTa and CTb. Relatively little is known about how alternate splicing of the receptor affects the interaction between calcitonin receptors and RAMPs. We have examined the effect of RAMP truncation, through use of mutant constructs that delete the last 8 amino acids of each of the 3 known human RAMPs, and characterised these for interaction with CTb receptors through co-expression in COS-7 cells. As seen with the CTa receptor isoform, RAMP truncation caused a marked loss in induction of AMYb receptor phenotypes as characterised by (125)I-rat amylin radioligand binding assays and cAMP accumulation assays; the latter as a marker of receptor signalling. The effect was most pronounced for RAMP1 and RAMP2 deletion mutants, but attenuated responses were also observed with co-expressed RAMP3 deletion mutants. These data support a direct role for the RAMP C-terminus in the interaction of RAMP/calcitonin receptor complexes with intracellular accessory proteins involved in signalling and/or receptor trafficking. 相似文献
2.
Christopoulos A Christopoulos G Morfis M Udawela M Laburthe M Couvineau A Kuwasako K Tilakaratne N Sexton PM 《The Journal of biological chemistry》2003,278(5):3293-3297
The receptor activity-modifying proteins (RAMPs) comprise a family of three accessory proteins that heterodimerize with the calcitonin receptor-like receptor (CL receptor) or with the calcitonin receptor (CTR) to generate different receptor phenotypes. However, RAMPs are more widely distributed across cell and tissue types than the CTR and CL receptor, suggesting additional roles for RAMPs in cellular processes. We have investigated the potential for RAMP interaction with a number of Class II G protein-coupled receptors (GPCRs) in addition to the CL receptor and the CTR. Using immunofluorescence confocal microscopy, we demonstrate, for the first time, that RAMPs interact with at least four additional receptors, the VPAC1 vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating peptide receptor with all three RAMPs; the glucagon and PTH1 parathyroid hormone receptors with RAMP2; and the PTH2 receptor with RAMP3. Unlike the interaction of RAMPs with the CL receptor or the CTR, VPAC1R-RAMP complexes do not show altered phenotypic behavior compared with the VPAC1R alone, as determined using radioligand binding in COS-7 cells. However, the VPAC1R-RAMP2 heterodimer displays a significant enhancement of agonist-mediated phosphoinositide hydrolysis with no change in cAMP stimulation compared with the VPAC1R alone. Our findings identify a new functional consequence of RAMP-receptor interaction, suggesting that RAMPs play a more general role in modulating cell signaling through other GPCRs than is currently appreciated. 相似文献
3.
RAMPs (receptor activity-modifying proteins) were discovered in 1998 as accessory proteins needed to the functionnal activity of CGRP (calcitonin gene-related peptide) receptors. Three RAMPs generated by three different genes are known in human, rat and mice. The coding sequences of such genes are described, but as yet, regulation sequences are unknown. RAMPs interact with GPCR (G protein-coupled receptors) of class II. In the case of the calcitonin/CGRP peptide family, RAMPs determine the functionnal specificity of the receptor, glycosylate and translocate the receptor to the cell surface. CGRP receptors are observed in presence of the RAMP1/calcitonin receptor-like receptor (CRLR), but the association of RAMP2 or RAMP3 with CRLR generates an adrenomedullin receptor. The calcitonin receptor (CTR) is translocated alone to the cell surface, but interactions of RAMPs with CTR forms amylin receptors. If RAMPs can interact with glucagon, parathyroid hormone and VIP/PACAP (vasoactive intestinal peptide/pituitary adenylate cyclase activating polypeptide (VPACR1)) receptors, the functionnal specificity of these receptors remains unaltered. However, the complex VPACR1/RAMP2 enhances specifically the phosphoinoside signaling pathway. 相似文献
4.
降钙素基因相关肽家族是一类多功能的激素家族 ,参与人体的多种生物学功能 ,与多种疾病有关。降钙素基因相关肽受体包括降钙素受体 (CTR)和降钙素受体样受体 (CRLR) ,CTR可以独自与降钙素结合 ,而CRLR必须与一组称作受体活性修饰蛋白 (RAMPs)的蛋白质共同作用才能发挥生物学功能。综述CTR的研究概况及CRLR与RAMPs相互作用的机制和表达调控 ,以期为人们设计新型药物提供参考。 相似文献
5.
受体活性修饰蛋白研究进展 总被引:1,自引:0,他引:1
受体活性修饰蛋白(receptor activity-modifying proteins,RAMPs)属于单跨膜蛋白家族,分三个结构域,RAMP的N端和跨膜区决定本身的功能和受体表型,胞内C端对于配体的信号传导和受体循环有重要作用。目前发现有三个成员:RAMP1、RAMP2和RAMP3。RAMPs通过改变G蛋白偶联受体的糖基化,作用于配体结合区域来调节受体表型。RAMP1与降钙素受体样受体(calcitonin receptor like receptor,CRLR)结合表现出降钙素基因相关肽(calcitonin gene-related peptide,CGRP)受体表型:RAMP2和RAMP3与CRLR结合则对肾上腺髓质素(adrenomedullin,AM)表现高亲和力,与降钙素受体(calcitonin receptor,CTR)结合则作为胰淀粉样酶(amylin,AMY)受体。由此可见,RAMPs不仅调节受体与配体结合,还影响细胞内的蛋白相互作用调节细胞内信号传导来影响细胞的增殖、迁移、分化等生物学特性。RAMPs还对心血管系统的病理生理有重要调节作用。 相似文献
6.
Multiple ramp domains are required for generation of amylin receptor phenotype from the calcitonin receptor gene product 总被引:4,自引:0,他引:4
Zumpe ET Tilakaratne N Fraser NJ Christopoulos G Foord SM Sexton PM 《Biochemical and biophysical research communications》2000,267(1):368-372
Calcitonin (CT), calcitonin gene-related peptide (CGRP), amylin, and adrenomedullin constitute a family of structurally related peptides that signal via either the calcitonin receptor-like receptor or the CT receptor, with receptor phenotype determined by coexpression of one of the three receptor activity-modifying proteins (RAMPs). The nature of the interaction between the receptor and RAMP was investigated using chimeras between RAMP1 and RAMP2 where the amino-terminal domain of RAMP1 was attached to the transmembrane domain and carboxy terminus of RAMP2 and called RAMP1/2, and vice versa for RAMP2/1. Cotransfection of wild-type or chimeric RAMPs with the insert-negative isoform of the human CT receptor (hCTR(I1-)) into COS-7 cells resulted in the expression of (125)I-rat amylin binding sites. Highest specific binding was observed when either RAMP1 or RAMP2/1 were cotransfected, indicating the importance of the RAMP transmembrane domain and/or carboxy terminus for the degree to which amylin receptors are expressed. In contrast, the phenotype generated was primarily determined by the amino terminus, with similar RAMP1- and RAMP1/2-induced receptor phenotypes that had higher affinity for human CGRPalpha and lower affinity for human calcitonin than the RAMP2- and RAMP2/1-induced receptors. 相似文献
7.
The expressions of the calcitonin receptor (CTR), the calcitonin receptor-like receptor (CLR), the receptor activity-modifying proteins (RAMP) 1-3, and of the receptor component protein (RCP) have been studied in mouse bone marrow macrophages (BMM) during osteoclast differentiation, induced by treatment with M-CSF and RANKL. Analyses of mRNA showed that CLR and RAMP1-3, but not CTR, were expressed in M-CSF stimulated BMM. RANKL gradually increased CTR mRNA, transiently enhanced CLR and transiently decreased RAMP1 mRNA, but did not affect RAMP2, RAMP3, or RCP mRNA. However, RANKL did not affect protein levels of CLR or RAMP1-3 as assessed by Western blots or FACS analyses, whereas immunocytochemistry showed enhanced CTR protein. Analyses of cAMP production showed that BMM cells expressed functional receptors for calcitonin gene-related peptide (CGRP), amylin, adrenomedullin, and intermedin, but not for calcitonin and calcitonin receptor stimulating peptide (CRSP), but that RANKL induced the expression of receptors for calcitonin and CRSP as well. Calcitonin, CGRP, amylin, adrenomedullin, intermedin, and CRSP all down regulated the CTR mRNA, but none of the peptides caused any effects on the expression of CLR or any of the RAMPs. Our data show that BMM cells express receptors for CGRP, amylin, adrenomedullin, and intermedin and that RANKL induces the formation of receptors for calcitonin and CRSP in these cells. We also show, for the first time, that the CTR is not only down regulated by signaling through the CTR but also by the peptides signaling through CLR/RAMPs. 相似文献
8.
Kuwasako K Shimekake Y Masuda M Nakahara K Yoshida T Kitaura M Kitamura K Eto T Sakata T 《The Journal of biological chemistry》2000,275(38):29602-29609
Expression of the calcitonin receptor-like receptor (CRLR) and its receptor activity modifying proteins (RAMPs) can produce calcitonin gene-related peptide (CGRP) receptors (CRLR/RAMP1) and adrenomedullin (AM) receptors (CRLR/RAMP2 or -3). A chimera of the CRLR and green fluorescent protein (CRLR-GFP) was used to study receptor localization and trafficking in stably transduced HEK 293 cells, with or without co-transfection of RAMPs. CRLR-GFP failed to generate responses to CGRP or AM without RAMPs. Furthermore, CRLR-GFP was not found in the plasma membrane and its localization was unchanged after agonist exposure. When stably coexpressed with RAMPs, CRLR-GFP appeared on the cell surface and was fully active in intracellular cAMP production and calcium mobilization. Agonist-mediated internalization of CRLR-GFP was observed in RAMP1/CGRP or AM, RAMP2/AM, and RAMP3/AM, which occurred with similar kinetics, indicating the existence of ligand-specific regulation of CRLR internalization by RAMPs. This internalization was strongly inhibited by hypertonic medium (0.45 m sucrose) and paralleled localization of rhodamine-labeled transferrin, suggesting that CRLR endocytosis occurred predominantly through a clathrin-dependent pathway. A significant proportion of CRLR was targeted to lysosomes upon binding of the ligands, and recycling of the internalized CRLR was not efficient. In HEK 293 cells stably expressing CRLR-GFP and Myc-RAMPs, these rhodamine-labeled RAMPs were co-localized with CRLR-GFP in the presence and absence of the ligands. Thus, the CRLR is endocytosed together with RAMPs via clathrin-coated vesicles, and both the internalized molecules are targeted to the degradative pathway. 相似文献
9.
Kenji Kuwasako Kazuo Kitamura Johji Kato 《Biochemical and biophysical research communications》2009,384(2):249-385
The three receptor activity-modifying proteins (RAMPs1, -2, and -3) associate with a wide variety of G protein-coupled receptors (GPCRs), including calcitonin receptor-like receptor (CRLR). In this study, we used flow cytometry to measure RAMP translocation to the cell surface as a marker of RAMP-receptor interaction. Because VPAC2 does not interact with RAMPs, although, like CRLR, it is a Family B peptide hormone receptor, we constructed a set of chimeric CRLR/VPAC2 receptors to evaluate the trafficking interactions between CRLR domains and each RAMP. We found that CRLR regions extending from transmembrane domain 1 (TM1) through TM5 are necessary and sufficient for the transport of RAMPs to the plasma membrane. In addition, the extracellular N-terminal domain of CRLR, its 3rd intracellular loop and/or TM6 were also important for the cell-surface translocation of RAMP2, but not RAMP1 or RAMP3. Other regions within CRLR were not involved in trafficking interactions with RAMPs. These findings provide new insight into the trafficking interactions between accessory proteins such as RAMPs and their receptor partners. 相似文献
10.
Calcitonin, calcitonin gene-related peptide (CGRP), adrenomedullin (ADM), and amylin belong to a unique group of peptide hormones important for homeostasis in diverse tissues. Calcitonin is essential for calcium balance, whereas CGRP and ADM are important for neurotransmission and cardiovascular and respiratory regulation. Based on phylogenetic analysis, we identified intermedin as a novel member of the calcitonin/CGRP peptide family. Analysis of intermedin expression indicated that intermedin is expressed primarily in the pituitary and gastrointestinal tract. Intermedin increased cAMP production in SK-N-MC and L6 cells expressing endogenous CGRP receptors and competed with labeled CGRP for binding to its receptors in these cells. In addition, treatment of 293T cells expressing recombinant calcitonin receptor-like receptor (CRLR) and one of the three receptor activity-modifying proteins (RAMPs) showed that a CRLR/RAMP receptor complex is required for intermedin signaling. In contrast to CGRP and ADM, which exhibited a preferential stimulation of CRLR when co-expressed with RAMP1 and RAMP2 or RAMP3, respectively, intermedin represents a nonselective agonist for the RAMP coreceptors. In vivo studies demonstrated that intermedin treatment led to blood pressure reduction in both normal and spontaneously hypertensive rats via interactions with the CRLR/RAMP receptor complexes. Furthermore, in vivo treatment in mice with intermedin led to suppression of gastric emptying activity and food intake. Thus, identification of intermedin as a novel member of the calcitonin/CGRP peptide family capable of signaling through CRLR/RAMP receptor complexes provides an additional player in the regulation of peripheral tissues by CRLR and will allow development of new therapeutic agents for pathologies associated with diverse vascular and gastrointestinal disorders. 相似文献
11.
Functional expression of heteromeric calcitonin gene-related peptide and adrenomedullin receptors in yeast. 总被引:3,自引:0,他引:3
Juan J Miret Larissa Rakhilina Lauren Silverman Bert Oehlen 《The Journal of biological chemistry》2002,277(9):6881-6887
The ability of G protein-coupled receptors (GPCRs) to form homo- and heteromeric complexes has important implications for the regulation of cellular events. A notable example of heteromer formation is the interaction of the calcitonin receptor-like receptor (CRLR) with different members of the receptor activity modifying protein (RAMP) family, which results in the formation of two different receptors, a calcitonin gene-related peptide (CGRP) receptor and an adrenomedullin receptor. To analyze the role of RAMPs in determining ligand specificity, we have co-expressed CRLR and RAMP proteins in the yeast Saccharomyces cerevisiae, which provides a null system to study the function of mammalian receptors. Co-expression of RAMP1 and CRLR reconstituted a CGRP receptor that was able to activate the pheromone-signaling pathway with pharmacological properties similar to those observed previously in mammalian cells. Co-expression of CRLR with RAMP2 or RAMP3 resulted in a response with the pharmacological properties of an adrenomedullin receptor. These data indicate that RAMPs are necessary and sufficient to determine ligand specificity of CRLR. Contrary to observations in mammalian cells, the glycosylation of CRLR was not affected by the presence of RAMPs in yeast, indicating that glycosylation of CRLR is not the prime determinant of ligand specificity. The first functional reconstitution of a heteromeric seven transmembrane receptor in yeast suggests this organism as a useful research tool to study the molecular nature of other heteromeric receptors. 相似文献
12.
Toshio Sekiguchi Kenji Kuwasako Michio Ogasawara Hiroki Takahashi Shin Matsubara Tomohiro Osugi Ikunobu Muramatsu Yuichi Sasayama Nobuo Suzuki Honoo Satake 《The Journal of biological chemistry》2016,291(5):2345-2356
The calcitonin (CT)/CT gene-related peptide (CGRP) family is conserved in vertebrates. The activities of this peptide family are regulated by a combination of two receptors, namely the calcitonin receptor (CTR) and the CTR-like receptor (CLR), and three receptor activity-modifying proteins (RAMPs). Furthermore, RAMPs act as escort proteins by translocating CLR to the cell membrane. Recently, CT/CGRP family peptides have been identified or inferred in several invertebrates. However, the molecular characteristics and relevant functions of the CTR/CLR and RAMPs in invertebrates remain unclear. In this study, we identified three CT/CGRP family peptides (Bf-CTFPs), one CTR/CLR-like receptor (Bf-CTFP-R), and three RAMP-like proteins (Bf-RAMP-LPs) in the basal chordate amphioxus (Branchiostoma floridae). The Bf-CTFPs were shown to possess an N-terminal circular region typical of the CT/CGRP family and a C-terminal Pro-NH2. The Bf-CTFP genes were expressed in the central nervous system and in endocrine cells of the midgut, indicating that Bf-CTFPs serve as brain and/or gut peptides. Cell surface expression of the Bf-CTFP-R was enhanced by co-expression with each Bf-RAMP-LP. Furthermore, Bf-CTFPs activated Bf-CTFP-R·Bf-RAMP-LP complexes, resulting in cAMP accumulation. These results confirmed that Bf-RAMP-LPs, like vertebrate RAMPs, are prerequisites for the function and translocation of the Bf-CTFP-R. The relative potencies of the three peptides at each receptor were similar. Bf-CTFP2 was a potent ligand at all receptors in cAMP assays. Bf-RAMP-LP effects on ligand potency order were distinct to vertebrate CGRP/adrenomedullin/amylin receptors. To the best of our knowledge, this is the first molecular and functional characterization of an authentic invertebrate CT/CGRP family receptor and RAMPs. 相似文献
13.
Smith DM Coppock HA Withers DJ Owji AA Hay DL Choksi TP Chakravarty P Legon S Poyner DR 《Biochemical Society transactions》2002,30(4):432-437
Adrenomedullin is a vascular tissue peptide and a member of the calcitonin family of peptides, which includes calcitonin, calcitonin-gene-related peptide (CGRP) and amylin. Its many biological actions are mediated via CGRP type 1 (CGRP(1)) receptors and by specific adrenomedullin receptors. Although the pharmacology of these receptors is distinct, they are both represented in molecular terms by the type II family G-protein-coupled receptor, calcitonin-receptor-like receptor (CRLR). The specificity here is defined by co-expression of receptor-activity-modifying proteins (RAMPs). CGRP(1) receptors are represented by CRLR and RAMP1, and specific adrenomedullin receptors by CRLR and RAMP2 or 3. Here we discuss how CRLR/RAMP2 relates to adrenomedullin binding, pharmacology and pathophysiology, and how chemical cross-linking of receptor-ligand complexes in tissue relates to that in CRLR/RAMP2-expressing cells. CRLR, like other type II family G-protein-coupled receptors, signals via G(s) and adenylate cyclase activation. We demonstrated that adrenomedullin signalling in cell lines expressing specific adrenomedullin receptors followed this expected pattern. 相似文献
14.
Receptor activity modifying proteins 总被引:15,自引:0,他引:15
Our understanding of G protein-coupled receptor (GPCR) function has recently expanded to encompass novel protein interactions that underlie both cell-surface receptor expression and the exhibited phenotype. The most notable examples are those involving receptor activity modifying proteins (RAMPs). RAMP association with the calcitonin (CT) receptor-like receptor (CRLR) traffics this receptor to the cell surface where individual RAMPs dictate the expression of unique phenotypes. A similar function has been ascribed to RAMP interaction with the CT receptor (CTR) gene product. This review examines our current state of knowledge of the mechanisms underlying RAMP function. 相似文献
15.
Bomberger JM Parameswaran N Hall CS Aiyar N Spielman WS 《The Journal of biological chemistry》2005,280(10):9297-9307
RAMPs (1-3) are single transmembrane accessory proteins crucial for plasma membrane expression, which also determine receptor phenotype of various G-protein-coupled receptors. For example, adrenomedullin receptors are comprised of RAMP2 or RAMP3 (AM1R and AM2R, respectively) and calcitonin receptor-like receptor (CRLR), while a CRLR heterodimer with RAMP1 yields a calcitonin gene-related peptide receptor. The major aim of this study was to determine the role of RAMPs in receptor trafficking. We hypothesized that a PDZ type I domain present in the C terminus of RAMP3, but not in RAMP1 or RAMP2, leads to protein-protein interactions that determine receptor trafficking. Employing adenylate cyclase assays, radioligand binding, and immunofluorescence microscopy, we observed that in HEK293 cells the CRLR-RAMP complex undergoes agonist-stimulated desensitization and internalization and fails to resensitize (i.e. degradation of the receptor complex). Co-expression of N-ethylmaleimide-sensitive factor (NSF) with the CRLR-RAMP3 complex, but not CRLR-RAMP1 or CRLR-RAMP2 complex, altered receptor trafficking to a recycling pathway. Mutational analysis of RAMP3, by deletion and point mutations, indicated that the PDZ motif of RAMP3 interacts with NSF to cause the change in trafficking. The role of RAMP3 and NSF in AM2R recycling was confirmed in rat mesangial cells, where RNA interference with RAMP3 and pharmacological inhibition of NSF both resulted in a lack of receptor resensitization/recycling after agonist-stimulated desensitization. These findings provide the first functional difference between the AM1R and AM2R at the level of post-endocytic receptor trafficking. These results indicate a novel function for RAMP3 in the post-endocytic sorting of the AM-R and suggest a broader regulatory role for RAMPs in receptor trafficking. 相似文献
16.
Rat receptor-activity-modifying proteins (RAMPs) for adrenomedullin/CGRP receptor: cloning and upregulation in obstructive nephropathy 总被引:7,自引:0,他引:7
Nagae T Mukoyama M Sugawara A Mori K Yahata K Kasahara M Suganami T Makino H Fujinaga Y Yoshioka T Tanaka I Nakao K 《Biochemical and biophysical research communications》2000,270(1):89-93
Adrenomedullin (AM) is a potent vasorelaxing peptide originally isolated pheochromocytoma. Recently, a family of receptor-activity-modifying proteins (RAMPs 1-3) were identified in humans. Associated with the calcitonin receptor-like receptor (CRLR), RAMP2 or RAMP3 may function as the AM receptor. Here we cloned rat RAMP family, analyzed their distribution in rat tissues, and examined regulation of their expression in the kidney using an obstructive nephropathy model. Northern blot analyses revealed that the RAMP family genes are expressed in various tissues with different tissue specificity; RAMP1 is abundantly expressed in the brain, fat, thymus, and spleen, RAMP2 in the lung, spleen, fat, and aorta, while RAMP3 is most abundant in the kidney and lung. After ureteral obstruction, RAMP1, RAMP2, and CRLR gene expressions in the obstructed kidney were markedly upregulated, whereas RAMP3 expression was unchanged. Thus, RAMPs are regulated differently in obstructive nephropathy, suggesting their distinct roles in renal pathophysiology. 相似文献
17.
Sian Bailey Matthew Harris Kerry Barkan Ian Winfield Matthew Thomas Harper John Simms Graham Ladds Mark Wheatley David Poyner 《生物化学与生物物理学报:生物膜》2019,1861(5):997-1003
Corticotrophin releasing factor (CRF) acts via two family B G-protein-coupled receptors, CRFR1 and CRFR2. Additional subtypes exist due to alternative splicing. CRFR1α is the most widely expressed subtype and lacks a 29-residue insert in the first intracellular loop that is present in CRFR1β. It has been shown previously that co-expression of CRFR1β with receptor activity modifying protein 2 (RAMP2) in HEK 293S cells increased the cell-surface expression of both proteins suggesting a physical interaction as seen with RAMPs and calcitonin receptor-like receptor (CLR). This study investigated the ability of CRFR1α, CRFR1β and CRFR2β to promote cell-surface expression of FLAG-tagged RAMP2. Four different cell-lines were utilised to investigate the effect of varying cellular context; COS-7, HEK 293T, HEK 293S and [ΔCTR]HEK 293 (which lacks endogenous calcitonin receptor). In all cell-lines, CRFR1α and CRFR1β enhanced RAMP2 cell-surface expression. The magnitude of the effect on RAMP2 was dependent on the cell-line ([ΔCTR]HEK 293 > COS-7 > HEK 293T > HEK 293S). RT-PCR indicated this variation may relate to differences in endogenous RAMP expression between cell types. Furthermore, pre-treatment with CRF resulted in a loss of cell-surface FLAG-RAMP2 when it was co-expressed with CRFR1 subtypes. CRFR2β co-expression had no effect on RAMP2 in any cell-line. Molecular modelling suggests that the potential contact interface between the extracellular domains of RAMP2 and CRF receptor subtypes is smaller than that of RAMP2 and CRL, the canonical receptor:RAMP pairing, assuming a physical interaction. Furthermore, a specific residue difference between CRFR1 subtypes (glutamate) and CRFR2β (histidine) in this interface region may impair CRFR2β:RAMP2 interaction by electrostatic repulsion. 相似文献
18.
The calcitonin gene-related peptide (CGRP) receptor and adrenomedullin (ADM) receptor are generated by the concomitant expression of a calcitonin receptor-like receptor (CL receptor) and a specific receptor activity-modifying protein (RAMP) in mammals. We have identified the sequence encoding the salmon CL receptor (sCL receptor) and studied its function after co-expression with the human RAMPs in Cos-7 cells. The potential open-reading frame encoded a 465-amino-acid protein which is 72% identical to the human CL receptor and 85.8% identical to the flounder CL receptor. Function was assessed by measuring the cyclic adenosine monophosphate (cAMP) produced by Cos-7 cells transiently transfected with recombinant vectors for the sCL receptor and human RAMP. Co-expression of the CL receptor and RAMP1, formed a CGRP receptor, as in mammals. This CGRP receptor responded to selective analogs as a type 1 CGRP receptor. Cells co-expressing the CL receptor and RAMP2 did not produce increased cAMP in response to human ADM. Cells co-expressing the CL receptor and RAMP3, produced such a response, as in mammals, indicating that the human ADM molecule is not the cause of the previous unresponsiveness. We suggest that the human RAMP2 molecule does not interact with the sCL receptor because of major differences in the sequences of the salmon CL receptor and the mammalian CL receptor. The availability of this receptor must allow to further study their structural basis. This identification of a non-mammalian CL receptor, and characterization of its function, give insight in the evolution of the CL receptor molecule. 相似文献
19.
Adrenomedullins (AM) form a multifunctional subfamily of the calcitonin gene-related peptide (CGRP) superfamily, the members of which exert their physiological roles through a 1:1 combination of calcitonin receptor-like receptors (CLRs) and receptor activity-modifying proteins (RAMPs). It has been shown that RAMPs can modify the biochemical properties of CLRs; for example, RAMP escorts CLR to the plasma membrane, affects glycosylation state of CLR, and transforms the ligand selectivity of CLR, but on the other hand the effects of CLRs on the biochemical and functional properties of the partner RAMPs are not well established. In this study, using pufferfish (mefugu, mf) homolog, we revealed that mfCLR1 could affect the post-translational modification and trafficking pathway of mfRAMP1. In addition, mfCLRs boosted mfRAMP1, mfRAMP2b, and mfRAMP3 translocation to cell surface. We further revealed that mfRAMPs, except mfRAMP1 and mfRAMP3, could be expressed as multimers on the plasma membrane. However, only monomeric form of mfRAMP2a, mfRAMP4, and mfRAMP5 could heteromerize with mfCLR1 but not with mfCLR2 or mfCLR3, which was consistent with their abilities to induce cAMP response. Collectively our results indicate that the glycosylation, subcellular trafficking, and pharmacological properties of the components of RAMP-CLR receptor complexes are regulated in an interdependent manner. 相似文献
20.
Identification of the human receptor activity-modifying protein 1 domains responsible for agonist binding specificity 总被引:2,自引:0,他引:2
Kuwasako K Kitamura K Nagoshi Y Cao YN Eto T 《The Journal of biological chemistry》2003,278(25):22623-22630
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. 相似文献