Identification of the Human Receptor Activity-modifying Protein 1 Domains Responsible for Agonist Binding Specificity

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 [ 125 I]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 CGRPinduced 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.CGRP 1 and AM belong to the calcitonin family of regulatory peptides and are both highly potent vasodilators (1, 2). Although they share very little sequence identity, both contain ring structures comprised of six amino acids linked by a disulfide bridge and an amidated C terminus that are required for biological activity (3). Both of these peptides and their specific or common receptors are widely distributed among peripheral tissues and in the central nervous system, enabling them to exert a wide variety of biological effects (4, 5).RAMPs are a recently identified group of single transmembrane domain accessory proteins that serve to transport CRLR to the cell surface where they form functional CGRP and AM receptors (6). The three RAMP isoforms (RAMP1, RAMP2, and RAMP3), which share only 30% sequence identity and differ in their tissue distributions, are all comprised of ϳ160 amino acids that make up a large extracellular N-terminal domain, a single membrane-spanning domain, and a very short cytoplasmic domain (6, 7). Co-expression of CRLR with RAMP1 leads to both proteins being presented at the plasma membrane as a heterodimeric CGRP receptor, whereas co-expression of CRLR with RAMP2 or RAMP3 enables the resultant heterodimer to function as a AM receptor (6,8,9). Upon binding their respective agonist, both receptors mediate a rise...

Functions of the Cytoplasmic Tails of the Human Receptor Activity-modifying Protein Components of Calcitonin Gene-related Peptide and Adrenomedullin Receptors

“…This discrepancy could be due in part to interference by the extra N-terminal tyrosine residue (Tyr 0 ). In any event, the contribution of the RAMP1 C-tail to CGRP potency is much smaller than that made by its extracellular domain (13,17,32,34,35).…”

“…They then mediate the transport of CRLR to the cell surface, where the heterodimers form functional CGRP or AM receptors: CRLR/RAMP1 forms the CGRP 1 receptor (4), which can also be activated by high concentrations of AM (7,8); CRLR/RAMP2 forms an AM-specific receptor that is sensitive to the AM receptor antagonist AM-(22-52) (AM 1 receptor) (7,9); and CRLR/RAMP3 forms an AM receptor that is sensitive to both the CGRP 1 receptor antagonist CGRP-(8 -37) and AM-(22-52) (AM 2 receptor) (7,9). It is the RAMP extracellular domain that mediates agonist binding to CRLR/RAMP heterodimers (11)(12)(13), which in turn mediate intracellular cAMP production and Ca 2ϩ mobilization (4,10).…”

Evidence for Conservation of the Calcitonin Superfamily and Activity-regulating Mechanisms in the Basal Chordate Branchiostoma floridae

“…These data suggest that a single orthologous gene of CTR/CLR existed in a common ancestor of chordates and that this gene was duplicated to CTR and CLR in the vertebrate lineage. Bf-RAMP-LPs possessed four Cys residues and a transmembrane domain, which are crucial for the biochemical functions of vertebrate RAMPs (43)(44)(45)(46). Furthermore, prediction of the secondary structure of Bf-RAMP-LPs demonstrated that, similar to vertebrate RAMPs, they possess three ␣-helical regions (Fig.…”

Role of Nitric Oxide in Mediating In Vivo Vascular Responses to Calcitonin Gene-Related Peptide in Essential and Peripheral Circulations in the Fetus

“…The RAMPs interact via their NH 2 terminus 23 with the calcitonin receptor-like receptors to produce a different receptor specificity by influencing the formation of the receptor pocket by allosteric modulation. 24 The biological actions of CGRP are mediated predominantly through the CGRP 1 receptor, which is made up of the calcitonin receptor-like receptors and RAMP1 proteins, both of which have been demonstrated within the endothelium and underlying smooth muscle cells of human fetoplacental vessels. 13,14 Unlike classic vasodilators, CGRP elicited cardiovascular responses, which were particularly long-lasting.…”