The Seven Amino Acids of Human RAMP2 (86) and RAMP3 (59) Are Critical for Agonist Binding to Human Adrenomedullin Receptors

Abstract

When co-expressed with a receptor activity-modifying protein (RAMP) accessory protein, calcitonin receptorlike receptor (CRLR) can function as a calcitonin generelated peptide receptor (CRLR-RAMP1) or an adrenomedullin (AM) receptor (CRLR-RAMP2/3). Here we report on the structural domain(s) involved in selective AM binding that were examined using various RAMP chimeras and deletion mutants. Co-expression of chimeric RAMPs and CRLR in HEK293 cells revealed that residues 77-101, situated in the extracellular N-terminal domain of human RAMP2 (hRAMP2), were crucial for selective AM-evoked cAMP production. More detailed analysis showed that deletion of hRAMP2 residues 86 -92 significantly attenuated high-affinity 125 I-AM binding and AM-evoked cAMP production despite full cell surface expression of the receptor heterodimer and that deletion of hRAMP3 residues 59 -65 had a similar effect. There is little sequence identity between hRAMP3 residues 59 -65 and hRAMP2 residues 86 -92; moreover, substituting alanine for of hRAMP2 had no effect on AM-evoked cAMP production. It thus seems unlikely that any one amino acid residue is responsible for determining selective AM binding or that AM binds directly to these peptide segments. Instead these findings suggest that the respective seven-amino acid sequences confer selectivity either by directly contributing to the structure of ligand binding pocket or by allosteric modulation of the conformation of CRLR. CGRP1 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 proteins contain ring structures comprised of six or seven amino acids linked by a disulfide bridge and an amidated C terminus required for biological activity (3). Both peptides and their specific or common binding sites are widely distributed among peripheral tissues and in the central nervous system and mediate a wide variety of biological effects (4 -6).In 1998, McLatchie et al. (7) identified and cloned the RAMP family of accessory proteins that includes three isoforms (RAMP1, RAMP2, and RAMP3) and serves to transport the CRLR to the cell surface, thereby forming functional CGRP and AM receptors. RAMPs consist of ϳ160 amino acids and possess a large extracellular N-terminal domain, a single transmembrane-spanning domain, and a short cytoplasmic domain. Coexpression of RAMP1 with CRLR leads to both proteins being presented at the plasma membrane as a CGRP receptor, while co-expression of RAMP2 or -3 allows CRLR to function as an AM receptor (7-10). Interestingly, although RAMP2 and -3 share only 30% sequence identity and differ in their tissue distributions, they generate essentially equivalent AM receptors when co-expressed with CRLR in HEK293T cells (9).It was initially believed that mature glycosylation of CRLR in the presence of RAMP1 determined CGRP specificity, while core glycosylation in the presence of RAMP2 or -3 determined AM specificity (7, 9). However, unlike in mammalian cells, co-expressi...

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

“…Neither 10 nM h␣CGRP nor hAM elicited significant increases in cAMP in nontransfected HEK-293 cells or cells transfected with HA-hRAMP1 or hCRLR alone. On the other hand, the coexpression of hCRLR with HA-hRAMP1 enabled CGRP and AM to each elicit significant increases in cAMP, which is consistent with previous reports that CGRP and AM bind to the CRLR/RAMP1 heterodimer with similar affinities (8,9,12,24,25). Similar CGRPand AM-evoked increases in cAMP levels were seen when hCRLR was co-expressed with D74-76, D105-107, or D109-112.…”

“…We also found that the same seven-residue segment situated between Trp-Cys and Tyr was conserved in rat RAMP2 and rRAMP3 (amino acids 93-99 and 58 -64, respectively) was also involved in agonist binding specificity (13). However, these findings did not enable us to draw any conclusions as to which structural domain(s) of human RAMP1 confer agonist specificity despite the use of the eight RAMP chimeras (RAMP1/2 and RAMP2/1) (12). Therefore, we decided to construct a group of deletion mutants that would enable a more detailed analysis of the extracellular domain of hRAMP1.…”

“…Moreover, Hilairet et al (11) showed that when complexed with CRLR, RAMPs are situated close to the agonist binding pocket because ificity by contributing to the structure of the ligand-binding pocket or by allosteric modulation of the conformation of the receptor. Consistent with that idea, we recently used various RAMP chimeras and deletion mutants to show that a sevenresidue segment situated between the residues (Trp-Cys and Tyr) in hRAMP2 (amino acids 86 -92) and hRAMP3 (amino acids 59 -65) is essential for high affinity agonist binding to hAM receptors but not for the interaction of RAMP with CRLR (12). We also found that the same seven-residue segment situated between Trp-Cys and Tyr was conserved in rat RAMP2 and rRAMP3 (amino acids 93-99 and 58 -64, respectively) was also involved in agonist binding specificity (13).…”

“…Since the discovery of RAMPs, various RAMP chimeras have been used to investigate the structural determinants of CGRP and AM receptor specificity (8,11,12,27). When RAMP chimeras were co-transfected into cells together with CRLR, their binding characteristics and functionality revealed that the determinants of CGRP and AM selectivity reside in the extracellular domains of RAMP.…”

“…A more detailed analysis using various deletion mutants showed that a seven-residue segment situated between the residues (Trp-Cys and Tyr) conserved in human, rat, and mouse RAMP2 and RAMP3 is essential for high affinity agonist binding to AM receptors and that RAMP mutants in which these segments were deleted act as negative regulators of AM receptor function (12,13). By contrast, there has been no detailed analysis of the extracellular domain(s) of RAMP1 that confer agonist specificity, although sequential truncation mutation of hRAMP1 showed the transmembrane domain to be critical for the functional expression of a CGRP receptor (14).…”