Protein-Protein Interaction and Not Glycosylation Determines the Binding Selectivity of Heterodimers between the Calcitonin Receptor-like Receptor and the Receptor Activity-modifying Proteins

Abstract

The receptor activity-modifying proteins (RAMPs) and the calcitonin receptor-like receptor (CRLR) are both required to generate adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) receptors. A mature, fully glycosylated, form of CRLR was associated with 125 I-CGRP binding, upon co-expression of RAMP1 and CRLR. In contrast, RAMP2 and -3 promoted the expression of smaller, core-glycosylated, CRLR forms, which were linked to AM receptor pharmacology. Since core glycosylation is classically a trademark of immature proteins, we tested the hypothesis that the coreglycosylated CRLR forms the AM receptor. Although significant amounts of core-glycosylated CRLR were produced upon co-expression with RAMP2 or -3, crosslinking experiments revealed that 125 I-AM only bound to the fully glycosylated forms. Similarly, 125 I-CGRP selectively recognized the mature CRLR species upon co-expression with RAMP1, indicating that the glycosylation does not determine ligand-binding selectivity. Our results also show that the three RAMPs lie close to the peptide binding pocket within the CRLR-RAMP heterodimers, since 125 I-AM and 125 I-CGRP were incorporated in RAMP2, -3, and -1, respectively. Cross-linking also stabilized the peptide-CRLR-RAMP ternary complexes, with the expected ligand selectivity, indicating that the fully processed heterodimers represent the functional receptors. Overall, the data indicate that direct protein-protein interactions dictate the pharmacological properties of the CRLR-RAMP complexes.In 1998, McLatchie et al. (1) reported that co-expression of two classes of polypeptides, a seven-transmembrane receptor known as the calcitonin receptor-like receptor (CRLR) 1 and accessory proteins termed receptor activity-modifying proteins (RAMPs), are required to generate functional receptors for calcitonin gene-related peptide (CGRP) and adrenomedullin (AM). The formation of heterodimers between RAMPs and CRLR was found to be essential for the proper cell surface targeting and pharmacological characteristics of both CGRP and AM receptors (2, 3). The RAMP family comprises three members (RAMP1, -2, and -3) that share less than 30% sequence identity but a common topological organization. They are small intrinsic membrane proteins (predicted sizes: M r 14,000 -17,000) with a large extracellular N terminus (ϳ100 amino acids), a single transmembrane domain, and a very short intracellular domain (10 amino acids). Co-expression of RAMP1 with CRLR led to the formation of a CGRP receptor, whereas RAMP2 and -3 promoted the expression of an AM receptor (1).The distinct ligand-binding specificity imposed by the RAMP expressed with CRLR was accompanied by apparent differences in the receptor maturation pathways. The major CRLR species obtained upon co-expression with RAMP1 corresponded to a mature glycoprotein of M r 66,000 containing fully processed complex oligosaccharides. This species was associated with the binding of 125 I-h␣CGRP to the cell surface. In contrast, the major CRLR product observed with RAMP2 and -3 was a...

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).…”

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

“…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. 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).…”

“…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.…”

“…The extracellular domain of RAMP proteins is known to be crucially involved in defining CGRP and AM selectivity (8,11). Therefore, to assess which regions of the CRLR/RAMP1 heterodimer are involved in binding hCGRP or hAM and mediating cAMP accumulation, we constructed a group of hRAMP1 mutants with various deletions in their extracellular N-terminal domains (Fig.…”

“…An analysis of various RAMP1/2 chimeras showed the extracellular N-terminal domain to be crucial for defining CGRP and AM selectivity (11), which is consistent with earlier radioligand binding and functional assays (8). 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).…”