P-660: Identification of dominant-negative human RAMP1 able to inhibit endogenous CGRP₁ receptor function

Abstract

Calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) belong to the calcitonin family of regulatory peptides and are both highly potent vasodilators. 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. The newly identified accessory proteins, receptor activity-modifying protein (RAMP)-1, -2 and -3, form heterodimers with an orphan receptor, calcitonin receptor-like receptor (CRLR), and mediate its translocation to the cell surface. CRLR/RAMP2 or CRLR/RAMP3 comprises an AM receptor. On the other hand, CRLR/RAMP1 comprises the CGRP₁ receptor, which also responds to higher concentrations of 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 human embryonic kidney (HEK)-293 cells revealed that deletion of residues 91-94, 96-100, or 101-103 blocked [¹²⁵I]CGRP binding and completely abolished intracellular cAMP accumulations 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 theses cases, the receptor heterodimers were fully expressed at the cell surface. Substituting alanine (A) 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 highly expressed in various cultured cells.

CGRP, CGRP₁ Receptor, Dominant-Negative RAMP1