Mapping interaction sites within the N-terminus of the calcitonin gene-related peptide receptor; the role of residues 23-60 of the calcitonin receptor-like receptor

Abstract

The calcitonin receptor-like receptor (CLR) acts as a receptor for the calcitonin gene-related peptide (CGRP) but in order to recognize CGRP, it must form a complex with an accessory protein, receptor activity modifying protein 1 (RAMP1). Identifying the protein/protein and protein/ligand interfaces in this unusual complex would aid drug design. The role of the extreme N-terminus of CLR (Glu23-Ala60) was examined by an alanine scan and the results were interpreted with the help of a molecular model. The potency of CGRP at stimulating cAMP production was reduced at Leu41Ala, Gln45Ala, Cys48Ala and Tyr49Ala; furthermore, CGRP-induced receptor internalization at all of these receptors was also impaired. Ile32Ala, Gly35Ala and Thr37Ala all increased CGRP potency. CGRP specific binding was abolished at Leu41Ala, Ala44Leu, Cys48Ala and Tyr49Ala. There was significant impairment of cell surface expression of Gln45Ala, Cys48Ala and Tyr49Ala. Cys48 takes part in a highly conserved disulfide bond and is probably needed for correct folding of CLR. The model suggests that Gln45 and Tyr49 mediate their effects by interacting with RAMP1 whereas Leu41 and Ala44 are likely to be involved in binding CGRP. Ile32, Gly35 and Thr37 form a separate cluster of residues which modulate CGRP binding. The results from this study may be applicable to other family B GPCRs which can associate with RAMPs.

Fig. 1

CGRP-stimulated cAMP responses of the mutated receptors where there is a decrease in potency. (a) I41A, (b) Q45A, (c) C48A and (d) Y49A. Cos-7 cells were transfected with WT/RAMP1 or mutant/RAMP1 and assayed for CGRP-stimulated cAMP production. WT type receptors; open squares. Mutant receptors; as indicated. Data are representative of three to five similar experiments.

Fig. 2

CGRP-stimulated cAMP response of the mutated receptors where there is either an increase in potency or constitutive activity. (a) I32A, (b) G35A, (c) T37A and (d) K51A. Cos-7 cells were transfected with WT/RAMP1 or mutant/RAMP1 and assayed for CGRP-stimulated cAMP production. WT type receptors; open squares. Mutant receptors; as indicated. Data are representative of three to five similar experiments.

Fig. 3

Model of the CLR: RAMP1 complex. (a) RAMP1 and CLR ECD-A interface residues in italics are on RAMP1, those in normal type are on CLR. (b) Side view of the predicted orientations of the subunits of CGRP receptor ECD; the proposed interaction of K51 with D96 is also shown for one of the CLR pairs.