Receptor Activity-modifying Protein 1 Determines the Species Selectivity of Non-peptide CGRP Receptor Antagonists

Abstract

. This observation provided an opportunity to map the determinants of receptor affinity exhibited by BIBN4096BS and the truncated analogs, Compounds 1 and 2. All three compounds exhibited higher affinity for the human receptor, human CRLR/human RAMP1, than for the rat receptor, rat CRLR/rat RAMP1. We have now demonstrated that this species selectivity was directed exclusively by RAMP1. By generating recombinant human/rat CRLR/RAMP1 receptors, we demonstrated that co-expression of human CRLR with rat RAMP1 produced rat receptor pharmacology, and vice versa. Moreover, with rat/human RAMP1 chimeras and site-directed mutants, we have identified a single amino acid at position 74 of RAMP1 that modulates the affinity of small molecule antagonists for CRLR/RAMP1. Replacement of lysine 74 in rat RAMP1 with tryptophan (the homologous amino acid in the human receptor) resulted in a >100-fold increase in antagonist affinities, similar to the K i values for the human receptor. These observations suggest that important determinants of small molecule antagonist affinity for the CGRP receptor reside within the extracellular region of RAMP1 and provide evidence that this receptor accessory protein may participate in antagonist binding.CGRP is a 37-amino acid neuropeptide that is expressed in a variety of cell types in both the central and peripheral nervous systems (5). In many tissues, CGRP-containing fibers are closely associated with blood vessels (6). Among the various physiological functions reported for CGRP, the most pronounced is vasodilation. CGRP is the most powerful of the vasodilator transmitters (7), and its vasoactive effects have been demonstrated in a variety of blood vessels (8), including those in the cerebral, coronary, and mesenteric vasculature.Mounting evidence suggests that CGRP is involved in the pathophysiology of migraine headache (9). Migraine is thought to be associated with dilation of cerebral blood vessels and activation of the trigeminovascular system (10). During the headache phase of a migraine, CGRP levels are elevated in the cranial circulation (11, 12). Successful treatment of the headache with sumatriptan resulted in normalization of CGRP levels (12), thus implicating CGRP in the pathophysiology of this disorder. Moreover, intravenous administration of CGRP to migraineurs induced a delayed migrainous headache in some patients (13). These observations suggest that inhibition of CGRP-mediated vasodilation may have therapeutic utility in the treatment of migraine.Research in the area of CGRP has intensified in recent years due in large part to the identification of its receptor, CRLR 1 (14), and the cloning of the receptor activity-modifying proteins (RAMPs) (1) and receptor component protein (15). CGRP activity is mediated by the G s -coupled G-protein-coupled receptor (GPCR), CRLR, which shares 55% homology with the calcitonin receptor. In an elegant study, McLatchie et al. (1) demonstrated that functional CGRP and adrenomedullin receptors are both derived from CRLR and that the phenotype i...

Differential distribution of calcitonin gene-related peptide and its receptor components in the human trigeminal ganglion

“…Transfections were performed by combining 60 µg/dish DNA with 180 µg/dish Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Rat CLR and RAMP1 cDNAs in the mammalian expression construct pcDNA3.1, described earlier (Mallee et al, 2002), were co-transfected in equal amounts. Transfection cocktail was added directly to the medium and the cells were harvested for membranes 48 h post-transfection.…”

The Nonpeptide Calcitonin Gene-Related Peptide Receptor Antagonist BIBN4096BS Lowers the Activity of Neurons with Meningeal Input in the Rat Spinal Trigeminal Nucleus

“…In rats, no changes in heart rate, mean arterial pressure, systemic vascular conductance, or cardiac output were observed with BIBN4096BS at 3 mg/kg (Arulmani et al, 2004). Compared with rat CGRP receptors, human CGRP receptors have Ͼ100-fold the affinity for BIBN4096BS (Mallee et al, 2002). In the first clinical trial published by Olesen et al (2004), the maximum response rate after 2 h of drug administration occurred at a dose of ϳ70 g/kg.…”

Targeting CGRP: A New Era for Migraine Treatment

“…According to the authors, no intrinsic cardiovascular effects were reported. The affinity of olcegepant for the CGRP receptor was RAMP1 related, directly competing with CGRP for its binding site [73]. In 2004, Olesen et al [74] reported the positive outcomes of CGRP-RA as a migraine-abortive therapy with similar effectiveness to that of oral sumatriptan.…”