Received for publication June 14, 2006.
Revised August 27, 2006.
Accepted for publication September 6, 2006.

Determinants of BIBN4096BS affinity for CGRP and amylin receptors; the role of RAMP1

Abstract

BIBN4096BS, a calcitonin gene-related peptide (CGRP) receptor antagonist, can alleviate the symptoms of migraine, and is highly selective for CGRP over adrenomedullin (AM) receptors. These receptors are heterodimers of the calcitonin receptor-like receptor (CL) and receptor activity modifying proteins (RAMPs), with the pharmacological properties determined by the RAMP subunit. BIBN4096BS-sensitive CGRP₁ receptors are CL/RAMP1 whereas BIBN4096BS-insensitive AM receptors are CL/RAMP2 or CL/RAMP3 (AM₁ and AM₂, respectively), implicating RAMP1 in conferring BIBN4096BS sensitivity. As calcitonin receptors (CT_(a)) also interact with RAMP1 (AMY_1(a) receptors), BIBN4096BS could also have affinity for these receptors. To test this, receptors were transfected into COS-7 cells and agonist-stimulated cAMP levels measured in the presence and absence of antagonists. We found that AMY_1(a) receptors were ~150-fold less sensitive to BIBN4096BS antagonism than CGRP₁ receptors. In contrast, AMY_3(a) (CT_(a)/RAMP3) or AM₂ receptors were not sensitive to BIBN4096BS antagonism. We investigated Trp74 in RAMP1, a residue implicated in the species selectivity of BIBN4096BS. BIBN4096BS affinity was reduced at AMY_1(a) and CGRP₁ receptors when this residue was mutated to Lys or Ala. The equivalent residue in RAMP3, Glu74, when mutated to Trp (E74W), induced BIBN4096BS sensitivity at AM₂ and AMY_3(a) receptors. Interestingly, a selective reduction in AM potency was observed at E74W AM₂ receptors, implicating this residue in AM interactions with this receptor. These data support the importance of Trp74 in RAMP1 in the interaction of BIBN4096BS with CGRP₁ and AMY_1(a) receptors and identified Glu74 in RAMP3 as the first amino acid in RAMP important for agonist interactions with calcitonin-family receptors.

Key words: CGRP/amylin, Gs family, cAMP, Peptide hormones

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