The selectivity and structural determinants of peptide antagonists at the CGRP receptor of rat, L6 myocytes

1. Potency orders were determined for a series of agonists and antagonists on the calcitonin gene-related peptide (CGRP) receptor of rat L6 myocytes. The agents tested were all shown to have been active against CGRP, amylin or adrenomedullin receptors. 2. AC187 had a PIC₅₀ Of 6.8 ± 0.10, making it 14 fold less potent as an antagonist than CGRP_8-37 (PIC₅₀, 7.95 ± 0.14). Amyline_8-37 was equipotent to AC187 (PIC₅₀, 6.6 ± 0.16) and CGRP_19-37 was a fold less potent than either (pIC₅₀ 6.1 ± 0.24). 3. [Ala¹¹]-CGRP_8-37 was 6 fold less potent than CGRP₈-₃₇, (pIC₅₀ 7.13 ± 0.14), whereas [Ala¹⁸] CGRP_8-37 was approximately equipotent to CGRP_8-37 (pIC₅₀, 7.52 ± 0.15). However, [Ala¹¹,Ala¹⁸]- CGRP_8-37 was over 300 fold less potent than CGRP_8-37 (pIC₅₀, 5.30 ± 0.04). 4. [Tyr⁰]-CGRP_28-37, amylin_19-37 and adrenomedullin_22-52 were inactive as antagonists at concentrations of up to 1 μM. 5. Biotinyl-human α-CGRP was 150 fold less potent than human α-CGRP itself (EC₅₀ values of 48 ± 17 nM and 0.31 ± 0.13 nM, respectively). At 1 μM, [Cys(acetomethoxy)(2'7)]-CGRP was inactive as an agonist. 6. These results confirm a role for Arg¹¹ in maintaining the high affinity binding of CGRP_8-37. Arg¹⁸ is of less direct significance for high affinity binding, but it may be important in maintaining the amphipathic nature of CGRP and its analogues.