DIVERGENT STRUCTURAL REQUIREMENTS EXIST FOR CALCITONIN RECEPTOR-BINDING SPECIFICITY AND ADENYLATE-CYCLASE ACTIVATION

The basis of the high potency of salmon calcitonin (sCT) in radioligan d binding competition and cAMP accumulation studies with cloned calcit onin (CT) receptors from rats, pigs, and humans was examined using two sets of CT analogues, i.e., chimeric sCT/human CT (hCT) analogues and analogues of sCT with differing capacities to form an amphipathic alp ha-helix. In competition for I-125-sCT binding the following relative specificities were observed for the chimeric peptides: rat C1a CT rece ptor, sCT greater than or equal to (1-16)hCT/(17-32)sCT (ACT-15) > (1- 16)sCT/ (17-32)hCT (ACT-27); rat C1b CT receptor, sCT much greater tha n ACT-15 > ACT-27; hCT receptor, sCT = ACT-15 > ACT-27; porcine CT rec eptor, sCT > ACT-27 > ACT-15. In contrast, in ligand-induced cAMP accu mulation studies the relative efficacies were as follows: rat C1a CT r eceptor, sCT = ACT-15 > ACT-27; rat C1b CT receptor, sCT = ACT-15 > AC T-27; hCT receptor, sCT = ACT-15 greater than or equal to ACT-27; porc ine CT receptor, sCT = ACT-15 = ACT-27. The data demonstrate that resi dues present in the carboxyl-terminal half of sCT are more important f or binding competition with the rat C1a, rat C1b, and human CT recepto rs, whereas residues in the amino-terminal half of sCT are more import ant for binding competition with the porcine CT receptor. Carboxyl-ter minal sCT residues are also important for full potency in adenylate cy clase activation with the rat C1a and rat C1b CT receptors but are les s important for activation via the hCT receptor. The disparity in the relative potencies of the peptides in studies of binding competition a nd cAMP accumulation is suggestive of significant differences in the r elative affinities of the peptides for active and inactive conformatio ns of the CT receptor. The use of sCT analogues with varying capacitie s to form alpha-helices also revealed divergence in the responses of d ifferent receptors. This was most apparent for the stimulation of cAMP production by the rat receptor isoforms C1a and C1b. In cells express ing the C1a receptor, the helical analogues sCT and des-Ser(2)-sCT wer e equipotent with [Gly(8)]-des-Leu(19)-sCT and des-1-amino-[Ala(1,7),G ly(8)]-des-Leu(19) sCT, analogues that have reduced or absent helical structure, respectively. In contrast, the nonhelical analogues were 10 0-1000-fold less potent than sCT and des-Ser(2)-sCT at the C1b recepto r. In general, reduction in the ability of sCT analogues to form helix structures had a greater impact on the potency of the analogues in co mpetition for I-125-sCT binding than in cAMP accumulation. The absence or reduction of helix-forming potential had much less impact on the i nteraction of the analogues with the cloned hCT receptor in both assay systems but also had minimal impact on the activation of the rat C1a receptor, as assessed by cAMP responses.