CHIMERIC HUMAN CALCITONIN AND GLUCAGON RECEPTORS REVEAL 2 DISSOCIABLE CALCITONIN INTERACTION SITES

Two chimeric receptors were constructed by transposing the coding regions for the putative N-terminal domains of the human calcitonin (hCTR) and glucagon (hGGR) receptors. These receptors were stably expressed as glycosylated proteins with molecular masses of 80 kDa for the calcitonin receptor N-terminus chimera (NtCTr) and 65 kDa for the glucagon receptor N-terminus chimera (NtGGr). The NtCTr chimera binds salmon calcitonin (sCT) with an apparent K-d of 12 nM relative to 0.3 nM for the native hCTR. However, this chimera does not mediate a cAMP response even with a transfectant expressing 1.8 x 10(6) cell surface receptors. Stable transfectants expressing the NtGGr chimera show no detectable binding of I-125-sCT or I-125-human glucagon. Surprisingly, adenylate cyclase is activated through the NtGGr chimera by sCT, pCT, and hCT with half-maximal activation at 2.2+/-0.6, 5.8+/-2.1, and 810+/-151 nM, respectively, and the maximum response is similar to that induced by 25 mu M forskolin. The rank-order of competition for sCT binding to the NtCTr chimera is similar to the hCTR (sCT > pCT > hCT), but the concentrations required for half-maximal competition are 100- to >2000-fold higher. In addition, salmon calcitonin binds with a much more rapid on-rate and off-rate to the NtCTr chimera relative to the hCTR which binds hormone irreversibly. Cross-linking of I-125-sCT to the NtCTr chimera with bis(sulfosuccinimidyl) suberate is much greater than to the hCTR, suggesting unique conformations for the two receptor-hormone complexes. These studies identify two physically dissociable hormone sites on the calcitonin receptor that likely cooperate in complexing the hormone on the native receptor. Their principal functions are demonstrated in the high-affinity binding of sCT at site-one in the receptor N-terminus and activation of adenylate cyclase at site-two in the remaining receptor C-terminus. Moreover, both sites confer a specificity for ligand interaction similar to the native receptor.