CHIMERIC HUMAN CALCITONIN AND GLUCAGON RECEPTORS REVEAL 2 DISSOCIABLECALCITONIN INTERACTION SITES

Two chimeric receptors were constructed by transposing the coding regi ons 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 calci tonin receptor N-terminus chimera (NtCTr) and 65 kDa for the glucagon receptor N-terminus chimera (NtGGr). The NtCTr chimera binds salmon ca lcitonin (sCT) with an apparent K-d of 12 nM relative to 0.3 nM for th e 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 w ith 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 th e NtCTr chimera is similar to the hCTR (sCT > pCT > hCT), but the conc entrations required for half-maximal competition are 100- to >2000-fol d 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 b inds hormone irreversibly. Cross-linking of I-125-sCT to the NtCTr chi mera with bis(sulfosuccinimidyl) suberate is much greater than to the hCTR, suggesting unique conformations for the two receptor-hormone com plexes. These studies identify two physically dissociable hormone site s on the calcitonin receptor that likely cooperate in complexing the h ormone on the native receptor. Their principal functions are demonstra ted 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 remain ing receptor C-terminus. Moreover, both sites confer a specificity for ligand interaction similar to the native receptor.