DETERMINANTS FOR CALCITONIN ANALOG INTERACTION WITH THE CALCITONIN RECEPTOR N-TERMINUS AND TRANSMEMBRANE-LOOP REGIONS

High affinity binding was characterized for a number of salmon calcito nin (sCT) analogs to a chimeric receptor (NtCTr) constructed by splici ng the N-terminal domain of the human CT receptor onto the C-terminal, transmembrane loop region of the receptor for glucagon. Another chime ric receptor (NtGGr) with the N-terminal domain of the glucagon recept or spliced onto the C-terminal regions of the CT receptor shows no hig h affinity binding of sCT. Nevertheless, sCT and a number of analogs o f the hormone are able to elevate cAMP levels in cells transfected wit h NtGGr. The least helical analog, des-1-amino-[Ala(1,7),Gly(8)]des-Le u(19)-sCT, is one of the most active in this regard. Two hormone analo gs with modifications in the amino-terminal region, des-Ser(2)-sCT and [Gly(2,3,4,5,6)]sCT, show reduced or no activity, respectively, for e levating cAMP in cells expressing the NtGGr. In addition, a 15-fold ex cess of the peptide sCT-(8-32) antagonizes sCT activation of this rece ptor. In contrast, these calcitonin analogs exhibited a different rank order for binding to the NtCTr receptor. In fact, des-Ser(2)-sCT and [Gly(8)]-des-Leu(19)-sCT along with the native hormone had the highest helical content as well as the highest binding affinities to the NtCT r receptor. These studies suggest that the helical portion of the horm one within residues 8-22 of sCT is the principal determinant for bindi ng to the receptor N-terminus. Residues 2-6 of sCT interact with the r eceptor transmembrane loop region and are critical for activation of a denylate cyclase; however, residues 8-32, including Leu(16), are respo nsible for most of the hormone interaction with the transmembrane loop region. Thus, unique requirements exist for CT interaction at the rec eptor N-terminus relative to the receptor transmembrane loop region, y et there is significant overlap in the hormone determinants that facil itate these interactions.