Determinants for calcitonin analog interaction with the calcitonin receptor N-terminus and transmembrane-loop regions.
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High affinity binding was characterized for a number of salmon calcitonin (sCT) analogs to a chimeric receptor (NtCTr) constructed by splicing the N-terminal domain of the human CT receptor onto the C-terminal, transmembrane loop region of the receptor for glucagon. Another chimeric receptor (NtGGr) with the N-terminal domain of the glucagon receptor spliced onto the C-terminal regions of the CT receptor shows no high affinity binding of sCT. Nevertheless, sCT and a number of analogs of the hormone are able to elevate cAMP levels in cells transfected with NtGGr. The least helical analog, des-1-amino-[Ala1,7,Gly8]des-Leu19-sCT, is one of the most active in this regard. Two hormone analogs with modifications in the amino-terminal region, des-Ser2-sCT and [Gly2,3,4,5,6]sCT, show reduced or no activity, respectively, for elevating cAMP in cells expressing the NtGGr. In addition, a 15-fold excess of the peptide sCT-(8-32) antagonizes sCT activation of this receptor. In contrast, these calcitonin analogs exhibited a different rank order for binding to the NtCTr receptor. In fact, des-Ser2-sCT and [Gly8]-des-Leu19-sCT along with the native hormone had the highest helical content as well as the highest binding affinities to the NtCTr receptor. These studies suggest that the helical portion of the hormone within residues 8-22 of sCT is the principal determinant for binding to the receptor N-terminus. Residues 2-6 of sCT interact with the receptor transmembrane loop region and are critical for activation of adenylate cyclase; however, residues 8-32, including Leu16, are responsible for most of the hormone interaction with the transmembrane loop region. Thus, unique requirements exist for CT interaction at the receptor N-terminus relative to the receptor transmembrane loop region, yet there is significant overlap in the hormone determinants that facilitate these interactions.
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