Recently, a high affinity amylin binding site was identified in the mouse α-TSH thyrotroph cell line. In this study, we have characterized binding sites for 125 I-salmon calcitonin ( 125 I-sCT), 125 I-rat α- calcitonin gene-related peptide ( 125 I-CGRP), and 125 I-rat amylin in α- TSH cells. Using 125 I-CGRP or 125 I-rat amylin, equilibrium was rapidly reached, and binding was fully reversible. Competition binding revealed the relative potency of peptides was sCT>amylin, CGRP>>rCT, which is similar to the specificity profile of amylin receptors characterized in rat brain. Furthermore, specific binding of 125 I-rat amylin and 125 I-CGRP to membrane preparations was reduced by 52% and 39%, respectively, in the presence of 20 μM GTP-γ-s, indicating a requirement of G protein coupling for high affinity binding. In contrast, 125 I-sCT binding reached equilibrium more slowly was essentially irreversible, and was unaltered by GTP-γ-s. Competition binding studies using 125 I-sCT as radioligand demonstrated only weak interaction by CGRP or amylin; consistent with other described CT receptors. Assessment of ligand-induced cAMP accumulation and intracellular calcium signaling revealed a relative specificity profile of sCT>rCT with little or no second messenger signaling stimulated by amylin or CGRP, consistent with a C1-CT receptor phenotype. RT-PCR amplification of messenger RNA indicated that the predominant isoform was the C1a CT receptor. In cross-linking studies, 125 I-rat amylin and 125 I-CGRP specifically labeled a major band of relative molecular mass (M(r)) approximately 80K, being approximately 10 kDa higher than the major 125 I-sCT binding protein. Full deglycosylation of N-linked carbohydrates with endoglycosidase F reduced the M(r) of each of the labeled proteins to approximately 50K. Cross-linked amylin or CT receptors were immunoprecipitated with C-terminally directed antimouse or antirat CT receptor antibodies but were not immunoprecipitated with nonimmune sera or antihuman CT receptor antibodies. The current data demonstrate expression of two biochemically distinct receptor phenotypes in mouse α-TSH cells, a CT receptor phenotype and an amylin receptor phenotype that have highly similar protein backbones.