Coexistence of novel amylin-binding sites with calcitonin receptors in human breast carcinoma MCF-7 cells

Amylin, calcitonin (CT) and calcitonin gene-related peptide (CGRP) share limited structural homology including amino-terminal ring structures linked by a disulfide bridge and amidated carboxy-termini. Here, we have compared [I-125]Bolton-Hunter-[Lys(1)] rat amylin ([I-125]amylin) binding and the stimulation of cyclic AMP accumulation by human (h) amylin, hCT and hCGRP-I in the human breast carcinoma cell lines MCF-7 and T47D, which predominantly express hCT(1a) and hCT(1b) receptor isoforms (hCTR(1a), hCTR(1b)) at a similar total number of hCT-binding sites. In MCF-7 cells, half-maximal inhibition (IC50) of [I-125]amylin binding by human amylin was observed at 3.6 +/- 0.8 nM (n=6). hCT and hCGRP-I displaced [I-125]amylin binding with 22 and 66 times higher IC50. [I-125]hCT binding was inhibited by hCT with an IC50 of 8.1 +/- 1.9 nM (n=5), and human amylin and hCGRP-I were over 100 times less potent. In T47D cells, on the other hand, specific binding of [I-125]amylin was not observed, but hCT inhibited [I-125]hCT binding with an IC50 of 3.2 +/- 0.4 nM (n=3), and human amylin and hCGRP-I had over 200 times higher IC50. In MCF-7 cells, half-maximal stimulation (EC50) of cyclic AMP accumulation by human amylin, hCT and hCGRP-I occurred at 1.4 +/- 0.2, 1.7 +/- 0.4 and 6.3 +/- 1.3 nM respectively. In T47D cells, the EC50 of hCT was C.32 +/- 0.02 nM (n=3), and 30- and 1900-fold higher with human amylin and hCGRP-I. In conclusion, the expression of hCTR(1a) and hCTR(1b) and [I-125]hCT binding were indistinguishable in MCF-7 and T47D cells. Yet, [I-125]amylin binding was only recognized in MCF-7 cells, consistent with a distinct amylin receptor.