Abstract

Human breast cell carcinoma MCF-7 cells were found to bind¹²⁵I-labeled rat amylin (rAmylin) and the peptide amylin antagonist radioligand ¹²⁵I-AC512 with high affinity. This high affinity binding possessed characteristics unique to the already defined high affinity binding site for amylin in the rat nucleus accumbens [Mol. Pharmacol. 44:493–497 (1993);J. Pharmacol. Exp. Ther. 270:779–787 (1994);Eur. J. Pharmacol. 262:133–141 (1994)]. To further define this receptor, we report results of expression cloning studies from an MCF-7 cell library. We isolated two variants of a seven-transmembrane receptor that were identical to two previously described human calcitonin receptors (hCTR1 and hCTR2). These receptors were characterized by expression in different surrogate host cell systems. Transient expression of hCTR1 in COS cells yielded membranes that bound ¹²⁵I-AC512 and ¹²⁵I-salmon calcitonin with high affinity, but no high affinity binding was observed with ¹²⁵I-human calcitonin (hCAL) or¹²⁵I-rAmylin. Stable expression of hCTR1 in HEK 293 cells produced similar data. In contrast, expression of hCTR2 in COS cells yielded membranes that bound ¹²⁵I-AC512,¹²⁵I-hCAL, and ¹²⁵I-rAmylin with high affinity. The agonists ¹²⁵I-hCAL and ¹²⁵I-rAmylin bound 65% and 1.5%, respectively, of the sites bound by the antagonist radioligand ¹²⁵I-AC512 in this expression system. This pattern of binding was repeated in HEK 293 cells stably transfected with hCTR2 (¹²⁵I-hCAL = 24.8%B _max, ¹²⁵I-rAmylin = 8%B _max). In both expression systems, the agonists hCAL and rAmylin were much more potent in displacing their radioligand counterparts than was the antagonist radioligand¹²⁵I-AC512. For example, the pK _i value for displacement of¹²⁵I-AC512 by rAmylin was 7.2 in HEK 293 cells but rose to 9.1 when displacing ¹²⁵I-rAmylin. Finally, hCTR2 was expressed in baculovirus-infected Ti ni cells. In this system, only specific binding to the antagonist ¹²⁵I-AC512 and agonist ¹²⁵I-hCAL was observed; no binding to¹²⁵I-rAmylin could be detected. These data are discussed in terms of two working hypotheses. The first is that amylin is a weak agonist for hCTR2 and that this receptor is unrelated to the amylin receptor found in this cell line. The second is that hCTR2 couples to different G proteins for calcitonin and amylin function in different cells. At present, these data cannot be used to disprove conclusively either hypothesis.