EXPRESSION CLONING AND RECEPTOR PHARMACOLOGY OF HUMAN CALCITONIN RECEPTORS FROM MCF-7 CELLS AND THEIR RELATIONSHIP TO AMYLIN RECEPTORS

Human breast cell carcinoma MCF-7 cells were found to bind I-125-label ed rat amylin (rAmylin) and the peptide amylin antagonist radioligand I-125-AC512 with high affinity. This high affinity binding possessed c haracteristics unique to the already defined high affinity binding sit e 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 resu lts of expression cloning studies from an MCF-7 cell library. We isola ted two variants of a seven-transmembrane receptor that were identical to two previously described human calcitonin receptors (hCTR1 and hCT R2). These receptors were characterized by expression in different sur rogate host cell systems, Transient expression of hCTR1 in COS cells y ielded membranes that bound I-125-AC512 and I-125-salmon calcitonin wi th high affinity, but no high affinity binding was observed with I-125 -human calcitonin (hCAL) or I-125-rAmylin. Stable expression of hCTR1 in HEK 293 cells produced similar data. In contrast, expression of hCT R2 in COS cells yielded membranes that bound I-125-AC512, I-125-hCAL, and I-125-rAmylin with high affinity. The agonists I-125-hCAL and I-12 5-rAmylin bound 65% and 1.5%, respectively, of the sites bound by the antagonist radioligand I-125-AC512 in this expression system. This pat tern of binding was repealed in HEK 293 cells stably transfected with hCTR2 (I-125-hCAL = 24.8% B-max, I-125-rAmylin = 8% B-max). In both ex pression systems, the agonists hCAL and rAmylin were much more potent in displacing their radioligand counterparts than was the antagonist r adioligand I-125-AC512. For example, the pK(i) value for displacement of I-125-AC512 by rAmylin was 7.2 in HEK 293 cells but rose to 9.1 whe n displacing I-125-rAmylin. Finally, hCTR2 was expressed in baculoviru s-infected Ti ni cells. In this system, only specific binding to the a ntagonist I-125-AC512 and agonist I-125-hCAL was observed; no binding to I-125-rAmylin could be detected. These data are discussed in terms of two working hypotheses. The first is that amylin is a weak agonist far hCTR2 and that this receptor is unrelated to the amylin receptor f ound in this cell line. The second is that hCTR2 couples to different G proteins for calcitonin and amylin function in different cells. At p resent, these data cannot be used to disprove conclusively either hypo thesis.