Expression Cloning and Receptor Pharmacology of Human Calcitonin Receptors from MCF-7 Cells and Their Relationship to Amylin Receptors WEN-JI CHEN, SUSAN ARMOUR, JAMES WAY, GRACE CHEN, CHRIS WATSON, PAUL IRVING, JEFF COBB, SUE KADWELL, KEVIN BEAUMONT, TOM RIMELE, and TERRY KENAKIN Departments of Receptor Biochemistry (G.C., C.W., P.I., T.R., T.K.), Molecular Biology (W.-J.C., S.A., J.W., S.K.), and Medicinal Chemistry (J.C.), Glaxo Wellcome, Research Triangle Park, North Carolina 27709, and Amylin Pharmaceuticals, San Diego, California 92121 (K.B.) Received February 26, 1997; Accepted July 28, 1997 SUMMARY Human breast cell carcinoma MCF-7 cells were found to bind 125 I-labeled rat amylin (rAmylin) and the peptide amylin antag- onist radioligand 125 I-AC512 with high affinity. This high affinity binding possessed characteristics unique to the already de- fined 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 charac- terized by expression in different surrogate host cell systems. Transient expression of hCTR1 in COS cells yielded mem- branes that bound 125 I-AC512 and 125 I-salmon calcitonin with high affinity, but no high affinity binding was observed with 125 I-human calcitonin (hCAL) or 125 I-rAmylin. Stable expression of hCTR1 in HEK 293 cells produced similar data. In contrast, expression of hCTR2 in COS cells yielded membranes that bound 125 I-AC512, 125 I-hCAL, and 125 I-rAmylin with high affin- ity. The agonists 125 I-hCAL and 125 I-rAmylin bound 65% and 1.5%, respectively, of the sites bound by the antagonist radio- ligand 125 I-AC512 in this expression system. This pattern of binding was repeated in HEK 293 cells stably transfected with hCTR2 ( 125 I-hCAL = 24.8% B max , 125 I-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 radioligand 125 I-AC512. For example, the pK i value for displacement of 125 I-AC512 by rAmylin was 7.2 in HEK 293 cells but rose to 9.1 when displacing 125 I- rAmylin. Finally, hCTR2 was expressed in baculovirus-infected Ti ni cells. In this system, only specific binding to the antagonist 125 I-AC512 and agonist 125 I-hCAL was observed; no binding to 125 I-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. Amylin is a peptide hormone that is synthesized and se- creted from pancreatic  cells. There is evidence that an increase in the cosecretion ratio of amylin to insulin exacer- bates insulin tolerance, and in general, there are data to suggest that this peptide may be important in the pathology of type II diabetes (1–5). High affinity binding for 125 I-rAmy- lin has been reported in the rat nucleus accumbens (6 – 8), thus defining an operational and experimentally accessible amylin receptor. Similar high affinity binding of both 125 I- rAmylin and an sCAL antagonist analogue radiolabel 125 I- AC512 has been described (9) in human MCF-7 cells. These data raise the possibility that these cells contain a human amylin receptor, and this information would be of value in the study of the role of amylin in human type II diabetes. We describe the expression cloning of the 125 I-AC512 bind- ing site from an MCF-7 cell cDNA library and the subsequent identification of the gene products as previously classified CTRs (10). The receptor pharmacology of these gene products in various host cells is described, as are data to suggest a relationship between the hCTR and the responses of human systems to amylin. Materials and Methods Cell culture. MCF-7 human breast adenocarcinoma cells from pleural effusion (HTB 22; American Type Culture Collection, Rock- ville, MD) were cultured in Eagle’s minimal essential medium with nonessential amino acids, sodium pyruvate [1 mM (90%)], and fetal ABBREVIATIONS: rAmylin, rat amylin; CTR, calcitonin receptor; hCTR, human calcitonin receptor; HEK, human embryonic kidney; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; sCAL, salmon calcitonin; hCAL, human calcitonin; Ti ni, Trichoplusia ni; rCGRP, rat calcitonin gene-related product; hCGRP, human calcitonin gene-related product; CI, confidence interval; sCAL, salmon calcitonin; hCAL, human calcitonin. 0026-895X/97/061164-12$3.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 52:1164 –1175 (1997). 1164 at ASPET Journals on October 13, 2017 molpharm.aspetjournals.org Downloaded from