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Vol. 52, Issue 6, 1164-1175, 1997
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.)
Human breast cell carcinoma MCF-7 cells were found to bind
125I-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) or
125I-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%
Bmax, 125I-rAmylin = 8%
Bmax). In both expression systems, the
agonists hCAL and rAmylin were much more potent in displacing their
radioligand counterparts than was the antagonist radioligand
125I-AC512. For example, the
pKi value for displacement of
125I-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 to
125I-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.
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