Expression Cloning and Receptor Pharmacology of Human Calcitonin Receptors from MCF-7 Cells and Their Relationship to Amylin Receptors

Abstract

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.

Molecular Mechanisms and Therapeutical Implications of Intramembrane Receptor/Receptor Interactions among Heptahelical Receptors with Examples from the Striatopallidal GABA Neurons

“…Based on this work it seems as if the functional meaning of the various RAMPs is severalfold, namely to transport the CRL receptor to the cell surface, to differentially glycosylate the CRL receptor and to interact differentially with the CRL receptor on the cell surface, which may involve heteromeric complexes. All these processes may lead to the development of receptor diversity with markedly different ligand specificities, and CGRP, ADM, and amylin receptor subtypes (see Chen et al, 1997;Perry et al, 1997) can be formed based on the CRL receptor and calcitonin receptor interactions with different types of RAMPs. Thus, CRL receptor and possibly other GPCR can markedly alter its binding pocket by interactions with single TM domain proteins.…”

Structural Determinants of Salmon Calcitonin Bioactivity

“…Alternative RNA splicing yields multiple CTR mRNA isoforms. In man, at least six potential variants exist (3,17,18); however, the most common human CTR isoforms differ by the presence (hCTRb) or absence (hCTRa) of a 16-amino acid insert between amino acids 174 and 175, within the first intracellular loop of the receptor (19). Of these, the hCTRa is the major human receptor isoform and is expressed in essentially all tissues known to express the CTR.…”

Class II G Protein-Coupled Receptors and Their Ligands in Neuronal Function and Protection

“…These amylin receptors have similar and high affinities for CT and amylin and lower affinities for CGRP. In this respect, the CT receptor/RAMP combination represents a similar amylin receptor to that characterized in native tissues (Chen et al, 1997).…”