Previous studies have shown that stimulation of cultured beating cardiac myocytes with calcitonin gene-related peptide (CGRP) produces increased beating frequency, increased cellular cAMP concentration, and a homologous desensitization of the cAMP-elevating action of CGRP. In the present study, the characteristics and regulation of [125I]CGRP binding sites in cultured cardiac myocytes were investigated. Binding of [125I] CGRP to membranes prepared from these cells was selective, saturable, and of high affinity. Scatchard transformation of the saturation isotherm generated a linear plot suggesting the existence of a homogeneous population of binding sites with an equilibrium binding constant of 41 ± 7 pM and maximum binding capacity of 31 ± 5 fmol/mg protein. Binding of [125I]CGRP to membranes was inhibited completely by guanosine 5’-(3-O- thio)triphosphate (250 μM), suggesting association of the binding sites with a G protein. Consistent with the saturation binding data, association kinetic studies indicated that [125I]CGRP associated with a single population of binding sites. Dissociation kinetic data, in contrast, indicated that [125I]CGRP dissociated from two affinity component sites on membranes, suggesting the existence of multiple affinity states of the G protein-coupled forms of the CGRP receptor. Nonequilibrium dissociation kinetic experiments revealed a time-dependent conversion of [125I] CGRP binding sites from a fast- to a slow-dissociating state. Desensitization of cells to CGRP by prior exposure to CGRP (10 nM) for 5 min reduced the maximal cAMP response of cells to further CGRP challenge and the number of [125I]CGRP binding sites in membranes prepared from these cells approximately 90% and 80%, respectively. These results demonstrate the existence of high affinity CGRP receptors in cardiac myocytes which appear coupled to G proteins and which undergo ligand-induced affinity alterations and desensitization-induced loss of receptor activity. The present findings also suggest the existence of multiple affinity states of the CGRP:receptor: G protein ternary complex.
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