Characteristics and regulation of high affinity ( sup 125 )calcitonin gene-related peptide (*CGRP) binding sites in cultured neonatal rat cardiac myocytes

In the present study, the characteristics and regulation of *CGRP binding sites in cultured cardiac myocytes were investigated. Binding of *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 a KD of 41 {plus minus} 7 pM and Bmax of 31 {plus minus} 5 fmol/mg protein. Binding of CGRP to membranes was inhibited completely by guanosine 5'-(3-O-thio)triphosphate (GTPS) (250 {mu}M) suggesting association of the binding sites with a G protein. Consistent with the saturation binding data, association kinetic studies indicated that *CGRP associated with a single population of binding sites. Dissociation kinetic data, in contrast, indicated that *CGRP dissociated from two affinity component sites on membranes suggesting the existence of multiple affinity states of the G protein-coupled forms of the receptor. Non-equilibrium dissociation kinetic experiments revealed a time-dependent alteration in the fraction of the *CGRP binding sites dissociating with a fast and slow rate. Prior exposure of cells to CGRP (10 nM) for 5 min caused nearly 80% attenuation of the maximal cellular cAMP response to further CGRP challenge and a 75% reduction ofmore » the number of *CGRP binding sites in membranes prepared from these cells.« less