Multiple affinity forms of the calcitonin gene-related peptide receptor in rat cerebellum

Binding of 125I-calcitonin gene-related peptide (125I-CGRP) to rat cerebellum membranes and the sensitivity to guanine nucleotides of binding were investigated. Cerebellum binding sites labeled by 125I-CGRP appear to be highly specific, inasmuch as CGRP inhibited binding with an IC50 of 100 pM but other peptides were inactive or much less active in displacing 125I-CGRP from these sites. 125I-CGRP binding sites in cerebellum membranes were saturable and of high affinity. Scatchard analysis of the saturation binding data revealed a homogeneous population of binding sites, with a KD of 224 +/- 28 pM and Bmax of 131 +/- 15 fmol/mg of protein. In the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) (100 microM), a single population of binding sites, with a KD of 464 +/- 77 pM and Bmax of 100 +/- 14 fmol/mg of protein, was observed. The kinetics of association of 125I-CGRP with cerebellum membranes were monophasic at all ligand concentrations tested. However, the observed association rate constant (kobs) was not dependent on [125I-CGRP] in a linear fashion in either the absence or the presence of GTP gamma S (100 microM). The kinetics of dissociation of 125I-CGRP from cerebellum membranes were multiexponential, with fast and slow dissociating components having rate constants of 0.34 +/- 0.01 and 0.025 +/- 0.001 min-1, respectively. The fast dissociating component represented 60 +/- 2% of the total specific binding sites. Dissociation of 125I-CGRP from cerebellum sites was much faster in the presence of GTP gamma S (100 microM) but still exhibited dissociation from two affinity components. The rate constants for these components of dissociation were 0.67 +/- 0.03 and 0.077 +/- 0.007 min-1, with the faster dissociating component representing 66 +/- 1% of the total specific binding sites. These findings provide the first evidence that CGRP receptors exist in multiple affinity states and that cerebellum CGRP receptors are regulated by guanine nucleotides. Our results also suggest the existence of two affinity states of the CGRP-receptor-guanine nucleotide-binding protein ternary complex.