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Bethesda, MD: American Society for Pharmacology and Experimental Therapeutics
Erscheinungsjahr
1991
Quelle
MEDLINE
Beschreibungen/Notizen
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.