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

T. K. Chatterjee, R. A. Fisher

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

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 K(D) of 224 ± 28 pM and B(max) of 131 ± 15 fmol/mg of protein. In the presence of guanosine 5'-O-(3-thiotriphosphate) (GTPγS) (100 μM), a single population of binding sites, with a K(D) of 464 ± 77 pM and B(max) 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 (k(obs)) was not dependent on [125I-CGRP] in a linear fashion in either the absence or the presence of GTPγS (100 μM). 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 GTPγS (100 μM) 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.

Original languageEnglish (US)
Pages (from-to)798-804
Number of pages7
JournalMolecular Pharmacology
Volume39
Issue number6
StatePublished - Jan 1 1991

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Calcitonin Gene-Related Peptide Receptors
Calcitonin Gene-Related Peptide
Cerebellum
Binding Sites
Guanine Nucleotides
Membranes
Guanosine 5'-O-(3-Thiotriphosphate)
Population
Inhibitory Concentration 50
Carrier Proteins
Proteins
Ligands
Peptides

ASJC Scopus subject areas

  • Medicine(all)
  • Molecular Medicine
  • Pharmacology

Cite this

Multiple affinity forms of the calcitonin gene-related peptide receptor in rat cerebellum. / Chatterjee, T. K.; Fisher, R. A.

In: Molecular Pharmacology, Vol. 39, No. 6, 01.01.1991, p. 798-804.

Research output: Contribution to journalArticle

Chatterjee, T. K. ; Fisher, R. A. / Multiple affinity forms of the calcitonin gene-related peptide receptor in rat cerebellum. In: Molecular Pharmacology. 1991 ; Vol. 39, No. 6. pp. 798-804.
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abstract = "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 K(D) of 224 ± 28 pM and B(max) of 131 ± 15 fmol/mg of protein. In the presence of guanosine 5'-O-(3-thiotriphosphate) (GTPγS) (100 μM), a single population of binding sites, with a K(D) of 464 ± 77 pM and B(max) 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 (k(obs)) was not dependent on [125I-CGRP] in a linear fashion in either the absence or the presence of GTPγS (100 μM). 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 GTPγS (100 μM) 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.",
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