Solubilization and characterization of a guanine nucleotide-sensitive form of the calcitonin gene-related peptide receptor

Tapan K. Chatterjee, Jadine A. Moy, John J. Cai, Hon Chi Lee, Rory A. Fisher

Research output: Contribution to journalArticlepeer-review

26 Scopus citations


Calcitonin gene-related peptide (CGRP) receptors were solubilized from rat cerebellum membranes in an active, stable, and guanine nucleotide-sensitive form by using digitonin. Nearly 90% of membrane CGRP receptors and 50% of membrane protein were solubilized by digitonin treatment of cerebellum membranes. Binding of 125ICGRP to soluble receptors was specific, saturable, of high affinity, and reversible. Scatchard analysis of the saturation binding data revealed a homogeneous population of binding sites with a Kd of 178 ± 42 pM and a Bmax of 201 ± 17 fmol/mg of protein. Binding of 125ICGRP to soluble receptors was inhibited nearly 60% by guanosine-5′-O-(3-thio)triphosphate (GTPγS) (100 μM), suggesting coupling of receptors with guanine nucleotide-binding proteins (G proteins) to form high affinity binding sites. Antiserum against the amino-terminal region of G immunoprecipitated a significant portion of soluble CGRP receptors, indicating association of receptors with G. In agreement with the saturation binding data, association kinetic studies with soluble receptors indicated binding of 125ICGRP to a single population of sites. Dissociation kinetic data, in contrast, demonstrated that 125ICGRP dissociated from labeled receptors with fast- and slow-dissociating components. GTPγS significantly accelerated dissociation of 125ICGRP from labeled receptors; however, dissociation still occurred from two distinct affinity components, with rate constants significantly different from those observed in the absence of GTPγS. These observations suggest that soluble CGRP receptors, like native membrane-bound receptors, exist in two distinct affinity states in both G protein-coupled and -uncoupled receptor states. Soluble receptors were retained specifically on a wheat germ lectin column, and affinity cross-linking of receptors specifically labeled with 125ICGRP demonstrated labeling of a 67-kDa protein, suggesting that the rat cerebellum CGRP receptor is a 67-kDa glycoprotein. This study is the first to report solubilization of CGRP receptors retaining the native ability of the receptor to undergo functional coupling with G proteins and to provide direct evidence for association of these receptors with G.

Original languageEnglish (US)
Pages (from-to)167-175
Number of pages9
JournalMolecular pharmacology
Issue number2
StatePublished - Feb 1993

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology


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