Open AccessSelective reconstitution of gastrin-releasing peptide receptor with Gα q
Author(s) -
Mark R. Hellmich,
James F. Battey,
John K. Northup
Publication year - 1997
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.94.2.751
Subject(s) - bombesin , g protein , receptor , gtp' , chemistry , peptide , heterotrimeric g protein , binding site , g protein coupled receptor , agonist , ligand (biochemistry) , microbiology and biotechnology , biochemistry , biology , neuropeptide , enzyme
Identification of the molecular mechanisms that determine specificity of coupling interactions between gastrin-releasing peptide receptors (GRPrs) and their cognate heterotrimeric GTP-binding proteins is a fundamental step in understanding the signal transduction cascade initiated by receptor–ligand interaction. To explore these mechanisms in greater detail, we have developed anin situ reconstitution assay in chaotrope-extracted membranes from mouse fibroblasts expressing the GRPr, and we have used it to measure GRPr-catalyzed binding of GTPγS to purified G protein α subunits. Binding studies with125 I-labeled [d -Tyr6 ]bombesin(6–13) methyl ester (125 I-Tyr-ME), a GRPr specific antagonist, show a single binding site with aK d = 1.4 nM ± 0.4 (mean ± SD,n = 3) and capacity of 15–22 pmol of receptor per mg of protein in the extracted membrane preparations, representing a 2- to 3-fold enrichment of binding sites compared with the membranes before extraction. Quantitative ligand displacement analysis using various unlabeled GRPr agonists shows a rank order of potency characteristic of the GRPr: bombesin ≥ GRP ≫ neuromedin B. Reconstitution of urea extracted membranes with a purified Gαq showed that receptor-catalyzed binding of GTPγS was dependent on agonist (GRP) and Gβγ subunits. The EC50 for GRP was 3.5 nM, which correlates well with the reportedK d of 3.1 nM for GRP binding to GRPr expressed in mouse fibroblasts [Benya, R. V.,et al. (1994)Mol. Pharmacol. 46, 235–245]. The apparentK d for bovine brain Gβγ in this assay was 60 nM, and theK m for squid retinal Gαq was 90 nM. The GRPr-catalyzed binding of GTPγS is selective for Gαq , since we did not detect receptor-catalyzed exchange using either Gαi/o or Gαt . These data demonstrate that GRPr can functionally couple to Gαq but not to the pertussis toxin-sensitive Gαi/o or retinal specific Gαt . Thisin situ receptor reconstitution method will allow molecular characterization of G protein coupling to other heptahelical receptors.