期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1997
卷号：94
期号：2
页码：751-756
DOI：10.1073/pnas.94.2.751
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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 an in situ reconstitution assay in chaotrope-extracted membranes from mouse fibroblasts expressing the GRPr, and we have used it to measure GRPr-catalyzed binding of GTP{gamma}S to purified G protein subunits. Binding studies with 125I-labeled [D-Tyr6]bombesin(6-13) methyl ester (125I-Tyr-ME), a GRPr specific antagonist, show a single binding site with a Kd = 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 Gq showed that receptor-catalyzed binding of GTP{gamma}S was dependent on agonist (GRP) and G{beta}{gamma} subunits. The EC50 for GRP was 3.5 nM, which correlates well with the reported Kd 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 apparent Kd for bovine brain G{beta}{gamma} in this assay was 60 nM, and the Km for squid retinal Gq was 90 nM. The GRPr-catalyzed binding of GTP{gamma}S is selective for Gq, since we did not detect receptor-catalyzed exchange using either Gi/o or Gt. These data demonstrate that GRPr can functionally couple to Gq but not to the pertussis toxin-sensitive Gi/o or retinal specific Gt. This in situ receptor reconstitution method will allow molecular characterization of G protein coupling to other heptahelical receptors.
