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 initialed 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 GTPγS to purified G protein α 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 K(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 Gα(q) 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 reported K(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 apparent K(d) for bovine brain Gβγ in this assay was 60 nM, and the K(m) for squid retinal Gα(q) was 90 nM. The GRPr- catalyzed binding of GTPγS is selective for Gα(q), since we did not detect receptor-catalyzed exchange using either Gα(i/o) or Gα(t). These data demonstrate that GRPr can functionally couple to Gα(q) but not to the pertussis toxin-sensitive Gα(i/o) or retinal specific Gα(t). This in situ receptor reconstitution method will allow molecular characterization of G protein coupling to other heptahelical receptors.
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