Selective reconstitution of gastrin-releasing peptide receptor with Gaq

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 GTPgS to purified G protein a 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 5 1.4 nM 6 0.4 (mean 6 SD, n 5 3) and capacity of 15–22 pmol of receptor per mg of protein in the extracted membrane preparations, representing a 2to 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 Gaq showed that receptor-catalyzed binding of GTPgS was dependent on agonist (GRP) and Gbg 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 Gbg in this assay was 60 nM, and the Km for squid retinal Gaq was 90 nM. The GRPr-catalyzed binding of GTPgS is selective for Gaq, since we did not detect receptorcatalyzed exchange using either Gai/o or Gat. These data demonstrate that GRPr can functionally couple to Gaq but not to the pertussis toxin-sensitive Gai/o or retinal specific Gat. This in situ receptor reconstitution method will allow molecular characterization of G protein coupling to other hepta-