Selective reconstitution of gastrin-releasing peptide receptor with Gα_q

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-Iigand 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 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 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 a GRP ≫ neuromedin B. Reconstitution of urea extracted membranes with a purified Ga_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 A_d for bovine brain Gβγ in this assay was 60 nM, and the K_m, for squid retinal Gaq was 90 nM. The GRPr-catalyzed binding of GTPγS is selective for Gα_q, since we did not detect receptorcatalyzed 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.