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. 1997 Jan 21;94(2):751-6.
doi: 10.1073/pnas.94.2.751.

Selective reconstitution of gastrin-releasing peptide receptor with G alpha q

M R Hellmich  1 , J F Battey, J K Northup
Affiliations

Selective reconstitution of gastrin-releasing peptide receptor with G alpha q

M R Hellmich et al. Proc Natl Acad Sci U S A. .

Abstract

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 alpha 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 > or = GRP > > neuromedin B. Reconstitution of urea extracted membranes with a purified G alpha q 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 G alpha q was 90 nM. The GRPr-catalyzed binding of GTP gamma S is selective for G alpha q, since we did not detect receptor-catalyzed exchange using either G alpha i/o or G alpha t. These data demonstrate that GRPr can functionally couple to G alpha q but not to the pertussis toxin-sensitive G alpha i/o or retinal specific G alpha t. This in situ receptor reconstitution method will allow molecular characterization of G protein coupling to other heptahelical receptors.

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Figures

Figure 1
Figure 1
Competition of binding of the antagonist 125I-Tyr-ME to membranes pretreated with agonist and extracted with 6 M urea. The relative potency of different Bn receptor agonists (Bn, ▴; GRP, ▵; NMB, ▪) and a GRPr-specific antagonist (ME, •) were compared by competition binding to membranes pretreated with agonist before extraction with 6 M urea. The binding reaction was initiated by adding 25 μl of membranes (0.41 μg of protein) to 25 μl of binding solution containing 125I-Tyr-ME (87 pM final concentration) and various concentrations of unlabeled competitor. Binding proceeded for 45 min at 30°C, and bound radioligand was measured as described.
Figure 2
Figure 2
Reconstitution of GRPr membranes with Gαq and/or βγ. A P2 GRPr membrane fraction was assayed for agonist-catalyzed GTPγS binding directly (Left), or after the membranes were pretreated with agonist and extracted with 6 M urea (Right). Either GRPr containing membranes alone (None) or membranes reconstituted with either Gαq or βγ alone, or Gαq and βγ were assayed with (stippled bars) or without (solid bars) 1 μM GRP. The GTPγS binding assay proceeded for 10 min at 30°C as described.
Figure 3
Figure 3
GRP saturation of GRPr-catalyzed exchange of GTPγS for GDP. GTPγS binding was measured in a reaction containing 3 nM GRPr, 1 μM βγ, 280 nM Gαq, and the indicated amounts of GRP. Binding reactions proceeded for 3.5 min at 30°C as described.
Figure 4
Figure 4
Saturation of the rate of agonist-stimulated, GRPr-catalyzed GDP/GTPγS exchange by Gαq and Gβγ. (A) The concentrations of GRP (1 μM), GRPr (0.4 nM), and βγ (1.2 μM) were fixed, and the concentration of Gαq varied from 28 nM to 426 nM as indicated. (B) The concentration of GRP (1 μM), GRPr (3 nM), and Gαq (284 nM) were fixed, and the concentration of βγ was varied from 34 nM to 1200 nM. Binding reactions proceeded for 15 min at 30°C, and the binding was determined as described.
Figure 5
Figure 5
Gαq coupling selectivity of the GRPr. Membranes containing GRPr (0.4 nM) were mixed with Gαq (280 nM) and βγ (1 μM), Gi/o (1 μM), or Gαt (1 μM) and βγ (1 μM). GTPγS exchange for bound GDP in the presence (stippled bars) or absence (solid bars) of 1 μM GRP proceeded for 15 min at 30°C as described. Gα subunit concentrations were determined by GTPγS binding with or without addition of βγ. Membrane-independent background binding of GTPγS was subtracted from total binding to give the values presented in the figure (membrane-independent backgrounds were as follows: 635 cpm for Gαq and βγ; 716 cpm for Gi/o; and 3173 cpm for Gαt and βγ).
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