Real-time Analysis of G Protein-coupled Receptor Reconstitution in a Solubilized System

Abstract

Receptor based signaling mechanisms are the primary source of cellular regulation. The superfamily of G protein-coupled receptors is the largest and most ubiquitous of the receptor mediated processes. We describe here the analysis in real-time of the assembly and disassembly of soluble G protein-coupled receptor-G protein complexes. A fluorometric method was utilized to determine the dissociation of a fluorescent ligand from the receptor solubilized in detergent. The ligand dissociation rate differs between a receptor coupled to a G protein and the receptor alone. By observing the sensitivity of the dissociation of a fluorescent ligand to the presence of guanine nucleotide, we have shown a time- and concentration-dependent reconstitution of the N-formyl peptide receptor with endogenous G proteins. Furthermore, after the clearing of endogenous G proteins, purified Gα subunits premixed with bovine brain Gβγ subunits were also able to reconstitute with the solubilized receptors. The solubilized N-formyl peptide receptor and Gαi3 protein interacted with an affinity of ∼ 10-6 M with other α subunits exhibiting lower affinities (Gαi3 < Gαi2 ≤ Gαi1 ≫ Gαo). The N-formyl peptide receptor-G protein interactions were inhibited by peptides corresponding to the Gα i C-terminal regions, by Gαi mAbs, and by a truncated form of arrestin-3. This system should prove useful for the analysis of the specificity of receptor-G protein interactions, as well as for the elucidation and characterization of receptor molecular assemblies and signal transduction complexes.