Real-time analysis of G protein-coupled receptor reconstitution in a solubilized system

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 a 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 G alpha subunits premixed with bovine brain G beta gamma subunits were also able to reconstitute with the solubilized receptors. The solubilized N-formyl peptide receptor and G(alpha i3) protein interacted with an affinity of similar to 10(-6) M with other alpha subunits exhibiting lower affinities (G(alpha i3) > G(alpha i2) > G(alpha i1) much greater than G(alphao)). The N-formyl peptide receptor-G protein interactions were inhibited by peptides corresponding to the G(alphai) C-terminal regions, by G(alphai) mAbs, and by a truncated form of arrestin-3. This system should prove useful for the analysis of the specificity of receptor a protein interactions, as well as for the elucidation and characterization of receptor molecular assemblies and signal transduction complexes.