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-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 Galpha subunits premixed with bovine brain Gbetagamma subunits were also able to reconstitute with the solubilized receptors. The solubilized N-formyl peptide receptor and Galpha(i3) protein interacted with an affinity of approximately 10(-6) m with other alpha subunits exhibiting lower affinities (Galpha(i3) > Galpha(i2) > Galpha(i1) Galpha(o)). The N-formyl peptide receptor-G protein interactions were inhibited by peptides corresponding to the Galpha(i) C-terminal regions, by Galpha(i) 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.
Bennett
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Cancer Center, Department of Pathology, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, USA.
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Gurevich
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Neubig
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Prossnitz
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Sklar
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D000818:Animals; D019393:Arrestin / Q000378:metabolism; D001921:Brain / Q000378:metabolism; D002417:Cattle; D005456:Fluorescent Dyes; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012995:Solubility; D013050:Spectrometry, Fluorescence; D020298:U937 Cells
D019393:Arrestin; D005456:Fluorescent Dyes; D011956:Receptors, Cell Surface; D020962:Heterotrimeric GTP-Binding Proteins