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Biotech / Medical : Cadus Pharmaceutical Corp. (KDUS) -- Ignore unavailable to you. Want to Upgrade?

To: scaram(o)uche who wrote (568)	7/14/2001 6:39:07 PM
From: scaram(o)uche	Read Replies (1) \| Respond to of 1833

The latest from Sklar et al........ interesting overlap, huh? More than coincidence, but..... don't recognize any Axiom names among the authors, so could mean zip......... J Biol Chem 2001 Jun 22;276(25):22453-60 Real-time analysis of G protein-coupled receptor reconstitution in a solubilized system. Bennett TA, Key TA, Gurevich VV, Neubig R, Prossnitz ER, Sklar LA. Cancer Center, Department of Pathology, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, USA. 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.