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A.K. Gross, S. Madabushi, T.G. Wensel, O. Lichtarge; Evolution Guided Mutagenesis of Rhodopsin . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3634.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the universal elements of the ligand–induced structural reorganization of G–protein coupled receptor (GPCR) activation. Methods: The data mining technique of Evolutionary Tracing (ET) was used to identify amino acid positions globally important in diverse GPCRs and map them onto the structure of rhodopsin. Residues important for rhodopsin family–specific functions were identified by a differential ET analysis. Experimental tests of residue importance were obtained by reviewing published accounts of mutations in various GPCRs, and by introducing novel mutations into bovine rhodopsin at residues L79 and W175. The mutant rhodopsins were assayed by difference spectrophotometry and by catalysis of nucleotide exchange by the G protein transducin. Results: The global trace residues cluster into a network of contacts from the retinal binding site to the G–protein coupling loops. Their roles in transduction were verified by functional defects in 211 of 239 published mutations. Opsin–specific ET revealed additional positions, including 11 linked to functional defects. Both the globally important trace residue, L79 and the opsin–specific trace residue W175 were found to be functionally critical, as revealed by constitutive G protein activation of the mutants and rapid loss of regeneration after photobleaching. Conclusions: These results define in GPCRs a canonical signal transduction mechanism whereby specific interactions with ligand induces conformational changes, and confirm that ET can be used to guide mutagenesis in transmembrane domains of GPCRs.
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