Mutation of the fourth cytoplasmic loop of rhodopsin affects binding of transducin and peptides derived from the carboxyl-terminal sequences of transducin alpha and gamma subunits

The role of the putative fourth cytoplasmic loop of rhodopsin in the bindin g and catalytic activation of the heterotrimeric G protein, transducin (G(t )), is not well defined. We developed a novel assay to measure the ability of G(t), or G(t)-derived peptides, to inhibit the photoregeneration of rhod opsin from its active metarhodopsin II state. We show that a peptide corres ponding to residues 340-350 of the alpha subunit of G(t), or a cysteinyl-th ioetherfarnesyl peptide corresponding to residues 50-71 of the gamma subuni t of G(t), are able to interact with metarhodopsin II and inhibit its photo conversion to rhodopsin. Alteration of the amino acid sequence of either pe ptide, or removal of the farnesyl group from the gamma-derived peptide, pre vents inhibition. Mutation of the amino-terminal region of the fourth cytop lasmic loop of rhodopsin affects interaction with G(t), (Marin, E, P,, Kris hna, A G;., Zvyaga T, A, Isele, J., Siebert, F,, and Sakmar, T. P. (2000) J . Biol Chem. 275, 1930-1936), Here, we provide evidence that this segment o f rhodopsin interacts with the carboxyl-terminal peptide of the cu subunit of G(t). We propose that the amino-terminal region of the fourth cytoplasmi c loop of rhodopsin is part of the binding site for the carboxyl terminus o f the alpha subunit of G(t) and plays a role in the regulation of beta gamm a subunit binding.