The amino terminus of the fourth cytoplasmic loop of rhodopsin modulates rhodopsin-transducin interaction

Rhodopsin is a seven-transmembrane helix receptor that binds and catalytica lly activates the heterotrimeric G protein transducin (G(t)). This interact ion involves the cytoplasmic surface of rhodopsin, which comprises four put ative loops and the carboxyl-terminal tail. The fourth loop connects the ca rboxyl end of transmembrane helix 7 with Cys(322) and Cys(323), which are b oth modified by membrane-inserted palmitoyl groups. Published data on the r oles of the fourth loop in the binding and activation of G(t) are contradic tory. Here, we attempt to reconcile these conflicts and define a role for t he fourth loop in rhodopsin-G(t) interactions. Fluorescence experiments dem onstrated that a synthetic peptide corresponding to the fourth loop of rhod opsin inhibited the activation of G(t) by rhodopsin and interacted directly with the alpha subunit of G(t). A series of rhodopsin mutants was prepared in which portions of the fourth loop were replaced with analogous sequence s from the beta(2)-adrenergic receptor or the mi muscarinic receptor. Chime ric receptors in which residues 310-312 were replaced could not efficiently activate G(t). The defect in G(t) interaction in the fourth loop mutants w as not affected by preventing palmitoylation of Cys(322) and CyS323. We sug gest that the amino terminus of the fourth loop interacts directly with G(t ), particularly with G alpha(t), and with other regions of the intracellula r surface of rhodopsin to support G(t) binding.