The recent report of the crystal structure of rhodopsin provides insights concerning structure-activity relationships in visual pigments and related G protein-coupled receptors (GPCRs). The seven transmembrane helices of rhodopsin are interrupted or kinked at multiple sites. An extensive network of interhelical interactions stabilizes the ground state of the receptor. The ligand-binding pocket of rhodopsin is remarkably compact, and several chromophore-protein interactions were not predicted from mutagenesis or spectroscopic studies. The helix movement model of receptor activation, which likely applies to all GPCRs of the rhodopsin family, is supported by several structural elements that suggest how light-induced conformational changes in the ligand-binding pocket are transmitted to the cytoplasmic surface. The cytoplasmic domain of the receptor includes a helical domain extending from the seventh transmembrane segment parallel to the bilayer surface. The cytoplasmic surface appears to be approximately large enough to bind to the transducin heterotrimer in a one-to-one complex. The structural basis for several unique biophysical properties of rhodopsin, including its extremely low dark noise level and high quantum efficiency, can now be addressed using a combination of structural biology and various spectroscopic methods. Future high-resolution structural studies of rhodopsin and other GPCRs will form the basis to elucidate the detailed molecular mechanism of GPCR-mediated signal transduction.

Rhodopsin is a seven-transmembrane helix receptor that binds and catalytically activates the heterotrimeric G protein transducin (G t ). This interaction involves the cytoplasmic surface of rhodopsin, which comprises four putative loops and the carboxyl-terminal tail. The fourth loop connects the carboxyl end of transmembrane helix 7 with Cys 322 and Cys 323 , which are both modified by membrane-inserted palmitoyl groups. Published data on the roles of the fourth loop in the binding and activation of G t are contradictory. Here, we attempt to reconcile these conflicts and define a role for the fourth loop in rhodopsin-G t interactions. Fluorescence experiments demonstrated that a synthetic peptide corresponding to the fourth loop of rhodopsin inhibited the activation of G t by rhodopsin and interacted directly with the ␣ subunit of G t . A series of rhodopsin mutants was prepared in which portions of the fourth loop were replaced with analogous sequences from the ␤ 2 -adrenergic receptor or the m1 muscarinic receptor. Chimeric 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 was not affected by preventing palmitoylation of Cys 322 and Cys 323 . We suggest that the amino terminus of the fourth loop interacts directly with G t , particularly with G␣ t , and with other regions of the intracellular surface of rhodopsin to support G t binding.Rhodopsin, the dim-light photoreceptor of the rod cell, is a prototypical member of the superfamily of G protein-coupled receptors (GPCRs) 1 (1, 2). Following exposure to light, rhodopsin assumes an active signaling conformation, metarhodopsin II (MII). MII can bind and catalytically activate the retinal heterotrimeric G protein, transducin (G t ). G t is composed of a guanine-nucleotide binding ␣ subunit (G␣ t ), and a functional heterodimer of ␤ and ␥ subunits (G␤␥ t ). Interaction of the trimer with MII promotes the release of GDP from G␣ t , leading to the formation of a stable MII-G␣ t (empty pocket) complex. The subsequent binding of GTP activates G␣ t , leading to its dissociation from the receptor and from G␤␥ t . The activated G␣ t binds and activates its effector, cyclic GMP phosphodiesterase.The molecular structure of the complex between rhodopsin and G t , and the mechanism by which rhodopsin catalyzes nucleotide exchange, are not understood in detail. Numerous studies have localized the G t -binding site to the cytoplasmic surface of rhodopsin. The cytoplasmic surface is composed of four loops (Fig. 1) and a carboxyl-terminal tail. The first (C1), second (C2), and third (C3) cytoplasmic loops connect adjacent transmembrane (TM) helices. The fourth cytoplasmic loop (C4) is unique in that it is bounded by a helix only at its amino terminus; its carboxyl terminus is formed by the insertion of two palmitoyl groups into the membrane bilayer (3). The palmitoyl groups are attached to Cys 322 and Cys 323 via thioester linkages (4, 5). The carboxyl-terminal tail is the region dist...

The second extracellular loop of rhodopsin folds back into the membrane-embedded domain of the receptor to form part of the binding pocket for the 11-cis-retinylidene chromophore. A carboxylic acid side chain from this loop, Glu181, points toward the center of the retinal polyene chain. We studied the role of Glu181 in bovine rhodopsin by characterizing a set of site-directed mutants. Sixteen of the 19 single-site mutants expressed and bound 11-cis-retinal to form pigments. The lambda(max) value of mutant pigment E181Q showed a significant spectral red shift to 508 nm only in the absence of NaCl. Other substitutions did not significantly affect the spectral features of the mutant pigments in the dark. Thus, Glu181 does not contribute significantly to spectral tuning of the ground state of rhodopsin. The most likely interpretation of these data is that Glu181 is protonated and uncharged in the dark state of rhodopsin. The Glu181 mutants displayed significantly increased reactivity toward hydroxylamine in the dark. The mutants formed metarhodopsin II-like photoproducts upon illumination but many of the photoproducts displayed shifted lambda(max) values. In addition, the metarhodopsin II-like photoproducts of the mutant pigments had significant alterations in their decay rates. The increased reactivity of the mutants to hydroxylamine supports the notion that the second extracellular loop prevents solvent access to the chromophore-binding pocket. In addition, Glu181 strongly affects the environment of the retinylidene Schiff base in the active metarhodopsin II photoproduct.