TRENDS in Biochemical Sciences Vol.26 No.5 May 2001 http://tibs.trends.com 0968-0004/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0968-0004(01)01799-6 318 Review Review Tetsuji Okada Dept of Ophthalmology, University of Washington, Seattle, WA 98195, USA and Dept of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan. Krzysztof Palczewski Depts of Ophthalmology, Pharmacology and Chemistry, University of Washington, Seattle, WA 98195, USA. Oliver P . Ernst Klaus Peter Hofmann* Institut für Medizinische Physik und Biophysik, Universitätsklinikum Charité, Humboldt Universität zu Berlin, D-10098 Berlin, Germany. * e-mail: kph@charite.de Significant progress in understanding the structure and function of rhodopsin has been made in recent years. Electron cryomicroscopy on two-dimensional crystals of bovine rhodopsin provided the first direct visualization of the seven transmembrane helices of a G-protein-coupled receptor (GPCR) 1,2 , and led to structural information about other members of the superfamily through homology-modeling studies 3 . In parallel to these advances, biochemical and biophysical studies, often combined with site-directed mutagenesis, have provided valuable information on the nature of rhodopsin structure and the mechanism of rhodopsin activation. In addition, the recent structural determination of bovine rhodopsin by X-ray crystallography 4 offers a new opportunity to assemble these related studies, providing further insight into the mechanism of activation of rhodopsin and GPCRs in general. G-protein-coupled receptors Signal detection and transmission across biological membranes is initiated by the interaction of a chemical or physical stimulus with a specific membrane receptor which, in turn, becomes activated and initiates a chain of intracellular reactions that result in modulation of target protein activity. GPCRs are a superfamily of such membrane proteins that transmit a signal by coupling to heterotrimeric guanine nucleotide-binding proteins (known as large G proteins), which consist of three subunits (α, β and γ). In the activated form, the cytoplasmic domain of a GPCR is competent for binding a G protein, leading to subsequent catalytic nucleotide exchange on the α subunit and G-protein activation. As can be predicted by their amino acid sequences, GPCRs share the structural motif of seven transmembrane α-helical (H) segments, and thus belong to an even larger family of proteins that serve a broad variety of functional properties, from ion translocation (e.g. bacteriorhodopsin) to signal transduction. In the absence of an activating ligand, the GPCR apoprotein (opsin in the case of rhodopsin) has low basal activity; this activity is greatly enhanced upon binding of an agonist and reduced by inverse agonists. An enhanced ability of GPCRs to activate G proteins in the absence of ligand is characteristic of constitutive activity and can be caused by, for example, specific mutations. The G protein in retinal rod cells is called transducin (or Gt), according to its tissue-specific expression of the α subunit (reviewed in Refs 5,6). In spite of the structural similarities between the receptor apoproteins, the ligands can be exceedingly diverse. The majority of ligands reach GPCRs by diffusion and bind to a site near the extracellular surface of the receptor 5 . In the case of rhodopsin, the chromophoric ligand 11-cis-retinal is permanently bound to the receptor as a prosthetic group that efficiently inactivates the receptor. This dormant visual pigment is activated by light-induced cis–trans isomerization of the bound retinal. During the activation process retinal plays a dual role, both as a chromophore in the initial rapid photochemistry and, after relaxation of the photoexcited state, as an agonist in producing the active state of the receptor (metarhodopsin II). It is this latter function for which we anticipate similarities between rhodopsin and other GPCRs, in particular for members of the so-called class I or rhodopsin subfamily of GPCRs. Class I GPCRs share sequence homology 5 and also key structural features (Fig. 1), such as a disulfide bond between H-III and the extracellular region, and a tripeptide Asp(Glu)-Arg-Tyr motif located at the intracellular end of H-III. There are several other highly conserved residues, such as an Asn–Asp pair located in H-I and H-II, respectively, Pro residues in H-V and H-VI, aromatic residues in H-IV and H-VI, and a common Asn-Pro-X-X-Tyr motif in H-VII. Topology of rhodopsin Vertebrate rhodopsin is located in the membranes of discs – flat vesicles that fill the outer segment of rod cells. The extracellular (intradiscal) and G-protein-coupled receptors (GPCRs) are involved in a vast variety of cellular signal transduction processes from visual, taste and odor perceptions to sensing the levels of many hormones and neurotransmitters. As a result of agonist-induced conformation changes, GPCRs become activated and catalyze nucleotide exchange w ithin the G proteins, thus detecting and amplifying the signal. GPCRs share a common heptahelical transmembrane structure as well as many conserved key residues and regions. Rhodopsins are prototypical GPCRs that detect photons in retinal photoreceptor cells and trigger a phototransduction cascade that culminates in neuronal signaling. Biophysical and biochemical studies of rhodopsin activation, and the recent crystal structure determination of bovine rhodopsin, have provided new information that enables a more complete mechanism of vertebrate rhodopsin activation to be proposed. In many aspects, rhodopsin might provide a structural and functional template for other members of the GPCR family. Activation of rhodopsin:new insights from structural and biochemical studies Tetsuji Okada, Oliver P . Ernst, Krzysztof Palczewski and Klaus Peter Hofmann