The changes that lead to activation of G protein-coupled receptors have not been elucidated at the structural level. In this work we report the crystal structures of both ground state and a photoactivated deprotonated intermediate of bovine rhodopsin at a resolution of 4.15 Å. In the photoactivated state, the Schiff base linking the chromophore and Lys-296 becomes deprotonated, reminiscent of the G protein-activating state, metarhodopsin II. The structures reveal that the changes that accompany photoactivation are smaller than previously predicted for the metarhodopsin II state and include changes on the cytoplasmic surface of rhodopsin that possibly enable the coupling to its cognate G protein, transducin. Furthermore, rhodopsin forms a potentially physiologically relevant dimer interface that involves helices I, II, and 8, and when taken with the prior work that implicates helices IV and V as the physiological dimer interface may account for one of the interfaces of the oligomeric structure of rhodopsin seen in the membrane by atomic force microscopy. The activation and oligomerization models likely extend to the majority of other G protein-coupled receptors.G protein-coupled receptor ͉ G protein-coupled receptor activation ͉ phototransduction ͉ membrane protein structure G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors in animals, accounting for Ϸ3% of the genome (1). GPCRs are involved in detecting a large variety of chemical and physical signals, and they are the targets of Ϸ50% of current therapeutics. Structural information on GPCRs has been limited because of difficulties with their expression, purification, intrinsic chemical heterogeneity, and instability. These biochemical problems were overcome by using rhodopsin as a model GPCR, as it is highly expressed in a homogeneous form in rod photoreceptors and stabilized in the ground state by its covalently bound chromophore, 11-cis-retinal (2).The first crystal structure of rhodopsin revealed the arrangement of helices, the interhelical connections, the chromophore binding site, the extracellular ''plug,'' interactions involved in ligand binding in other GPCRs, and cytoplasmic helix 8 (3). Further improvements in the rhodopsin crystals yielded higher-resolution diffraction data that provided details about the effects of water molecules located close to the chromophore and more precise descriptions of the cytoplasmic loops. However, the improved crystals did not elucidate the mechanism of activation (4, 5). The arrangement of the seven transmembrane helices of rhodopsin differs from that in the more completely structurally studied bacterial retinoid-binding protein, bacteriorhodopsin (6).Upon absorption of a single photon of light, rhodopsin's chromophore, 11-cis-retinal, isomerizes to form all-trans-retinal, a covalently bound, full agonist. Once all-trans-retinal is formed, the protein portion of rhodopsin progresses through a series of photostates, including bathorhodopsin, lumirhodopsin, and metarhodopsin I (Met...

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Structural water molecules may act as prosthetic groups indispensable for proper protein function. In the case of allosteric activation of G protein-coupled receptors (GPCRs), water likely imparts structural plasticity required for agonist-induced signal transmission. Inspection of structures of GPCR superfamily members reveals the presence of conserved embedded water molecules likely important to GPCR function. Coupling radiolytic hydroxyl radical labeling with rapid H 2O 18 solvent mixing, we observed no exchange of these structural waters with bulk solvent in either ground state or for the Meta II or opsin states. However, the radiolysis approach permitted labeling of selected side chain residues within the transmembrane helices and revealed activation-induced changes in local structural constraints likely mediated by dynamics of both water and protein. These results suggest both a possible general mechanism for water-dependent communication in family A GPCRs based on structural conservation, and a strategy for probing membrane protein structure.footprinting ͉ mass spectrometry ͉ signal transduction ͉ membrane proteins ͉ radiolysis

G protein-coupled receptors (GPCRs) are seven transmembrane domain proteins that transduce extracellular signals across the plasma membrane and couple to the heterotrimeric family of G proteins. Like most intrinsic membrane proteins, GPCRs are capable of oligomerization, the function of which has only been established for a few different receptor systems. One challenge in understanding the function of oligomers relates to the inability to separate monomeric and oligomeric receptor complexes in membrane environments. Here we report the reconstitution of bovine rhodopsin, a GPCR expressed in the retina, into an apolipoprotein A-I phospholipid particle, derived from high density lipoprotein (HDL). We demonstrate that rhodopsin, when incorporated into these 10 nm reconstituted HDL (rHDL) particles, is monomeric and functional. Rhodopsin⅐rHDL maintains the appropriate spectral properties with respect to photoactivation and formation of the active form, metarhodopsin II. Additionally, the kinetics of metarhodopsin II decay is similar between rhodopsin in native membranes and rhodopsin in rHDL particles. Photoactivation of monomeric rhodopsin⅐rHDL also results in the rapid activation of transducin, at a rate that is comparable with that found in native rod outer segments and 20-fold faster than rhodopsin in detergent micelles. These data suggest that monomeric rhodopsin is the minimal functional unit in G protein activation and that oligomerization is not absolutely required for this process.

Rhodopsin (Rho) is a G protein-coupled receptor that initiates phototransduction in rod photoreceptors. High expression levels of Rho in the disc membranes of rod outer segments and the propensity of Rho to form higher oligomeric structures are evident from atomic force microscopy, transmission electron microscopy, and chemical cross-linking experiments. To explore the structural and functional properties of Rho in n-dodecyl-␤-maltoside, frequently used to purify heterologously expressed Rho and its mutants, we used gel filtration techniques, blue native gel electrophoresis, and functional assays. Here, we show that in micelles containing n-dodecyl-␤-maltoside at concentrations greater than 3 mM, Rho is present as a single monomer per detergent micelle. In contrast, in 12 mM 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS), micelles contain mostly dimeric Rho. The cognate G protein transducin (Gt) appears to have a preference for binding to the Rho dimer, and the complexes fall apart in the presence of guanosine 5-3-O-(thio)triphosphate. Cross-linked Rho dimers release the chromophore at a slower rate than monomers and are much more resistant to heat denaturation. Both Rho* monomers and dimers are capable of activating Gt, and both of them are phosphorylated by Rho kinase. Rho expressed in HEK293 cells is also readily cross-linked by a bifunctional reagent. These studies provide an explanation of how detergent influences the oligomer-dimermonomer equilibrium of Rho and describe the functional characterization of Rho monomers and dimers in detergent.Rhodopsin (Rho), 1 the prototypical G protein-coupled receptor (GPCR), functions in the absorption of a photon in retinal rod photoreceptors (1-3). As are other GPCRs (4, 5), Rho is a seven-transmembrane-spanning helical protein (6). The high expression level of Rho, its specific localization in the internal discs of the structures termed rod outer segments (ROS), and the lack of other highly abundant membrane proteins allow Rho to be imaged in the native disc membranes by atomic force microscopy and transmission electron microscopy (7-9). These images have revealed rows of Rho dimers in native disc membranes. Subsequently, BN-and SDS-PAGE, chemical crosslinking, and proteolysis experiments corroborated that Rho consists mainly of dimers and higher oligomers in disc membranes.2 Medina et al. (7) reported that Rho and photoactivated Rho (Rho*) preserved a dimeric quaternary structure in detergent.These results are in conflict with a model of rapidly diffusing Rho in the "mosaic" fluid disc membrane (10) supported by measurements of Rho diffusion and rotation in disc membranes and by low resolution neutron diffraction studies (11)(12)(13)(14). The concept of rapidly diffusing monomeric molecules is at variance with the dimeric forms of other GPCRs (15,16). Moreover, the size of the G protein and arrestin surface interacting with Rho is almost twice as large as the exposed cytoplasmic surface of a single Rho molecule (17,18).Nonphysiological dimers of R...

RDH12 has been suggested to be one of the retinol dehydrogenases (RDH) involved in the vitamin A recycling system (visual cycle) in the eye. Loss of function mutations in the RDH12 gene were recently reported to be associated with autosomal recessive childhood-onset severe retinal dystrophy. Here we show that RDH12 localizes to the photoreceptor inner segments and that deletion of this gene in mice slows the kinetics of alltrans-retinal reduction, delaying dark adaptation. However, accelerated 11-cis-retinal production and increased susceptibility to light-induced photoreceptor apoptosis were also observed in Rdh12 ؊/؊ mice, suggesting that RDH12 plays a unique, nonredundant role in the photoreceptor inner segments to regulate the flow of retinoids in the eye. Thus, severe visual impairments of individuals with null mutations in RDH12 may likely be caused by light damage 1 .

A major question in G protein-coupled receptor signaling concerns the quaternary structure required for signal transduction. Do these transmembrane receptors function as monomers, dimers, or larger oligomers? We have investigated the oligomeric state of the model G protein-coupled receptor rhodopsin (Rho), which absorbs light and initiates a phototransduction-signaling cascade that forms the basis of vision. In this study, different forms of Rho were isolated using gel filtration techniques in mild detergents, including n-dodecyl-␤-D-maltoside, n-tetradecyl-␤-D-maltoside, and n-hexadecyl-␤-D-maltoside. The quaternary structure of isolated Rho was determined by transmission electron microscopy, demonstrating that in micelles containing n-dodecyl-␤-D-maltoside, Rho exists as a mixture of monomers and dimers whereas in n-tetradecyl-␤-D-maltoside and n-hexadecyl-␤-D-maltoside Rho forms higher ordered structures. Especially in n-hexadecyl-␤-D-maltoside, most of the particles are present in tightly packed rows of dimers. The oligomerization of Rho seems to be important for interaction with its cognate G protein, transducin. Although the activated Rho (Meta II) monomer or dimers are capable of activating the G protein, transducin, the activation process is much faster when Rho exists as organized dimers. Our studies provide direct comparisons between signaling properties of Meta II in different quaternary complexes.

Recently, a human ortholog of mouse calcyclin (S100A6)-binding protein (CacyBP) called SIP (Siah-1-interacting protein) was shown to be a component of a novel ubiquitinylation pathway regulating ␤-catenin degradation (Matsuzawa, S., and Reed, J. C. (2001) Mol. Cell 7, 915-926). In murine brain, CacyBP/SIP is expressed at a high level, but S100A6 is expressed at a very low level. Consequently we carried out experiments to determine if CacyBP/SIP binds to other S100 proteins in this tissue. Using CacyBP/SIP affinity chromatography, we found that S100B from the brain extract binds to CacyBP/SIP in a Ca 2؉ -dependent manner. Using a nitrocellulose overlay assay with 125 I-CacyBP/SIP and CacyBP/SIP affinity chromatography, we found that this protein binds purified S100A1, S100A6, S100A12, S100B, and S100P but not S100A4, calbindin D 9k , parvalbumin, and calmodulin.