Effects of phosphorylation on the structure of the G-protein receptor rhodopsin (original) (raw)
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Biochemistry, 2001
Activation of G-protein coupled receptors (GPCR) is not yet understood. A recent structure showed most of rhodopsin in the ground (not activated) state of the GPCR, but the cytoplasmic face, which couples to the G protein in signal transduction, was not well-defined. We have determined an experimental three-dimensional structure for rhodopsin in the unactivated state, which shows good agreement with the crystal structure in the transmembrane domain. This new structure defines the cytoplasmic face of rhodopsin. The G-protein binding site can be mapped. The same experimental approach yields a preliminary structure of the cytoplasmic face in the activated (metarhodopsin II) receptor. Differences between the two structures suggest how the receptor is activated to couple with transducin.
The crystallographic model of rhodopsin and its use in studies of other G protein-coupled receptors
2003
G protein-coupled receptors (GPCRs) are integral membrane proteins that respond to environmental signals and initiate signal transduction pathways activating cellular processes. Rhodopsin is a GPCR found in rod cells in retina where it functions as a photopigment. Its molecular structure is known from cryo-electron microscopic and X-ray crystallographic studies, and this has reshaped many structure/function questions important in vision science. In addition, this first GPCR structure has provided a structural template for studies of other GPCRs, including many known drug targets. After presenting an overview of the major structural elements of rhodopsin, recent literature covering the use of the rhodopsin structure in analyzing other GPCRs will be summarized. Use of the rhodopsin structural model to understand the structure and function of other GPCRs provides strong evidence validating the structural model.
Three-Dimensional Structure of the Cytoplasmic Face of the G Protein Receptor Rhodopsin †
Biochemistry, 1997
Rhodopsin is a G protein receptor from a many-membered family of membrane receptors. No high-resolution structure exists for any member of this family due to the insolubility of membrane proteins and the difficulty in crystallizing membrane proteins. Two new approaches to the structure of rhodopsin are described that circumvent these limitations: (1) individual solution structures of the four cytoplasmic domains of rhodopsin are fitted with the transmembrane domain; (2) the solution structure of a complex of the four cytoplasmic domains is determined from nuclear magnetic resonance data. The two structures are similar. To test the validity of these structures, specific site-to-site distances measured on intact membrane-bound rhodopsin are compared to the same distances on the structures reported here. Excellent agreement is obtained. Furthermore, the agreement is obtained with distances measured on the activated form of the receptor and not with distances on the dark-adapted form of rhodopsin. This approach may prove to have general applicability for the determination of the structure for membrane proteins.
2001
Physiological and sensory mechanisms are primarily controlled by membrane impermeant signals-which are usually detected by membrane-spanning receptor proteins that ultimately trigger intracellular responses. Similarly, development and repair in multi-cellular organisms are controlled by specific adhesion mechanisms that recognize macromolecular binding sites outside cells and control cytoplasmic responses. Understanding of signaling and adhesion mechanisms could be greatly advanced if the structures of the membrane-spanning receptor proteins and their non-covalent interaction partners could be determined at atomic resolution-but these structures can not typically be deduced by x-ray crystallography or NMR. Mass spectrometry is able to mass analyze high molecular weight species, however, non-covalent protein-ligand or protein-protein interactions are disrupted by the non-aqueous conditions used in conventional mass spectrometry. This thesis describes new mass spectrometric methodologies to analyze proteins directly from aqueous solutions, which is an important first step in mass spectrometry of non-covalent protein-ligand complexes. Insight is gained into fundamental mechanisms of laser desorption ionization mass spectrometry from aqueous solutions. This thesis also describes biochemical and mass spectrometric methods to identify non-covalent interactions between the rhodopsin visual receptor and the G protein, transducin. The contacts between rhodopsin and transducin were studied using synthetic peptides derived from transducin that were characterized by their ability to inhibit rhodopsin catalysis of nucleotide binding by transducin. The peptides were also characterized for their ability to inhibit the homologous formyl peptide receptor's catalysis of nucleotide binding by the Gi protein. Fluorescent photo-activatable analogs of the most potent peptides were synthesized. Non-covalent rhodopsin-peptide interactions were tested for specificity and covalently stabilized by photo-chemical cross-linking. Rhodopsin-peptide complexes (which are about 42,000 Da) must be cleaved into smaller peptides for mass spectrometric analysis of the amino acid sites cross-linked. Cyanogen bromide cleavage of rhodopsin reduced the size of the hydrophobic fragments to facilitate analysis. Methods are described for mass spectral analysis of all of the cyanogens bromide fragments of rhodopsin at the picomole level, which is an essential first step in identifying the peptide cross-linking
Rhodopsin: Structure, signal transduction and oligomerisation
International Journal of Biochemistry & Cell Biology, 2009
Rhodopsin was the first G protein-coupled receptor (GPCR) for which a high-resolution crystal structure was obtained. Several crystal structures have now been solved representing different activation states of the receptor. These structures, together with those from lower resolution techniques (e.g. electron microscopy), shed light on the stepwise process by which energy from an extracellular photon is transduced across the membrane to the intracellular compartment thereby activating signalling mechanisms responsible for very low-level light detection. Controversy remains in several areas including: (i) transmembrane helix movements responsible for the transduction process, (ii) the stoichiometry of coupling to G proteins and their mode of activation, (iii) the role, if any, of receptor oligomerisation and (iv) the suitability of using structures of this GPCR as templates for modelling the structures of other GPCRs, and their mechanisms of activation.
ChemBioChem, 2007
constitute one of the most attractive pharmaceutical targets, as around 40 % of clinically prescribed drugs and 25 % of the top-selling drugs act at these receptors. GPCRs are receptors for sensory signals of external origin such as odors, pheromones, or tastes; and for endogenous signals such as neurotransmitters, (neuro)peptides, divalent cations, proteases, glycoprotein hormones, and purine ligands. Phylogenetic analyses of the human genome have permitted GPCR sequences to be classified into five main families: rhodopsin (class A or family 1), secretin (class B or family 2), glutamate (class C or family 3), adhesion, and frizzled/ taste2. Specialized databases of GPCRs can be found at http://www.gpcr.org/7tm, http://gris.ulb.ac.be/, and http:// www.iuphar-db.org. Due to the low natural abundance of GPCRs and the difficulty in producing and purifying recombinant protein, only one member of this family, rhodopsin, the photoreceptor protein of rod cells, has been crystallized so far. Five structural models of inactive rhodopsin are available at the Protein Data Bank, at resolutions of 2.8 (PDB IDs: 1F88 and 1HZX), 2.65 (1GZM), 2.6 (1L9H), and 2.2 (1U19). Structural models of rhodopsin photointermediates such as bathorhodopsin (2G87), lumirhodopsin (2HPY), metarhodopsin I, and a photoactivated deprotonated intermediate reminiscent of metarhodopsin II (2I37) are also available. Rhodopsin is formed by an extracellular N terminus of four b-strands, seven transmembrane helices (TM1 to TM7) connected by alternating intracellular (I1 to I3) and extracellular (E1 to E3) hydrophilic loops, a disulfide bridge between E2 and TM3, and a cytoplasmic C terminus containing an a-helix (Hx8) parallel to the cell membrane. Statistical analysis of the residues forming the TM helices of the rhodopsin family of GPCRs shows a large number of conserved sequence patterns; this suggests a common TM structure. Thus, the availability of the rhodopsin structure allows the use of homology modeling techniques to build three-dimensional models of other homologous GPCRs. The putative structural homology between rhodopsin and other GPCRs probably does not extend to the extracellular domain, since the extracellular N terminus and loop [a] Prof.
Biochemistry, 2001
Membrane proteins, encoded by ∼20% of genes in almost all organisms, including humans, are critical for cellular communication, electrical and ion balances, structural integrity of the cells and their adhesions, and other functions. Atomic-resolution structures of these proteins furnish important information for understanding their molecular organization and constitute major breakthroughs in our understanding of how they participate in physiological processes. However, obtaining structural information about these proteins has progressed slowly (1, 2), mostly because of technical difficulties in the purification and handling of integral membrane proteins. Instability of the proteins in †
Journal of Molecular Biology, 1997
A model for the alpha-carbon positions in the seven transmembrane helices in the rhodopsin family of G-protein-coupled receptors is presented. The model incorporates structural information derived from the analysis of $500 sequences in this family. The location, relative to the centre of the lipid bilayer, of each of the seven helical sequence segments and their probable lengths are deduced from sequence analysis, along with the orientation, relative to the centre of the helix bundle, of each helical segment around its axis. The packing of the helices in the model is guided by the density in a three-dimensional map of frog rhodopsin determined by electron cryo-microscopy. The model suggests which of the residues that are highly conserved in this family of receptors interact with each other. Helices III, V and VI are predicted to protrude more than the others from the central lipid core towards the aqueous phase on the intracellular side of the membrane. This feature could be a property of the receptor structure in some but not all of the conformations that it adopts, since recent studies suggest that relative movement occurs between these helices on photoactivation of rhodopsin. Results from other techniques, including the creation of metal-binding sites and disulphide bridges, site-directed spin-labelling studies, the substituted-cysteine accessibility method and other site-directed mutagenesis studies, are discussed in terms of the model.
Molecular Pharmacology, 2001
The availability of a high-resolution structure of rhodopsin now allows us to reconsider research attempts to understand structure-function relationships in other G protein-coupled receptors (GPCRs). A comparison of the rhodopsin structure with the results of previous sequence analysis and molecular modeling that incorporated experimental results demonstrates a high degree of success for these methods in predicting the helix ends and protein-protein interface of GPCRs. Moreover, the amino acid residues inferred to form the surface of the bindingsite crevice based on our application of the substituted-cysteine accessibility method in the dopamine D 2 receptor are in remarkable agreement with the rhodopsin structure, with the notable exception of some residues in the fourth transmembrane segment. Based on our analysis of the data reviewed, we propose that the overall structures of rhodopsin and of amine receptors are very similar, although we also identified localized regions where the structure of these receptors may diverge. We