Disease relevance of RHO

• The naturally occurring mutations G51A and G51V in transmembrane helix I and G89D in the transmembrane helix II of rhodopsin are associated with the retinal degenerative disease autosomal dominant retinitis pigmentosa [1].
• Characterization of rhodopsin congenital night blindness mutant T94I [2].
• For rapid single-step purification of recombinant rhodopsin, a baculovirus expression vector was constructed containing the bovine opsin coding sequence extended at the 3'-end by a short sequence encoding six histidine residues [3].
• These results have implications for transgenic models of retinal degeneration and mechanisms of position-effect variegation and demonstrate the utility of rho-GFP as a probe for rhodopsin transport and temporal regulation of promoter function [4].
• This effect could have implications in the instability and functional changes seen for certain mutations in rhodopsin associated with retinal disease, and in the stability of the different conformers induced by mutations in other G protein-coupled receptors [5].

Psychiatry related information on RHO

• Extrapolation of the measured on-rates to physiological conditions yielded reaction times for the binding of p44 to activated rhodopsin [6].

High impact information on RHO

• RPE65 is essential for the biosynthesis of 11-cis-retinal, the chromophore of rhodopsin [7].
• We have isolated cDNA clones generated from the mRNA encoding the opsin apoprotein of bovine rhodopsin and used these cDNAs to isolate genomic DNA clones containing the complete opsin gene [8].
• Nucleotide sequence analysis of the cloned DNAs has yielded a complete amino acid sequence for bovine rhodopsin and provided an intron-exon map of its gene [8].
• The G protein transducin, Gt, couples photon-activated rhodopsin with the effector cyclic GMP phosophodiesterase (PDE) in the vertebrate phototransduction cascade [9].
• Here we determine the three-dimensional structure of 11-cis retinal bound to bovine rhodopsin in the ground state at atomic level using a new high-resolution solid-state NMR method [10].

Chemical compound and disease context of RHO

• The T94I mutant pigment (with a bound 11-cis-retinal chromophore), like the other known rhodopsin night blindness mutants, is not active in the dark and has wild-type activity upon exposure to light [2].
• However, T alpha' is unable to participate in T beta gamma-dependent activities such as the light-stimulated binding of guanine nucleotides, binding to photoexcited rhodopsin, and ADP-ribosylation catalyzed by pertussis toxin [11].
• While T alpha-bound TF16 does not inhibit either pertussis toxin-catalyzed ADP-ribosylation, rhodopsin binding, or transducin subunit interaction, it blocks both the light-activated uptake of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and the GTP-dependent elution of transducin from photolyzed rhodopsin [12].
• Antibodies directed against the cytoplasmic loop between transmembrane domains 1 and 2, as well as those directed against the serine/threonine-rich region of the COOH terminus of bovine rhodopsin, also recognized purified beta-adrenergic receptor isolated from mouse S49 lymphoma cells [13].
• The ADP-ribosylation of T alpha by pertussis toxin and binding of T alpha to rhodopsin, both of which are enhanced in the presence of T beta gamma, were inhibited by NaF and AlCl3 [14].

Biological context of RHO

• Opposite to the mostly extended structure of the unphosphorylated C-terminal domain of rhodopsin, the arrestin-bound C-terminal helix is a compact domain that occupies a central position between the cytoplasmic loops and occludes the key binding sites of transducin [15].
• In the phototransduction pathway of rhodopsin, the metarhodopsin (Meta) III retinal storage form arises from the active G-protein binding Meta II by a slow spontaneous reaction through the Meta I precursor or by light absorption and photoisomerization, respectively [16].
• Little is known about the molecular mechanism of Schiff base hydrolysis in rhodopsin [17].
• Mutations at position 51 (G51V and G51L) bound retinal like wild-type rhodopsin but had thermally destabilized structures in the dark, altered photobleaching behavior, destabilized metarhodopsin II active conformations, and were severely defective in signal transduction [1].
• These results prove the modification of cysteines 322 and 323 with palmitic acid in bovine rhodopsin, and illustrate the utility of mass spectrometry to characterize the post-translational modifications in G-protein coupled receptors [18].

Anatomical context of RHO

• The mammalian photoreceptor, rhodopsin, is the best-studied member of this superfamily [19].
• Rhodopsin, a well known member of the G-protein-coupled receptor family, is located in the disk membranes of the rod outer segment, where it is responsible for the visualization of dim light [20].
• Rhodopsin (Rho) has been extracted in n-dodecyl beta-D-maltoside (DM) from bovine retinal rod outer segments and purified to homogeneity by affinity chromatography on concanavalin A-Sepharose [21].
• Rhodopsin (Rho) is a G protein-coupled receptor that initiates phototransduction in rod photoreceptors [22].
• The role of glutamic acid 181 in the bovine rhodopsin retinylidene chromophore pocket was studied by expressing E181 mutants in COS cells and measuring, as a function of time, the absorbance changes produced after excitation of lauryl maltoside pigment suspensions with 7 ns laser pulses [23].

Associations of RHO with chemical compounds

• The rhodopsin crystal structure reveals that intradiscal loop E-2 covers the 11-cis-retinal, creating a "retinal plug." Recently, we noticed the ends of loop E-2 are linked by an ion pair between residues Arg-177 and Asp-190, near the highly conserved disulfide bond [24].
• Rhodopsin bears 11-cis-retinal covalently bound by a protonated Schiff base linkage [25].
• In one mutant, CysVII, all the 10 cysteine residues of rhodopsin were replaced by serines [26].
• 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 [22].
• The size of Rho was determined to be 65,300 and 69,800 Da, respectively, when the purified Rho.DM complex was either chromatographed on Sephacryl S-300 or ultracentrifuged on sucrose gradients in the absence of DM [21].

Physical interactions of RHO

• Inactivation of photolyzed rhodopsin requires phosphorylation of the receptor and binding of the 48-kDa regulatory protein arrestin [27].
• The fact that 7-cis-rhodopsin can be readily converted to rhodopsin and to 9-cis-rhodopsin shows that the identical retinal binding site of opsin is involved in the three isomeric rhodopsins [28].
• Early expression and localization of rhodopsin and interphotoreceptor retinoid-binding protein (IRBP) in the developing fetal bovine retina [29].
• We investigated the effect of increased VEGF expression in the retina using tissue-specific, gain-of-function transgenic mice in which the bovine rhodopsin promoter is coupled to the gene for human VEGF [30].
• Recoverin in every fraction bound Ca2+ as assessed by fluorescence spectroscopy and inhibited the light-dependent rhodopsin phosphorylation in the same range of free Ca2+ concentration (0.3-0.8 microM) [31].

Enzymatic interactions of RHO

• Our data suggest that 48-kDa protein binds to phosphorylated R* and thereby quenches its capacity to activate transducin and PDEase [32].
• The analog-reconstituted rhodopsin activated transducin and was phosphorylated by rhodopsin kinase on illumination [33].
• Purified GRK5 phosphorylates rhodopsin in a light-dependent manner and beta 2-adrenergic receptor in an agonist-dependent manner and phosphorylates the C-terminal tail regions of both receptor proteins [34].
• Finally, we observe that a kinase mutant lacking the N-terminal recoverin binding site is unable to phosphorylate light-activated rhodopsin [35].
• The beta-adrenergic receptor kinase (beta-ARK) is a recently discovered enzyme which specifically phosphorylates the agonist-occupied form of the beta-adrenergic receptor (beta-AR) as well as the light-bleached form of rhodopsin. beta-ARK is present in a wide variety of mammalian tissues [36].

Regulatory relationships of RHO

• The 48-kDa rod outer segment protein arrestin (S-antigen) was found to inhibit the dephosphorylation of freshly photolyzed rhodopsin by protein phosphatase 2A but did not inhibit the dephosphorylation of unbleached rhodopsin [37].
• The studies above support a mechanism for rhodopsin kinase that we have termed the 'kinase-activation hypothesis'. This requires that the kinase exists in an inactive form and is activated only after binding to photoactivated rhodopsin [38].
• The ability of rhodopsin to activate PDE may be inhibited by the phosphorylation of sites exposed on the opsin surface as a result of light-induced conformational changes [39].
• Supplementation with IRBP enhanced the formation of rhodopsin in both the ROS/RPE-eyecup and retina/RPE-eyecup preparations [40].
• We report that kinase activity toward either GPCR (rhodopsin) or a synthetic peptide substrate is enhanced in the presence of GST-GRK2 fusion proteins or peptides corresponding to either N- or C-terminal sequences of GRK2 [41].

Other interactions of RHO

• Both Rho(*) monomers and dimers are capable of activating Gt, and both of them are phosphorylated by Rho kinase [22].
• Bovine rhodopsin in disc membranes was digested with thermolysin to generate the C-terminal fragment (241-327), which was subsequently cleaved with cyanogen bromide to generate the peptide Val-Thr-Thr-Leu-Cys-Cys-Gly-Lys-Asn-Pro (318-327) [18].
• Arrestin (also called S-antigen or 48-kDa protein) binds to photoexcited and phosphorylated rhodopsin and, thereby, blocks competitively the activation of transducin [42].
• In light-reared Royal College of Surgeons (RCS) and RCS retinal dystrophy gene (rdy)+ rats, the amount of IRBP in the interphotoreceptor matrix increased in corresponding proportion to the amount of total rhodopsin through postnatal day 22 (P22) [43].
• The enzyme acts specifically on photobleached, not unbleached, rhodopsin and will not catalyze the phosphorylation of histones, phosvitin, or casein [44].

Analytical, diagnostic and therapeutic context of RHO

• Isolation, sequence analysis, and intron-exon arrangement of the gene encoding bovine rhodopsin [8].
• The molecular weights of the complexes between dark and photoexcited states of Rho and DM were determined by gel filtration chromatography on Sephacryl S-300, in the presence of 0.1% DM [21].
• 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 [22].
• The three-dimensional structure of bovine rhodopsin determined by electron cryomicroscopy [20].
• For these reasons, we anticipate that the mutant may be of use in detailed kinetic and mechanistic investigations of the ligand binding reaction and for crystallization trials involving recombinant rhodopsin, especially the unliganded opsin form of the protein [45].

References