The heterotrimeric G-protein binding site on G-protein coupled receptors remains relatively unexplored regarding its potential as a new target of therapeutic intervention or as a secondary site of ...action by the existing drugs. Tauroursodeoxycholic acid bears structural resemblance to several compounds that were previously identified to specifically bind to the light-activated form of the visual receptor rhodopsin and to inhibit its activation of transducin. We show that TUDCA stabilizes the active form of rhodopsin, metarhodopsin II, and does not display the detergent-like effects of common amphiphilic compounds that share the cholesterol scaffold structure, such as deoxycholic acid. Computer docking of TUDCA to the model of light-activated rhodopsin revealed that it interacts using similar mode of binding to the C-terminal domain of transducin alpha subunit. The ring regions of TUDCA made hydrophobic contacts with loop 3 region of rhodopsin, while the tail of TUDCA is exposed to solvent. The results show that TUDCA interacts specifically with rhodopsin, which may contribute to its wide-ranging effects on retina physiology and as a potential therapeutic compound for retina degenerative diseases.
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•A mechanism contributing to the anti-RD properties of TUDCA is proposed.•The mechanism includes TUDCA interaction with light-activated rhodopsin.•UV/Vis spectroscopy and computer modeling support TUDCA binding to R*.
The interaction between receptor and a heterotrimeric G protein (alpha beta gamma) is thought to involve the intracellular
loops of receptors and specific domains on the G protein. Here we show that ...a chemically farnesylated peptide (P5far) specific
to the carboxyl-terminal domain (amino acids 60-71: DKNPFKELKGGC) of the gamma subunit of the G protein, Gt, directly stabilizes
the active form of rhodopsin, metarhodopsin II (M II), and also uncouples rhodopsin-Gt interaction. Peptide activity is significantly
affected by the absence of the isoprenoid moiety. Moreover, we show that altering the amino acid sequence of the farnesylated
peptide by randomizing the sequence, substituting hydrophobic with hydrophilic residues (F64T; L67S) or deleting amino acids
60-66 significantly reduces the ability of the peptide to stabilize M II. This indicates that both the farnesyl moiety and
the structure of the gamma subunit tail are specific determinants of receptor-G protein interaction. These results also suggest
a general function for the family of G protein gamma subunits in signaling.
A large superfamily of transmembrane receptors control cellular responses to diverse extracellular signals by catalyzing activation of specific types of heterotrimeric GTP-binding proteins. How these ...receptors recognize and promote nucleotide exchange on G protein α subunits to initiate signal amplification is unknown. The three-dimensional structure of the transducin (Gt) α subunit C-terminal undecapeptide Gtα (340-350) IKENLKDCGLF was determined by transferred nuclear Overhauser effect spectroscopy while it was bound to photoexcited rhodopsin. Light activation of rhodopsin causes a dramatic shift from a disordered conformation of Gtα (340-350) to a binding motif with a helical turn followed by an open reverse turn centered at Gly-348, a helix-terminating C capping motif of an αLtype. Docking of the NMR structure to the GDP-bound x-ray structure of Gt reveals that photoexcited rhodopsin promotes the formation of a continuous helix over residues 325-346 terminated by the C-terminal helical cap with a unique cluster of crucial hydrophobic side chains. A molecular mechanism by which activated receptors can control G proteins through reversible conformational changes at the receptor-G protein interface is demonstrated.
Photoactivation of the retinal photoreceptor rhodopsin proceeds through a cascade of intermediates, resulting in protein-protein interactions catalyzing the activation of the G-protein transducin ...(Gt). Using stabilization and photoregeneration of the receptor's signaling state and Gt activation assays, we provide evidence for a two-site sequential fit mechanism of Gt activation. We show that the C-terminal peptide from the Gt γ -subunit, Gtγ (50-71)farnesyl, can replace the holoprotein in stabilizing rhodopsin's active intermediate metarhodopsin II (MII). However, the peptide cannot replace the Gtβ γ complex in direct activation assays. Competition by Gtγ (50-71) farnesyl with Gt for the active receptor suggests a pivotal role for Gtβ γ in signal transfer from MII to Gt. MII stabilization and competition is also found for the C-terminal peptide from the Gt α -subunit, Gtα (340-350), but the capacity of this peptide to interfere in MII-Gt interactions is paradoxically low compared with its activity to stabilize MII. Besides this disparity, the pH profiles of competition with Gt are characteristically different for the two peptides. We propose a two-site sequential fit model for signal transfer from the activated receptor, R*, to the G-protein. In the center of the model is specific recognition of conformationally distinct sites of R*by Gtα (340-350) and Gtγ (50-71)farnesyl. One matching pair of domains on the proteins would, on binding, lead to a conformational change in the G-protein and/or receptor, with subsequent binding of the second pair of domains. This process could be the structural basis for GDP release and the formation of a stable empty site complex that is ready to receive the activating cofactor, GTP.
Receptor-G protein interaction is characterized by cycles of association and dissociation. We present evidence which indicates that during receptor-G protein interaction, the C-terminal tail of the G ...protein gamma subunit, which is masked in the beta gamma complex, is exposed and establishes high-affinity contact with the receptor. This potential conformational switch provides a mechanism to regulate receptor-G protein coupling. This switch may also be significant for the role of the beta gamma complex in regulation of effector function.
Interaction with a receptor is the first step in the process of signal transduction by heterotrimeric (alpha beta gamma) G proteins. We have examined the role of the G protein gamma subunit in ...interaction between a receptor (rhodopsin) and a G protein, transducin (Gt). We have obtained recombinant beta gamma complexes containing the same beta subunit but three different gamma subunit types by expressing them in the baculovirus/insect cell system. We show that the different recombinant beta gamma complexes interact equally well with the alpha subunit of transducin (alpha t) but only the gamma subunit specific to rod photoreceptors (gamma 1) is able to support interaction of alpha t with rhodopsin. This indicates a direct role for the G protein gamma subunits, which are a family of proteins with diverse structures, in conferring specificity to receptor-G protein interaction.
To probe the interaction between transducin (G
t) and photoactivated rhodopsin (R*), 14 analog peptides were designed and synthesized restricting the backbone of the R*-bound structure of the ...C-terminal 11 residues of G
t
α derived by transferred nuclear Overhauser effect (TrNOE) NMR. Most of the analogs were able to bind R*, supporting the TrNOE structure. Improved affinities of constrained peptides indicated that preorganization of the bound conformation is beneficial. Cys347 was found to be a recognition site; particularly, the free sulfhydryl of the side chain seems to be critical for R* binding. Leu349 was another invariable residue. Both Ile and
tert-leucine (Tle) mutations for Leu349 significantly reduced the activity, indicating that the Leu side chain is in intimate contact with R*. The structure of R* was computer generated by moving helix 6 from its position in the crystal structure of ground-state rhodopsin (R) based on various experimental data. Seven feasible complexes were found when docking the TrNOE structure with R* and none with R. The analog peptides were modeled into the complexes, and their binding affinities were calculated. The predicted affinities were compared with the measured affinities to evaluate the modeled structures. Three models of the R*/G
t
α complex showed strong correlation to the experimental data.
The visual signaling pathway is initiated by photoactivation of the GPCR rhodopsin, which activates nucleotide exchange on the heterotrimeric G-protein transducin (Gt). Domains on both Gtα and Gtβγ ...subunits participate in coupling to rhodopsin. Previously, we have shown by high-resolution NMR that the farnesylated C-terminal peptide of Gtγ(60−71), DKNPFKELKGGC, assumes an amphipathic helical conformation during interaction with metarhodopsin II Kisselev, O. G., and Downs, M. A. (2003) Structure 11, 367−373. This conformation was docked to the structure of holo-Gt to create a model of rhodopsin−Gt interaction. Here we test this model by mutational analysis of Gt. To evaluate the contribution of specific amino acids of the Gtγ C-terminal region involved in binding and GTP-dependent release of transducin from native rhodopsin membranes, we have systematically substituted each of the amino acids in the C-terminal region of Gtγ for alanine. The mutants were co-expressed with six-histidine-tagged Gtβ subunits in Sf9 insect cells. The Gtβ-6-His-γ mutant proteins were purified and assayed in the presence of Gtα for the GTP-dependent interactions with light-activated rhodopsin. Several of the alanine mutants, N62A, P63A, and F64A, exhibited significant functional defects at the level of R*−Gt complex formation. These data show that the conserved N-terminal end of the helical domain in the Gtγ(60−71) region has the most significant effect on rhodopsin−Gt interactions, which places important constraints on the model of the rhodopsin−Gt complex.
Large CMS cathode strip chambers: design and performance Acosta, D; Apollinari, G; Blomquist, J ...
Nuclear instruments & methods in physics research. Section A, Accelerators, spectrometers, detectors and associated equipment,
10/2000, Letnik:
453, Številka:
1
Journal Article
Recenzirano
Presented are the main design features of the large Cathode Strip Chambers (CSCs) for the CMS Endcap Muon System as well as the performance results obtained with the two full-scale 3.4×1.5 m
2 ...six-plane prototypes. The prototype performance was within the baseline requirements: (a) higher than 99% efficiency of muon track finding at the trigger level with more than 92% probability for bunch crossing identification and better than 2
mm spatial resolution, and (b) better than 150
μm spatial resolution in off-line.
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