G protein-coupled receptors mediate biological signals by stimulating nucleotide exchange in heterotrimeric proteins (Galphabetagamma). Receptor dimers have been proposed as the functional unit responsible for catalytic interaction with Galphabetagamma. To investigate whether a receptor monomer can activate Galphabetagamma, we used retinal photoreceptor rhodopsin and its cognate protein transducin (G(t)) to determine stoichiometry of rhodopsin/G(t) binding rate catalyzed G(t). Purified was...

G-protein-coupled receptors (GPCRs) transmit extracellular signals to activate intracellular heterotrimeric G proteins (Gαβγ) and arrestins. For protein signalling, the Gα C-terminus (GαCT) binds a cytoplasmic crevice of receptor that opens upon activation. A consensus motif is shared among GαCT from Gi/Gt family ‘finger loop’ region (ArrFL1–4) all four Here we present 2.75 Å crystal structure ArrFL-1, peptide analogue finger loop rod photoreceptor arrestin, in complex with prototypical GPCR...

The G protein coupled receptor rhodopsin contains a pocket within its seven-transmembrane helix (TM) structure, which bears the inactivating 11-cis-retinal bound by protonated Schiff-base to Lys296 in TM7. Light-induced 11-cis-/all-trans-isomerization leads deprotonated active Meta II intermediate. With decay, bond is hydrolyzed, all-trans-retinal released from pocket, and apoprotein opsin reloaded with new 11-cis-retinal. crystal structure of Ops* conformation provides basis for...

G-protein-coupled receptors are universally regulated by arrestin binding. Here we show that rod induces uptake of the agonist all-trans-retinal [corrected] in only half population phosphorylated opsin native membrane. Agonist blocks subsequent entry inverse 11-cis-retinal (that is, regeneration rhodopsin), but is not blocked other aporeceptors. Environmentally sensitive fluorophores attached to reported conformational changes loop(V-VI) (N-domain) coupled agonist, while loop(XVIII-XIX)...

In the retinal binding pocket of rhodopsin, a Schiff base links ligand covalently to Lys296 side chain. Light transforms inverse agonist 11- cis -retinal into all- trans -retinal, leading active Meta II state. Crystal structures and conformation opsin apoprotein revealed two openings 7-transmembrane (TM) bundle towards hydrophobic core membrane, one between TM1/TM7 TM5/TM6, respectively. Computational analysis putative channel connecting traversing pocket. Identified constrictions within...

Deactivation of light-activated rhodopsin (metarhodopsin II) involves, after kinase and arrestin interactions, the hydrolysis covalent bond all-trans-retinal to apoprotein. Although long-lived storage form metarhodopsin III is transiently formed, eventually released from active site. Here we address question whether release results in a retinal that freely diffusible lipid phase photoreceptor membrane. The reaction accompanied by an increase intrinsic protein fluorescence (release signal),...

Vertebrate rhodopsin consists of the apoprotein opsin and chromophore 11-<i>cis</i>-retinal covalently linked via a protonated Schiff base. Upon photoisomerization to all-<i>trans</i>-retinal, retinylidene linkage hydrolyzes, all-<i>trans</i>-retinal dissociates from opsin. The pigment is eventually restored by recombining with enzymatically produced 11-<i>cis</i>-retinal. All-<i>trans</i>-retinal release occurs in parallel decay active form, metarhodopsin (Meta) II, which original base...

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTG-protein-effector coupling: A real-time light-scattering assay for transducin-phosphodiesterase interactionMartin Heck and Klaus Peter HofmannCite this: Biochemistry 1993, 32, 8220–8227Publication Date (Print):August 1, 1993Publication History Published online1 May 2002Published inissue 1 August 1993https://pubs.acs.org/doi/10.1021/bi00083a024https://doi.org/10.1021/bi00083a024research-articleACS PublicationsRequest reuse permissionsArticle...

Photoactivation of the retinal photoreceptor rhodopsin proceeds through a cascade intermediates, resulting in protein–protein interactions catalyzing activation G-protein transducin (Gt). Using stabilization and photoregeneration receptor’s signaling state Gt assays, we provide evidence for two-site sequential fit mechanism activation. We show that C-terminal peptide from γ-subunit, Gtγ(50–71)farnesyl, can replace holoprotein stabilizing rhodopsin’s active intermediate metarhodopsin II...

Guanine nucleotide exchange in heterotrimeric G proteins catalyzed by protein-coupled receptors (GPCRs) is a key event many physiological processes. The crystal structures of the GPCR rhodopsin and two as well binding sites on both catalytically interacting are known, but temporal sequence events leading to remains be elucidated. We employed time-resolved near infrared light scattering study order which Galpha Ggamma C-terminal holo-G protein interact with active state (R*) native membranes....

The G protein coupled receptor (GPCR) rhodopsin activates the heterotrimeric transducin (Gt) to transmit light signal into retinal rod cells. activity is virtually zero in dark and jumps by more than one billion fold after photon capture. Such perfect switching implies both high fidelity speed of rhodopsin/Gt coupling. We employed Fourier transform infrared (FTIR) spectroscopy supporting all-atom molecular dynamics (MD) simulations study conformational diversity membrane environment extend...

Among cyclic nucleotide phosphodiesterases (PDEs), PDE6 is unique in serving as an effector enzyme G protein-coupled signal transduction. In retinal rods and cones, membrane-bound activated to hydrolyse its substrate, cGMP, by binding of two active protein α-subunits (Gα*). To investigate the activation mechanism mammalian rod PDE6, we have collected functional structural data, analysed them reaction–diffusion simulations. Gα* titration reveals a strong asymmetry with respect affinity for...

Photoexcitation of rhodopsin activates a heterotrimeric G-protein cascade leading to cyclic GMP hydrolysis in vertebrate photoreceptors. Light-induced exchanges the visual transducin between outer and inner segment rod photoreceptors occur through narrow connecting cilium. Here we demonstrate that colocalizes with Ca(2+)-binding protein centrin 1 specific domain this Coimmunoprecipitation, centrifugation, overlay, size exclusion chromatography, kinetic light-scattering experiments indicate...

Extracellular signals prompt G protein-coupled receptors (GPCRs) to adopt an active conformation (R*) and catalyze GDP/GTP exchange in the alpha-subunit of intracellular proteins (Galphabetagamma). Kinetic analysis transducin (G(t)alphabetagamma) activation shows that intermediary R*xG(t)alphabetagamma.GDP complex is formed precedes GDP release formation nucleotide-free R*xG protein complex. Based on this reaction sequence, we explore dynamic interface between during these complexes. We...

In the rod cell of retina, arrestin is responsible for blocking signaling G-protein-coupled receptor rhodopsin. The general visual signal transduction model implies that must be able to interact with a single light-activated, phosphorylated rhodopsin molecule (Rho*P), as would generated at physiologically relevant low light levels. However, elongated bi-lobed structure suggests it might accommodate two molecules. this study, we directly addressed question binding stoichiometry by quantifying...

Catalysis of nucleotide exchange in heterotrimeric G proteins (Galphabetagamma) is a key step cellular signal transduction mediated by protein-coupled receptors. The Galpha N terminus with its helical stretch thought to be crucial for protein/activated receptor (R(*)) interaction. N-terminal fatty acylation important membrane targeting proteins. By applying biophysical techniques the rhodopsin/transducin model system, we studied effect truncations Galpha. In Galphabetagamma, lack acid and up...

Binding of arrestin to photoactivated phosphorylated rhodopsin terminates the amplification visual signals in photoreceptor cells. Currently, there is no crystal structure a rhodopsin−arrestin complex available, although structures unbound and have been determined. High-affinity receptor binding dependent on distinct sites responsible for recognition activation phosphorylation. The loop connecting β-strands V VI rod has implicated active rhodopsin. We report receptor-bound peptide Arr(67−77)...

Rhodopsin, a seven transmembrane helix (TM) receptor, binds its ligand 11-cis-retinal via protonated Schiff base. Coupling to the G-protein transducin (Gt) occurs after light-induced cis/trans-retinal isomerization, which leads through photoproducts into sequence of metarhodopsin (Meta) states: Meta I ⇌ IIa IIb IIbH+. The structural changes behind this three-step activation scheme are mediated by microswitch domains consisting conserved amino acids. Here we focus on Tyr2235.58 as part...

Rhodopsin bears 11-cis-retinal covalently bound by a protonated Schiff base linkage. 11-cis/all-trans isomerization, induced absorption of green light, leads to active metarhodopsin II, in which the is intact but deprotonated. The subsequent metabolic retinoid cycle starts with hydrolysis and release photolyzed all-trans-retinal from site ends uptake fresh 11-cis-retinal. To probe chromophore-protein interaction state, we have studied effects blue light on II using infrared time-resolved...

Transducin is a heterotrimer formed by fatty acylated alpha-subunit and farnesylated betagamma-subunit. The role of these two covalent modifications adjacent hydrophobic charged amino acid residues in reversible anchoring at disk model membranes investigated different pH values, salt concentrations, lipid packing densities using the monolayer expansion technique CD spectroscopy. only binds if acetylated transformed into its surface-active form divalent cations. In presence salts...