C-terminal peptides of rhodopsin. Determination of the optimum sequence for recognition of retinal transducin.
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ABSTRACT:
SUBMITTER: Takemoto DJ
PROVIDER: S-EPMC1146684 | biostudies-other | 1986 Apr
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Transducin is a multi-subunit guanine-nucleotide-binding protein that mediates signal coupling between rhodopsin and cyclic GMP phosphodiesterase in retinal rod outer segments. Whereas the T alpha subunit of transducin binds guanine nucleotides and is the activator of the phosphodiesterase, the T beta gamma subunit may function to link physically T alpha with photolysed rhodopsin. In order to determine the binding sites of rhodopsin to transducin, we have synthesized eight peptides (Rhod-1 etc.) that correspond to the C-terminal regions of rhodopsin and to several external and one internal loop region. These peptides were tested for their inhibition of restored GTPase activity of purified transducin reconstituted into depleted rod-outer-segment disc membranes. A marked inhibition of GTPase activity was observed when transducin was pre-incubated with peptides Rhod-1, Rhod-2 and Rhod-3. These peptides correspond to opsin amino acid residues 332-339, 324-331 and 317-321 respectively. Peptides corresponding to the three external loop regions or to the C-terminal residues 341-348 did not inhibit reconsituted GTPase activity. Likewise, Rhod-8, a peptide corresponding to an internal loop region of rhodopsin, did not inhibit GTPase activity. These findings support the concept that these specific regions of the C-terminus of rhodopsin serve as recognition sites for transducin.
| S-EPMC1152937 | biostudies-other
Project description:A novel combination of experimental data and extensive computational modeling was used to explore probable protein-protein interactions between photoactivated rhodopsin (R*) and experimentally determined R*-bound structures of the C-terminal fragment of alpha-transducin (Gt(alpha)(340-350)) and its analogs. Rather than using one set of loop structures derived from the dark-adapted rhodopsin state, R* was modeled in this study using various energetically feasible sets of intracellular loop (IC loop) conformations proposed previously in another study. The R*-bound conformation of Gt(alpha)(340-350) and several analogs were modeled using experimental transferred nuclear Overhauser effect data derived upon binding R*. Gt(alpha)(340-350) and its analogs were docked to various conformations of the intracellular loops, followed by optimization of side-chain spatial positions in both R* and Gt(alpha)(340-350) to obtain low-energy complexes. Finally, the structures of each complex were subjected to energy minimization using the OPLS/GBSA force field. The resulting residue-residue contacts at the interface between R* and Gt(alpha)(340-350) were validated by comparison with available experimental data, primarily from mutational studies. Computational modeling performed for Gt(alpha)(340-350) and its analogs when bound to R* revealed a consensus of general residue-residue interactions, necessary for efficient complex formation between R* and its Gt(alpha) recognition motif.
| S-EPMC1877773 | biostudies-literature
Project description:Upon illumination the visual receptor rhodopsin (Rho) transitions to the activated form Rho(?), which binds the heterotrimeric G protein, transducin (Gt) causing GDP to GTP exchange and Gt dissociation. Using succinylated concanavalin A (sConA) as a probe, we visualized native Rho dimers solubilized in 1mM n-dodecyl-?-d-maltoside (DDM) and Rho monomers in 5mM DDM. By nucleotide depletion and affinity chromatography together with crosslinking and size exclusion chromatography, we trapped and purified nucleotide-free Rho(?)·Gt and sConA-Rho(?)·Gt complexes kept in solution by either DDM or lauryl-maltose-neopentyl-glycol (LMNG). The 3 D envelope calculated from projections of negatively stained Rho(?)·Gt-LMNG complexes accommodated two Rho molecules, one Gt heterotrimer and a detergent belt. Visualization of triple sConA-Rho(?)·Gt complexes unequivocally demonstrated a pentameric assembly of the Rho(?)·Gt complex in which the photoactivated Rho(?) dimer serves as a platform for binding the Gt heterotrimer. Importantly, individual monomers of the Rho(?) dimer in the heteropentameric complex exhibited different capabilities for regeneration with either 11-cis or 9-cis-retinal.
| S-EPMC3633645 | biostudies-literature
Project description:The process of vision is initiated when the G protein-coupled receptor, rhodopsin (Rho), absorbs a photon and transitions to its activated Rho(?) form. Rho(?) binds the heterotrimeric G protein, transducin (G(t)) inducing GDP to GTP exchange and G(t) dissociation. Using nucleotide depletion and affinity chromatography, we trapped and purified the resulting nucleotide-free Rho(?)·G(t) complex. Quantitative SDS-PAGE suggested a 2:1 molar ratio of Rho(?) to G(t) in the complex and its mass determined by scanning transmission electron microscopy was 221±12kDa. A 21.6Å structure was calculated from projections of negatively stained Rho(?)·G(t) complexes. The molecular envelope thus determined accommodated two Rho molecules together with one G(t) heterotrimer, corroborating the heteropentameric structure of the Rho(?)·G(t) complex.
| S-EPMC3210324 | biostudies-literature
Project description:Activation of GPCRs (G-protein-coupled receptors) leads to conformational changes that ultimately initiate signal transduction. Activated GPCRs transiently combine with and activate heterotrimeric G-proteins resulting in GTP replacement of GDP on the G-protein alpha subunit. Both the detailed structural changes essential for productive GDP/GTP exchange on the G-protein alpha subunit and the structure of the GPCR-G-protein complex itself have yet to be elucidated. Nevertheless, transient GPCR-G-protein complexes can be trapped by nucleotide depletion, yielding an empty-nucleotide G-protein-GPCR complex that can be isolated. Whereas early biochemical studies indicated formation of a complex between G-protein and activated receptor only, more recent results suggest that G-protein can bind to pre-activated states of receptor or even couple transiently to non-activated receptor to facilitate rapid responses to stimuli. Efficient and reproducible formation of physiologically relevant, conformationally homogenous GPCR-G-protein complexes is a prerequisite for structural studies designed to address these possibilities.
| S-EPMC2875673 | biostudies-literature
Project description:Suramin, a polysulfonated naphthylurea, is under investigation for the treatment of several cancers. It interferes with signal transduction through G(s), G(i), and G(o), but structural and kinetic aspects of the molecular mechanism are not well understood. Here, we have investigated the influence of suramin on coupling of bovine rhodopsin to G(t), where G-protein activation and receptor structure can be monitored by spectroscopic in vitro assays. G(t) fluorescence changes in response to rhodopsin-catalyzed nucleotide exchange reveal that suramin inhibits G(t) activation by slowing down the rate of complex formation between metarhodopsin-II and G(t). The metarhodopsin-I/-II photoproduct equilibrium, GTPase activity, and nucleotide uptake by G(t) are unaffected. Attenuated total reflection Fourier transform infrared spectroscopy shows that the structure of rhodopsin, metarhodopsin-II, and the metarhodopsin-II G(t) complex is also not altered. Instead, suramin dissociates G(t) from disk membranes in the dark, whereas metarhodopsin-II G(t) complexes are stable. Förster resonance energy transfer suggests a suramin-binding site near Trp(207) on the G(t alpha) subunit (K(d) approximately 0.5 microM). The kinetic analyses and the structural data are consistent with a specific perturbation by suramin of the membrane attachment site on G(t alpha). Disruption of membrane anchoring may contribute to some of the effects of suramin exerted on other G-proteins.
| S-EPMC1301888 | biostudies-other
Project description:Rhodopsin (Rho), a prototypical G-protein-coupled receptor (GPCR) in vertebrate vision, activates the G-protein transducin (GT) by catalyzing GDP-GTP exchange on its ? subunit (G?T). To elucidate the determinants of GT coupling and activation, we obtained cryo-EM structures of a fully functional, light-activated Rho-GT complex in the presence and absence of a G-protein-stabilizing nanobody. The structures illustrate how GT overcomes its low basal activity by engaging activated Rho in a conformation distinct from other GPCR-G-protein complexes. Moreover, the nanobody-free structures reveal native conformations of G-protein components and capture three distinct conformers showing the G?T helical domain (?HD) contacting the G?? subunits. These findings uncover the molecular underpinnings of G-protein activation by visual rhodopsin and shed new light on the role played by G?? during receptor-catalyzed nucleotide exchange.
| S-EPMC6707884 | biostudies-literature
Project description: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. Rhodopsin.rHDL 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 rhodopsin.rHDL 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.
| S-EPMC2651572 | biostudies-literature
Project description:G protein coupled receptors (GPCRs) can be activated by various extracellular stimuli, including hormones, peptides, odorants, neurotransmitters, nucleotides, or light. After activation, receptors interact with heterotrimeric G proteins and catalyze GDP release from the G? subunit, the rate limiting step in G protein activation, to form a high affinity nucleotide-free GPCR-G protein complex. In vivo, subsequent GTP binding reduces affinity of the G? protein for the activated receptor. In this study, we investigated the biochemical and structural characteristics of the prototypical GPCR, rhodopsin, and its signaling partner, transducin (G(t)), in bicelles to better understand the effects of membrane composition on high affinity complex formation, stability, and receptor mediated nucleotide release. Our results demonstrate that the high-affinity complex (rhodopsin-G(t)(empty)) forms more readily and has dramatically increased stability when rhodopsin is integrated into bicelles of a defined composition. We increased the half-life of functional complex to 1 week in the presence of negatively charged phospholipids. These data suggest that a membrane-like structure is an important contributor to the formation and stability of functional receptor-G protein complexes and can extend the range of studies that investigate properties of these complexes.
| S-EPMC3119548 | biostudies-other
Project description:In order to investigate the possible roles of the intracellular domains of rhodopsin in the functional coupling of the photoreceptor to transducin, different peptides that correspond to parts of the known sequence of rhodopsin were synthesized. Since we have found tht the binding of rhodopsin to the alpha subunit of transducin (alpha T) increases the susceptibility of alpha T to phosphorylation by protein kinase C-beta 1, we used this phosphorylation reaction as an initial screen for peptides that mimic the actions of rhodopsin. The results of this screen indicated that a peptide from the C-terminal tail of rhodopsin (amino acids 325-338; KNPLGDDEASTTVS-amide; designated as peptide 3) was capable of interacting with the alpha T subunit. Evidence that peptide 3 binds to alpha T at a site that overlaps the rhodopsin-binding domain was obtained from experiments showing that peptide 3 inhibited the rhodopsin-stimulated GTPase activity of alpha T and that this inhibition was overcome at high levels of rhodopsin. A potentially important outcome of the peptide 3/alpha T interaction is the facilitation of the activation of the alpha T subunit. This was first demonstrated in fluorescence experiments where the binding of peptide 3 was shown to strongly promote the enhancement of the tryptophane emission of alpha T that is elicited by the addition of NaF. Specifically, the EC50 for NaF was shifted from approximately 4 mM in the absence of peptide 3 to below 0.5 mM in the presence of peptide 3. Further verification that peptide 3 facilitated the ability of NaF to activate the alpha T subunit was obtained from experiments measuring the alpha T GDP/NaF-stimulated hydrolysis of cyclic GMP by the cyclic GMP phosphodiesterase.
| S-EPMC1138279 | biostudies-other