Bacteriorhodopsin chimeras containing the third cytoplasmic loop of bovine rhodopsin activate transducin for GTP/GDP exchange

The mechanisms by which G-protein-coupled receptors (GPCRs) activate G-proteins are not well understood due to the lack of atomic structures of GPCRs in an active form or in GPCR/G-protein complexes. For study of GPCR/G-protein interactions, we have generated a series of chimeras by replacing the third cytoplasmic loop of a scaffold protein bacteriorhodopsin (bR) with various lengths of cytoplasmic loop 3 of bovine rhodopsin (Rh), and one such chimera containing loop 3 of the human beta2-adrenergic receptor. The chimeras expressed in the archaeon Halobacterium salinarum formed purple membrane lattices thus facilitating robust protein purification. Retinal was correctly incorporated into the chimeras, as determined by spectrophotometry. A 2D crystal (lattice) was evidenced by circular dichroism analysis, and proper organization of homotrimers formed by the bR/Rh loop 3 chimera Rh3C was clearly illustrated by atomic force microscopy. Most interestingly, Rh3C (and Rh3G to a lesser extent) was functional in activation of GTPgamma35S/GDP exchange of the transducin alpha subunit (Galphat) at a level 3.5-fold higher than the basal exchange. This activation was inhibited by GDP and by a high-affinity peptide analog of the Galphat C terminus, indicating specificity in the exchange reaction. Furthermore, a specific physical interaction between the chimera Rh3C loop 3 and the Galphat C terminus was demonstrated by cocentrifugation of transducin with Rh3C. This Galphat-activating bR/Rh chimera is highly likely to be a useful tool for studying GPCR/G-protein interactions.     

The receptor-bound "empty pocket" state of the heterotrimeric G-protein alpha-subunit is conformationally dynamic

Heterotrimeric G-protein activation by a G-protein-coupled receptor (GPCR) requires the propagation of structural signals from the receptor-interacting surfaces to the guanine nucleotide-binding pocket. To probe conformational changes in the G-protein alpha-subunit (G(alpha)) associated with activated GPCR (R*) interactions and guanine nucleotide exchange, high-resolution solution NMR methods are being applied in studying signaling of the G-protein, transducin, by light-activated rhodopsin. Using these methods, we recently demonstrated that an isotope-labeled G(alpha) reconstituted heterotrimer forms functional complexes under NMR experimental conditions with light-activated, detergent-solubilized rhodopsin and a soluble mimic of R*, both of which trigger guanine nucleotide exchange [Ridge, K. D., et al. (2006) J. Biol. Chem. 281, 7635-7648]. Here, it is shown that both light-activated rhodopsin and the soluble mimic of R form trapped intermediate complexes with a GDP-released "empty pocket" state of the heterotrimer in the absence of GTP (or GTPgammaS). In contrast to guanine nucleotide-bound forms of G(alpha), the NMR spectra of the GDP-released, R-bound empty pocket state of G(alpha) display severe line broadening suggestive of a dynamic intermediate state. Interestingly, the conformation of a GDP-depleted, Mg(2+)-bound state of G(alpha) generated in a manner independent of R* does not exhibit a similar degree of line broadening but rather appears structurally similar to the GDP/Mg(2+)-bound form of the protein. Taken together, these results suggest that R*-mediated changes in the receptor-interacting regions of G(alpha), and not the absence of bound guanine nucleotide, are the predominant factors which dictate G(alpha) conformation and dynamics in this R*-bound state of the heterotrimer.     

Differential expression and interaction with the visual G-protein transducin of centrin isoforms in mammalian photoreceptor cells

Photoisomerization of rhodopsin activates a heterotrimeric G-protein cascade leading to closure of cGMP-gated channels and hyperpolarization of photoreceptor cells. Massive translocation of the visual G-protein transducin, Gt, between subcellular compartments contributes to long term adaptation of photoreceptor cells. Ca(2+)-triggered assembly of a centrin-transducin complex in the connecting cilium of photoreceptor cells may regulate these transducin translocations. Here we demonstrate expression of all four known, closely related centrin isoforms in the mammalian retina. Interaction assays revealed binding potential of the four centrin isoforms to Gtbetagamma heterodimers. High affinity binding to Gtbetagamma and subcellular localization of the centrin isoforms Cen1 and Cen2 in the connecting cilium indicated that these isoforms contribute to the centrin-transducin complex and potentially participate in the regulation of transducin translocation through the photoreceptor cilium. Binding of Cen2 and Cen4 to Gbetagamma of non-visual G-proteins may additionally regulate G-proteins involved in centrosome and basal body functions.     

Structural studies of metarhodopsin II,the activated form of the G-protein coupled receptor,rhodopsin

The structural changes that accompany activation of a G-protein coupled receptor (GPCR) are not well understood. To better understand the activation of rhodopsin, the GPCR responsible for visual transduction, we report studies on the three-dimensional structure for the activated state of this receptor, metarhodopsin II. Differences between the three-dimensional structure of ground state rhodopsin and metarhodopsin II, particularly in the cytoplasmic face of the receptor, suggest how the receptor is activated to couple with transducin. In particular, activation opens a groove on the surface of the receptor that could bind the N-terminal helix of the G protein, transducin alpha.     

Hydrophobic amino acids at the cytoplasmic ends of helices 3 and 6 of rhodopsin conjointly modulate transducin activation

Rhodopsin is the visual photoreceptor responsible for dim light vision. This receptor is located in the rod cell of the retina and is a prototypical member of the G-protein-coupled receptor superfamily. The structural details underlying the molecular recognition event in transducin activation by photoactivated rhodopsin are of key interest to unravel the molecular mechanism of signal transduction in the retina. We constructed and expressed rhodopsin mutants in the second and third cytoplasmic domains of rhodopsin – where the natural amino acids were substituted by the human M3 acetylcholine muscarinic receptor homologous residues – in order to determine their potential involvement in G-protein recognition. These mutants showed normal chromophore formation and a similar photobleaching behavior than WT rhodopsin, but decreased thermal stability in the dark state. The single mutant V138^3.53 and the multiple mutant containing V227^5.62 and a combination of mutations at the cytoplasmic end of transmembrane helix 6 caused a reduction in transducin activation upon rhodopsin photoactivation. Furthermore, combination of mutants at the second and third cytoplasmic domains revealed a cooperative role, and partially restored transducin activation. The results indicate that hydrophobic interactions by V138^3.53, V227^5.62, V250^6.33, V254^6.37 and I255^6.38 are critical for receptor activation and/or efficient rhodopsin–transducin interaction.     

Chimeric microbial rhodopsins containing the third cytoplasmic loop of bovine rhodopsin

G-protein-coupled receptors transmit stimuli (light, taste, hormone, neurotransmitter, etc.) to the intracellular signaling systems, and rhodopsin (Rh) is the most-studied G-protein-coupled receptor. Rh possesses an 11-cis retinal as the chromophore, and 11-cis to all-trans photoisomerization leads to the protein structural changes in the cytoplasmic loops to activate G-protein. Microbial rhodopsins are similar heptahelical membrane proteins that function as bacterial sensors, light-driven ion-pumps, or light-gated channels. Microbial rhodopsins possess an all-trans retinal, and all-trans to 13-cis photoisomerization triggers protein structural changes for each function. Despite these similarities, there is no sequence homology between visual and microbial rhodopsins, and microbial rhodopsins do not activate G-proteins. However, it was reported that bacteriorhodopsin (BR) chimeras containing the third cytoplasmic loop of bovine Rh are able to activate G-protein, suggesting a common mechanism of protein structural changes. Here we design chimeric proteins for Natronomonas pharaonis sensory rhodopsin II (SRII, also called pharaonis phoborhodopsin), which has a two-orders-of-magnitude slower photocycle than BR. Light-dependent transducin activation was observed for most of the nine SRII chimeras containing the third cytoplasmic loop of bovine Rh (from Y223, G224, Q225 to T251, R252, and M253), but the activation level was 30,000–140,000 times lower than that of bovine Rh. The BR chimera, BR/Rh223-253, activates a G-protein transducin, whereas the activation level was 37,000 times lower than that of bovine Rh. We interpret the low activation by the chimeric proteins as reasonable, because bovine Rh must have been optimized for activating a G-protein transducin during its evolution. On the other hand, similar activation level of the SRII and BR chimeras suggests that the lifetime of the M intermediates is not the simple determinant of activation, because SRII chimeras have two-orders-of-magnitude's slower photocycle than the BR chimera. Activation mechanism of visual and microbial rhodopsins is discussed on the basis of these results.     

Rhodopsin activation exposes a key hydrophobic binding site for the transducin alpha-subunit C terminus

Conformational changes enable the photoreceptor rhodopsin to couple with and activate the G-protein transducin. Here we demonstrate a key interaction between these proteins occurs between the C terminus of the transducin alpha-subunit (G(Talpha)) and a hydrophobic cleft in the rhodopsin cytoplasmic face exposed during receptor activation. We mapped this interaction by labeling rhodopsin mutants with the fluorescent probe bimane and then assessed how binding of a peptide analogue of the G(Talpha) C terminus (containing a tryptophan quenching group) affected their fluorescence. From these and other assays, we conclude that the G(Talpha) C-terminal tail binds to the inner face of helix 6 in a retinal-linked manner. Further, we find that a "hydrophobic patch" comprising key residues in the exposed cleft is required for transducin binding/activation because it enhances the binding affinity for the G(Talpha) C-terminal tail, contributing up to 3 kcal/mol for this interaction. We speculate the hydrophobic interactions identified here may be important in other GPCR signaling systems, and our Trp/bimane fluorescence methodology may be generally useful for mapping sites of protein-protein interaction.     

Efficient coupling of transducin to monomeric rhodopsin in a phospholipid bilayer

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.     

A Monte Carlo study of the dynamics of G-protein activation.

To link quantitatively the cell surface binding of ligand to receptor with the production of cellular responses, it may be necessary to explore early events in signal transduction such as G-protein activation. Two different model frameworks relating receptor/ligand binding to G-protein activation are examined. In the first framework, a simple ordinary differential equation model is used to describe receptor/ligand binding and G-protein activation. In the second framework, the events leading to G-protein activation are simulated using a dynamic Monte Carlo model. In both models, reactions between ligand-bound receptors and G-proteins are assumed to be diffusion-limited. The Monte Carlo model predicts two regimes of G-protein activation, depending upon whether the lifetime of a receptor/ligand complex is long or short compared with the time needed for diffusional encounters of complexes and G-proteins. When the lifetime of a complex is relatively short compared with the diffusion time, the movement of ligand among free receptors by binding and unbinding ("switching") significantly enhances G-protein activation. Receptor antagonists dramatically reduce G-protein activation and, thus, signal transduction in this case, and significant clustering of active G-proteins near receptor/ligand complexes results. The simple ordinary differential equation model poorly predicts G-protein activation for this situation. In the alternative case, when diffusion is relatively fast, ligand movement among receptors is less important and the simple ordinary differential equation model and Monte Carlo model results are similar. In this case, there is little clustering of active G-proteins near receptor/ligand complexes. Results also indicate that as the GTPase activity of the alpha-subunit decreases, the steady-state level of alpha-GTP increases, although temporal sensitivity is compromised.     

Multiple mechanisms involving protein phosphorylation are linked to desensitization of muscarinic receptors

Agonists induce phosphorylation of m2 muscarinic receptors (mAChR) in several cell types. This phosphorylation correlates with desensitization. The mechanisms underlying mAChR phosphorylation have been investigated using several in vitro approaches. Protein kinase C phosphorylated the purified and reconstituted m2 mAChR to a stoichiometry of approximately 5 mols P/mol receptor; this phosphorylation resulted in the decreased ability of receptors to activate G-proteins. Although the phosphorylation by PKC was not modulated by agonist binding to the mAChR, heterotrimeric G-proteins were able to completely block the PKC-mediated effects. If significant receptor/G-protein coupling occurs in vivo, agonists would be required to promote dissociation of the G-proteins from the receptors and reveal the phosphorylation sites for PKC. Members of the G-protein coupled receptor kinase (GRK) family also phosphorylated the purified and reconstituted m2 mAChR. In contrast to PKC, the GRKs phosphorylated the m2 mAChR strictly in an agonist-dependent manner. GRK mediated phosphorylation perturbed receptor/G-protein coupling. In addition, phosphorylation allowed for arrestin binding to the m2 mAChR which should further contribute to desensitization. Using a new strategy that does not require purification and reconstitution of receptors for GRK studies, the m3 mAChR were revealed as substrates for the GRKs. For both the m2 and m3 receptor subtypes, the most effective kinases were GRK 2 and 3. Phosphorylation of the receptors by these enzymes was stimulated by low concentrations of G-proteins and by membrane phospholipids. Thus, multiple mechanisms involving protein phosphorylation appear to contribute to the overall process of mAChR desensitization.     

Characterization of rhodopsin kinase purified from bovine rod outer segments

Rhodopsin kinase was purified by sequential chromatography on DEAE-cellulose and blue-Sepharose. Kinase activity co-purified with a 62-kDa polypeptide, which bound light-dependently in the absence of ATP to purified vesicle-reconstituted rhodopsin. Purified rhodopsin kinase is free of any detectable arrestin or the retinal G-protein. Rhodopsin kinase is autophosphorylated on serine residues which is unaffected by the presence of bleached rhodopsin and results in a transition in molecular mass to 64 kDa. Autophosphorylation of the kinase did not appear to alter the overall rate of rhodopsin phosphorylation or the apparent KM (0.6 microM) for purified reconstituted rhodopsin. Peptides corresponding to sequences within opsin loops 3-4 and 5-6 and the COOH terminus inhibited kinase phosphorylation of bleached rhodopsin, suggesting at least three potential sites to account for the stable high affinity binding of rhodopsin kinase to the bleached photoreceptor molecule that are at least in part distinct from the substrate sites for phosphorylation. These sequences are similar to those proposed for receptor recognition of G-proteins and indicate that the domains involved in light-dependent binding of rhodopsin kinase and retinal G-protein are similar or overlapping.     

Studies on the structure of the G-protein-coupled receptor rhodopsin including the putative G-protein binding site in unactivated and activated forms

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.     

Interaction of rhodopsin with the G-protein,transducin

Rhodopsin, upon activation by light, transduces the photon signal by activation of the G-protein, transducin. The well-studied rhodopsin/transducin system serves as a model for the understanding of signal transduction by the large class of G-protein-coupled receptors. The interactive form of rhodopsin, R*, is conformationally similar or identical to rhodopsin's photolysis intermediate Metarhodopsin II (MII). Formation of MII requires deprotonation of rhodopsin's protonated Schiff base which appears to facilitate some opening of the rhodopsin structure. This allows a change in conformation at rhodopsin's cytoplasmic surface that provides binding sites for transducin. Rhodopsin's 2nd, 3rd and putative 4th cytoplasmic loops bind transducin at sites including transducin's 5 kDa carboxyl-terminal region. Site-specific mutagenesis of rhodopsin is being used to distinguish sites on rhodopsin's surface that are important in binding transducin from those that function in activating transducin. These observations are consistent with and extend studies on the action of other G-protein-coupled receptors and their interactions with their respective G proteins.     

Artificial membrane-like environments for in vitro studies of purified G-protein coupled receptors

Functional reconstitution of transmembrane proteins remains a significant barrier to their biochemical, biophysical, and structural characterization. Studies of seven-transmembrane G-protein coupled receptors (GPCRs) in vitro are particularly challenging because, ideally, they require access to the receptor on both sides of the membrane as well as within the plane of the membrane. However, understanding the structure and function of these receptors at the molecular level within a native-like environment will have a large impact both on basic knowledge of cell signaling and on pharmacological research. The goal of this article is to review the main classes of membrane mimics that have been, or could be, used for functional reconstitution of GPCRs. These include the use of micelles, bicelles, lipid vesicles, nanodiscs, lipidic cubic phases, and planar lipid membranes. Each of these approaches is evaluated with respect to its fundamental advantages and limitations and its applications in the field of GPCR research. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Coupling efficiency of rhodopsin and transducin in bicelles

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 Gα 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 Gα 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.     

Transducin activation by nanoscale lipid bilayers containing one and two rhodopsins

Nanodiscs are nanometer scale planar membranes of controlled size that are rendered soluble in aqueous solution via an encircling amphipathic membrane scaffold protein "belt" (Bayburt, T. H., Grinkova, Y. V., and Sligar, S. G. (2002) Nano. Lett. 2, 853-856). Integral membrane proteins can be self-assembled into the Nanodisc bilayer with defined stoichiometry, which allows an unprecedented opportunity to investigate the nature of the oligomerization state of a G-protein-coupled receptor and its coupling to heterotrimeric G-proteins. We generated Nanodiscs having one and two rhodopsins present in the 10-nm-diameter lipid bilayer domain. Efficient transducin activation and isolation of a high affinity transducin-metarhodopsin II complex was demonstrated for a monodisperse and monomeric receptor. A population of Nanodiscs containing two rhodopsins was generated using an increased ratio of receptor to membrane scaffold protein in the self-assembly mixture. The two-rhodopsin population was isolated and purified by density gradient centrifugation. Interestingly, in this case, only one of the two receptors present in the Nanodisc was able to form a stable metarhodopsin II-G-protein complex. Thus there is clear evidence that a monomeric rhodopsin is capable of full coupling to transducin. Importantly, presumably due to steric interactions, it appears that only a single receptor in the Nanodiscs containing two rhodopsins can interact with G-protein. These results have important implications for the stoichiometry of receptor-G-protein coupling and cross talk in signaling pathways.     

Rhodopsin and the retinal G-protein distinguish among G-protein beta gamma subunit forms

The beta gamma subunits of G-proteins are composed of closely related beta 35 and beta 36 subunits tightly associated with diverse 6-10 kDa gamma subunits. We have developed a reconstitution assay using rhodopsin-catalyzed guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding to resolved alpha subunit of the retinal G-protein transducin (Gt alpha) to quantitate the activity of beta gamma proteins. Rhodopsin facilitates the exchange of GTP gamma S for GDP bound to Gt alpha beta gamma with a 60-fold higher apparent affinity than for Gt alpha alone. At limiting rhodopsin, G-protein-derived beta gamma subunits catalytically enhance the rate of GTP gamma S binding to resolved Gt alpha. The isolated beta gamma subunit of retinal G-protein (beta 1, gamma 1 genes) facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha in a concentration-dependent manner (K0.5 = 254 +/- 21 nM). Purified human placental beta 35 gamma, composed of beta 2 gene product and gamma-placenta protein (Evans, T., Fawzi, A., Fraser, E.D., Brown, L.M., and Northup, J.K. (1987) J. Biol. Chem. 262, 176-181), substitutes for Gt beta gamma reconstitution of rhodopsin with Gt alpha. However, human placental beta 35 gamma facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha with a higher apparent affinity than Gt beta gamma (K0.5 = 76 +/- 54 nM). As an alternative assay for these interactions, we have examined pertussis toxin-catalyzed ADP-ribosylation of the Gt alpha subunit which is markedly enhanced in rate by beta gamma subunits. Quantitative analyses of rates of pertussis modification reveal no differences in apparent affinity between Gt beta gamma and human placental beta 35 gamma (K0.5 values of 49 +/- 29 and 70 +/- 24 nM, respectively). Thus, the Gt alpha subunit alone does not distinguish among the beta gamma subunit forms. These results clearly show a high degree of functional homology among the beta 35 and beta 36 subunits of G-proteins for interaction with Gt alpha and rhodopsin, and establish a simple functional assay for the beta gamma subunits of G-proteins. Our data also suggest a specificity of recognition of beta gamma subunit forms which is dependent both on Gt alpha and rhodopsin. These results may indicate that the recently uncovered diversity in the expression of beta gamma subunit forms may complement the diversity of G alpha subunits in providing for specific receptor recognition of G-proteins.     

Subtype differences in pre-coupling of muscarinic acetylcholine receptors

Based on the kinetics of interaction between a receptor and G-protein, a myriad of possibilities may result. Two extreme cases are represented by: 1/Collision coupling, where an agonist binds to the free receptor and then the agonist-receptor complex "collides" with the free G-protein. 2/Pre-coupling, where stable receptor/G-protein complexes exist in the absence of agonist. Pre-coupling plays an important role in the kinetics of signal transduction. Odd-numbered muscarinic acetylcholine receptors preferentially couple to G(q/11), while even-numbered receptors prefer coupling to G(i/o). We analyzed the coupling status of the various subtypes of muscarinic receptors with preferential and non-preferential G-proteins. The magnitude of receptor-G-protein coupling was determined by the proportion of receptors existing in the agonist high-affinity binding conformation. Antibodies directed against the C-terminus of the α-subunits of the individual G-proteins were used to interfere with receptor-G-protein coupling. Effects of mutations and expression level on receptor-G-protein coupling were also investigated. Tested agonists displayed biphasic competition curves with the antagonist [(3)H]-N-methylscopolamine. Antibodies directed against the C-terminus of the α-subunits of the preferential G-protein decreased the proportion of high-affinity sites, and mutations at the receptor-G-protein interface abolished agonist high-affinity binding. In contrast, mutations that prevent receptor activation had no effect. Expression level of preferential G-proteins had no effect on pre-coupling to non-preferential G-proteins. Our data show that all subtypes of muscarinic receptors pre-couple with their preferential classes of G-proteins, but only M(1) and M(3) receptors also pre-couple with non-preferential G(i/o) G-proteins. Pre-coupling is not dependent on agonist efficacy nor on receptor activation. The ultimate mode of coupling is therefore dictated by a combination of the receptor subtype and the class of G-protein.     

GPCR-induced dissociation of G-protein subunits in early stage signal transduction

G-protein coupled receptors (GPCRs) form a ternary complex of agonist, receptor and G-proteins during primary signal transduction at the cell membrane. Downstream signalling is thought to be preceded by the process of dissociation of Galpha and Gbetagamma subunits, thus exposing new surfaces to interact with downstream effectors. We demonstrate here for the first time, the dissociation of heterotrimeric G-protein subunits (i.e., Galpha and Gbetagamma) following agonist-induced GPCR (alpha(2A)-adrenergic receptor; alpha(2A)-AR) activation in a cell-free assay system. alpha(2A)-AR membranes were reconstituted with the G-proteins (+/-hexahistidine-tagged) Galpha(i1) and Gbeta1gamma2 and functional signalling was determined following activation of the reconstituted receptor:G-protein complex with the potent agonist UK-14304, and [35S]GTPgammaS. In the presence of Ni(2+)-coated agarose beads, the activated his-tagged Galpha(i1)his-[35S]GTPgammaS complex was captured on the Ni(2+)-presenting surface. When his-tagged Gbeta1gamma2 (Gbeta1gamma2his) was used with Galpha(i1), the [35S]GTPgammaS-bound Galpha(i1) was not present on the Ni(2+)-coated beads, but rather, it was separated from the beta1gamma2(his)-beads, demonstrating receptor-induced dissociation of Galpha and Gbetagamma subunits. Treatment of the reconstituted alpha(2A)-AR membranes containing Gbeta1gamma2his:Galpha(i1) with imidazole confirmed the specificity for the Ni2+:G-protein surface dissociation of Galpha(i1) from Gbeta1gamma2his. These data demonstrate for the first time, the complete dissociation of the G-protein subunits and extend observations on the role of G-proteins in the assembly and disassembly of the ternary complex in the primary events of GPCR signalling.     

Modeling of the complex between transducin and photoactivated rhodopsin, a prototypical G-protein-coupled receptor

Obtaining a reliable 3D model for the complex formed by photoactivated rhodopsin (R*) and its G-protein, transducin (Gtalphabetagamma), would significantly benefit the entire field of structural biology of G-protein-coupled receptors (GPCRs). In this study, we have performed extensive configurational sampling for the isolated C-terminal fragment of the alpha-subunit of transducin, Gtalpha 340-350, within cavities of photoactivated rhodopsin formed by different energetically feasible conformations of the intracellular loops. Our results suggested a new 3D model of the rhodopsin-transducin complex that fully satisfied all available experimental data on site-directed mutagenesis of rhodopsin and Gtalphabetagamma as well as data from disulfide-linking experiments. Importantly, the experimental data were not used as a priori constraints in model building. We performed a thorough comparison of existing computational models of the rhodopsin-transducin complex with each other and with current experimental data. It was found that different models suggest interactions with different molecules in the rhodopsin oligomer, that providing valuable guidance in design of specific novel experimental studies of the R*-Gtalphabetagamma complex. Finally, we demonstrated that the isolated Gtalpha 340-350 fragment does not necessarily bind rhodopsin in the same binding mode as the same segment in intact Gtalpha.