Structure and function in rhodopsin: effects of disulfide cross-links in the cytoplasmic face of rhodopsin on transducin activation and phosphorylation by rhodopsin kinase.

Six rhodopsin mutants containing disulfide cross-links between different cytoplasmic regions were prepared: disulfide bond 1, between Cys65 (interhelical loop I-II) and Cys316 (end of helix VII); disulfide bond 2, between Cys246 (end of helix VI) and Cys312 (end of helix VII); disulfide bond 3, between Cys139 (end of helix III) and Cys248 (end of helix VI); disulfide bond 4, between Cys139 (end of helix III) and Cys250 (end of helix VI); disulfide bond 5, between Cys135 (end of helix III) and Cys250 (end of helix VI); and disulfide bond 6, between Cys245 (end of helix VI) and Cys338 (C-terminus). The effects of local restrictions caused by the cross-links on transducin (G(T)) activation and phosphorylation by rhodopsin kinase (RK) following illumination were studied. Disulfide bond 1 showed little effect on either G(T) activation or phosphorylation by RK, suggesting that the relative motion between interhelical loop I-II and helix VII is not crucial for recognition by G(T) or by RK. In contrast, disulfide bonds 2-5 abolished both G(T) activation and phosphorylation by RK. Disulfide bond 6 resulted in enhanced G(T) activation but abolished phosphorylation by RK, suggesting the structure recognized by G(T) was stabilized in this mutant by cross-linking of the C-terminus to the cytoplasmic end of helix VI. Thus, the consequences of the disulfide cross-links depended on the location of the restriction. In particular, relative motions of helix VI, with respect to both helices III and VII upon light activation, are required for recognition of rhodopsin by both G(T) and RK. Further, the conformational changes in the cytoplasmic face that are necessary for protein-protein interactions need not be cooperative, and may be segmental.     

The transitory complex between photoexcited rhodopsin and transducin. Reciprocal interaction between the retinal site in rhodopsin and the nucleotide site in transducin

In the first step of the visual transduction cascade a photoexcited rhodopsin molecule, R*ret, binds to a GDP-carrying transducin molecule, TGDP. The R*-T interaction causes the opening of the nucleotide site in T and catalyzes the GDP/GTP exchange by allowing the release of the GDP. We have studied the influences on this R*-T transitory complex of the occupancies of the nucleotide site in T and the retinal site in rhodopsin. After elimination of the GDP released from the bound transducin, the complex, named R*ret-te (ret for retinal present, e for nucleotide site empty) remains stabilized almost indefinitely in a medium whose ionic composition is close to physiological. In this complex the bound Te retains a lasting ability to interact with GDP or GTP, and R*ret remains spectroscopically in the meta-II state, by contrast with free R*ret which decays to opsin and free retinal. Hence the R*-T interaction which opens the nucleotide site in T conversely blocks the retinal site in R*ret. Upon prolonged incubation in a low-ionic-strength medium the R*ret-Tc complex dissociates partially, but the liberated Te is then unable to rebind GDP or GTP, even in the presence of R*ret, it is probably denaturated. Upon treatment of the R*ret-Te complex by a high concentration of hydroxylamine, the retinal can be removed from the rhodopsin. The Re-Te complex remains stable and the complexed transducin keeps its capacity to bind GTP. TGTP then dissociates from Re. The liberated Re loses its capacity to interact with a new transducin. These data are integrated into a discussion of the development of the cascade. We stress that affinities, i.e. dissociation equilibrium constants, are insufficient to describe the flow of reactions triggered by one R*ret molecule. It depends on a few critical rapid binding and dissociation processes, and is practically insensitive to other slow ones, hence to the values of affinities that express only the ratio of kinetics constants. The effect of the R*-T interaction on the retinal site in rhodopsin is analogous to the effect of the binding of a G-protein on the apparent affinity of a receptor for its agonist.     

Functional interaction between bovine rhodopsin and G protein transducin

To elucidate the mechanisms of specific coupling of bovine rhodopsin with the G protein transducin (G(t)), we have constructed the bovine rhodopsin mutants whose second or third cytoplasmic loop (loop 2 or 3) was replaced with the corresponding loop of the G(o)-coupled scallop rhodopsin and investigated the difference in the activation abilities for G(t), G(o), and G(i) among these mutants and wild type. We have also prepared the Galpha(i) mutants whose C-terminal 11 or 5 amino acids were replaced with those of Galpha(t), Galpha(o), and Galpha(q) to evaluate the role of the C-terminal tail of the alpha-subunit on the specific coupling of bovine rhodopsin with G(t). Replacement of loop 2 of bovine rhodopsin with that of the scallop rhodopsin caused about a 40% loss of G(t) and G(o) activation, whereas that of loop 3 enhanced the G(o) activation four times with a 60% decrease in the G(t) activation. These results indicated that loop 3 of bovine rhodopsin is one of the regions responsible for the specific coupling with G(t). Loop 3 of bovine rhodopsin discriminates the difference of the 6-amino acid sequence (region A) at a position adjacent to the C-terminal 5 amino acids of the G protein, resulting in the different activation efficiency between G(t) and G(o). In addition, the binding of region A to loop 3 of bovine rhodopsin is essential for activation of G(t) but not G(i), even though the sequence of the region A is almost identical between Galpha(t) and Galpha(i). These results suggest that the binding of loop 3 of bovine rhodopsin to region A in Galpha(t) is one of the mechanisms of specific G(t) activation by bovine rhodopsin.     

A dynamic scaffolding mechanism for rhodopsin and transducin interaction in vertebrate vision

The early steps in vertebrate vision require fast interactions between Rh (rhodopsin) and Gt (transducin), which are classically described by a collisional coupling mechanism driven by the free diffusion of monomeric proteins on the disc membranes of rod and cone cells. Recent findings, however, point to a very low mobility for Rh and support a substantially different supramolecular organization. Moreover, Rh-G(t) interactions seem to possibly occur even prior to light stimuli, which is also difficult to reconcile with the classical scenario. We investigated the kinetics of interaction between native Rh and G(t) in different conditions by surface plasmon resonance and analysed the results in the general physiological context by employing a holistic systems modelling approach. The results from the present study point to a mechanism that is intermediate between pure collisional coupling and physical scaffolding. Such a 'dynamic scaffolding', in which prevalently dimeric Rh and G(t) interact in the dark by forming transient complexes (~25% of G(t) is precoupled to Rh), does not slow down the phototransduction cascade, but is compatible with the observed photoresponses on a broad scale of light stimuli. We conclude that Rh molecules and Rh-G(t) complexes can both absorb photons and trigger the visual cascade.     

Suramin affects coupling of rhodopsin to transducin.

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. F?rster 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.     

Mutation of the fourth cytoplasmic loop of rhodopsin affects binding of transducin and peptides derived from the carboxyl-terminal sequences of transducin alpha and gamma subunits

The role of the putative fourth cytoplasmic loop of rhodopsin in the binding and catalytic activation of the heterotrimeric G protein, transducin (G(t)), is not well defined. We developed a novel assay to measure the ability of G(t), or G(t)-derived peptides, to inhibit the photoregeneration of rhodopsin from its active metarhodopsin II state. We show that a peptide corresponding to residues 340-350 of the alpha subunit of G(t), or a cysteinyl-thioetherfarnesyl peptide corresponding to residues 50-71 of the gamma subunit of G(t), are able to interact with metarhodopsin II and inhibit its photoconversion to rhodopsin. Alteration of the amino acid sequence of either peptide, or removal of the farnesyl group from the gamma-derived peptide, prevents inhibition. Mutation of the amino-terminal region of the fourth cytoplasmic loop of rhodopsin affects interaction with G(t) (Marin, E. P., Krishna, A. G., Zvyaga T. A., Isele, J., Siebert, F., and Sakmar, T. P. (2000) J. Biol. Chem. 275, 1930-1936). Here, we provide evidence that this segment of rhodopsin interacts with the carboxyl-terminal peptide of the alpha subunit of G(t). We propose that the amino-terminal region of the fourth cytoplasmic loop of rhodopsin is part of the binding site for the carboxyl terminus of the alpha subunit of G(t) and plays a role in the regulation of betagamma subunit binding.     

Two-step mechanism of interaction of rhodopsin intermediates with the C-terminal region of the transducin alpha-subunit

Rhodopsin is a prototypical G-protein-coupled receptor that contains 11-cis-retinal as a light-absorbing chromophore. Light causes conformational changes in the protein moiety through cis-trans isomerization of the chromophore, which leads to the formation of G-protein-interacting states. Our previous studies indicated that there are two intermediate states of rhodopsin, Meta Ib and Meta II, which interact differently with retinal G-protein transducin (Gt) [S. Tachibanaki, H. Imai, T. Mizukami, T. Okada, Y. Imamoto, T. Matsuda, Y. Fukada, A. Terakita, and Y. Shichida (1997) Biochemistry 36, 14173-14180]. Here we demonstrate that the interactions of Gt with these intermediates in the absence of GTPgammaS can be mimicked by the C-terminus 11-amino acid peptide (340-350) of the alpha-subunit of Gt (Gt(alpha)), suggesting that the C-terminal region of Gt(alpha) plays important roles in the interaction with rhodopsin intermediates. Replacement of either of the two leucine residues (Leu344 and Leu349) in the peptide with alanine caused the loss of the interaction with Meta II. However, the interaction with Meta Ib was abolished only when both residues were replaced. These results indicate that rearrangement of the C-terminal region of Gt(alpha) after the binding of a rhodopsin intermediate is necessary for the GDP-GTP exchange reaction on Gt(alpha).     

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.     

Studies on the interaction between disulfiram and sheep liver cytoplasmic aldehyde dehydrogenase.

The effect of disulfiram, [1-14C]disulfiram and some other thiol reagents on the activity of cytoplasmic aldehyde dehydrogenase from sheep liver was studied. The results are consistent with a rapid covalent interaction between disulfiram and the enzyme, and inconsistent with the notion that disulfiram is a reversible competitive inhibitor of cytoplasmic aldehyde dehydrogenase. There is a non-linear relationship between loss of about 90% of the enzyme activity and amount of disulfiram added; possible reasons for this are discussed. The remaining approx. 10% of activity is relatively insensitive to disulfiram. It is found that modification of only a small number of groups (one to two) per tetrameric enzyme molecule is responsible for the observed loss of activity. The dehydrogenase activity of the enzyme is affected more severely by disulfiram than is the esterase activity. Negatively charged thiol reagents have little or no effect on cytoplasmic aldehyde dehydrogenase. 2,2'-Dithiodipyridine is an activator of the enzyme.     

Binding of rhodopsin and rhodopsin analogues to transducin,rhodopsin kinase and arrestin-1

AIM: To investigate the interaction of reconstituted rhodopsin, 9-cis-retinal-rhodopsin and 13-cis-retinal-rhodopsin with transducin, rhodopsin kinase and arrestin-1. METHODS: Rod outer segments(ROS) were isolated from bovine retinas. Following bleaching of ROS membranes with hydroxylamine, rhodopsin and rhodopsin analogues were generated with the different retinal isomers and the concentration of the reconstituted pigments was calculated from their UV/visible absorption spectra. Transducin and arrestin-1 were purified to homogeneity by column chromatography, and an enriched-fraction of rhodopsin kinase was obtainedby extracting freshly prepared ROS in the dark. The guanine nucleotide binding activity of transducin was determined by Millipore filtration using β,γ-imido-(3H)-guanosine 5'-triphosphate. Recognition of the reconstituted pigments by rhodopsin kinase was determined by autoradiography following incubation of ROS membranes containing the various regenerated pigments with partially purified rhodopsin kinase in the presence of(γ-32P) ATP. Binding of arrestin-1 to the various pigments in ROS membranes was determined by a sedimentation assay analyzed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis. RESULTS: Reconstituted rhodopsin and rhodopsin analogues containing 9-cis-retinal and 13-cis-retinal rendered an absorption spectrum showing a maximum peak at 498 nm, 486 nm and about 467 nm, respectively, in the dark; which was shifted to 380 nm, 404 nm and about 425 nm, respectively, after illumination. The percentage of reconstitution of rhodopsin and the rhodopsin analogues containing 9-cis-retinal and 13-cis-retinal was estimated to be 88%, 81% and 24%, respectively. Although only residual activation of transducin was observed in the dark when reconstituted rhodopsin and 9-cis-retinal-rhodopsin was used, the rhodopsin analogue containing the 13-cis isomer of retinal was capable of activating transducin independently of light. Moreover, only a basal amount of the reconstituted rhodopsin and 9-cis-retinal-rhodopsin was phosphorylated by rhodopsin kinase in the dark, whereas the pigment containing the 13-cis-retinal was highly phosphorylated by rhodopsin kinase even in the dark. In addition, arrestin-1 was incubated with rhodopsin, 9-cis-retinal-rhodopsin or 13-cis-retinal-rhodopsin. Experiments were performed using both phosphorylated and non-phosphorylated regenerated pigments. Basal amounts of arrestin-1 interacted with rhodopsin, 9-cis-retinal-rhodopsin and 13-cis-retinal-rhodopsin under dark and light conditions. Residual arrestin-1 was also recognized by the phosphorylated rhodopsin and phosphorylated 9-cis-retinal-rhodopsin in the dark. However, arrestin-1 was recognized by phosphorylated 13-cis-retinal-rhodopsin in the dark. As expected, all reformed pigments were capable of activating transducin and being phosphorylated by rhodopsin kinase in a lightdependent manner. Additionally, all reconstituted photolyzed and phosphorylated pigments were capable of interacting with arrestin-1. CONCLUSION: In the dark, the rhodopsin analogue containing the 13-cis isomer of retinal appears to fold in a pseudo-active conformation that mimics the active photointermediate of rhodopsin.     

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.     

Regulation of retinal transducin by C-terminal peptides of rhodopsin.

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.     

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.     

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.     

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.     

Fourier transform infrared studies of active-site-methylated rhodopsin. Implications for chromophore-protein interaction, transducin activation, and the reaction pathway.

Fourier transform infrared studies of active-site-methylated rhodopsin (ASMR) show that, as compared to unmodified rhodopsin, the photoreaction is almost unchanged up to the formation of lumirhodopsin. Especially, the deviations are much smaller than those observed for the corresponding intermediates of 13-desmethyl-rhodopsin. In metarhodopsin-I, larger alterations are present with respect to the three internal carboxyl groups. Similar deviations have been observed in meta-I of 13-desmethyl-rhodopsin. This indicates that, in agreement with our previous investigations, these carboxyl groups are located in close proximity to the chromophore. Because this latter pigment is capable, when bleached, of activating transducin, our data provide support for the earlier conclusion that deprotonation of the Schiff base is a prerequisite for transducin activation. The positions of the C = C and C - C stretching modes of the retinal suggest that the redshift observed in ASMR and its photoproducts can be explained by an increased distance of the Schiff base from the counterion(s). It is further shown that the photoreaction does not stop at metarhodopsin-I, but that this intermediate directly decays to a metarhodopsin-III-like species.     

Effect of detergents and lipids on transducin photoactivation by rhodopsin.

Rhodopsin samples, isolated using four different extraction procedures, were used to investigate the photodependent activation of the GTPase activity of transducin. A complete inhibition of transducin light-dependent GTP hydrolytic activity was observed when rhodopsin purified in the presence of 1% digitonin, following rod outer segment (ROS) solubilization with 1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate (CHAPS), was used for its activation [0 pmol of inorganic phosphate (Pi) released/min/pmol of rhodopsin]. Rhodopsin, isolated in the presence of 1% digitonin following ROS solubilization with 1% digitonin, was capable of stimulating slightly transducin GTPase activity, with an initial rate of 1 pmol of GTP hydrolyzed/min/pmol of rhodopsin. However, rhodopsin purified in the presence of 0.2% n-dodecyl-beta-D-maltoside (DM), following ROS solubilization with either 1% CHAPS or 1% DM, stimulated the enzymatic activity of transducin in a light-dependent manner, with an initial rate of 5 pmol of Pi released/min/pmol of rhodopsin. Addition of 0.075% egg phosphatidylcholine (PC) to the four different solubilized rhodopsin samples significantly enhanced light-stimulated GTP hydrolysis by transducin, with initial rates increasing from 0 to 1, 1 to 2, and 5 to 30 pmol of Pi released/min/pmol of rhodopsin, respectively. Furthermore, DM-solubilized rhodopsin induced the hydrolysis of the maximum amount of GTP by transducin at 0.0075% PC, while digitonin-solubilized rhodopsin only stimulated the GTPase activity of transducin to a similar value, when the amount of the photoreceptor protein was increased 4-fold and 0.15% PC was added to the assay mixture. These results suggest that the effective photoactivation of transducin by rhodopsin requires phospholipids, which seem to be differentially eliminated with the detergent extraction procedure utilized during ROS membranes solubilization and photopigment isolation.     

Functional interaction between the cytoplasmic leucine-zipper domain of HIV-1 gp41 and p115-RhoGEF.

The long cytoplasmic tail of the human immunodeficiency virus (HIV)-1 transmembrane protein gp41 (gp41C) is implicated in the replication and cytopathicity of HIV-1 [1]. Little is known about the specific functions of gp41C, however. HIV-1 or simian immunodeficiency virus (SIV) mutants with defective gp41C have cell-type- or species-dependent phenotypes [2] [3] [4] [5] [6]. Thus, host factors are implicated in mediating the functions of gp41C. We report here that gp41C interacted with the carboxy-terminal regulatory domain of p115-RhoGEF [7], a specific guanine nucleotide exchange factor (GEF) and activator of the RhoA GTPase, which regulates actin stress fiber formation, activation of serum response factor (SRF) and cell proliferation [8] [9]. We demonstrate that gp41C inhibited p115-mediated actin stress fiber formation and activation of SRF. An amphipathic helix region with a leucine-zipper motif in gp41C is involved in its interaction with p115. Mutations in gp41C leading to loss of interaction with p115 impaired HIV-1 replication in human T cells. These findings suggest that an important function of gp41C is to modulate the activity of p115-RhoGEF and they thus reveal a new potential anti-HIV-1 target.     

Effect of GDP on transducin interaction with cyclic nucleotide phosphodiesterase and rhodopsin from bovine retinal rods

In the presence of guanyl nucleotides and rhodopsin-containing retinal rod outer segment membranes, transducin stimulates the light-sensitive cyclic nucleotide phosphodiesterase 5.5-7 times. The activation constant (Ka) for GTP and Gpp(NH)p is 0.25 microM, that for GDP and GDP beta S is 14 and 110 microM, respectively. GDP purified from other nucleotide contaminations at concentrations up to 1 mM does not stimulate phosphodiesterase but binds to transducin and inhibits the Gpp(NH)p-dependent activation of phosphodiesterase. The mode of transducin interaction with bleached rhodopsin also depends on the nature of the bound guanyl nucleotide: in the presence of GDP rhodopsin-containing membranes bind 70-100% of transducin, whereas in the presence of Gpp(NH)p the membranes bind only 13% of the protein. The experimental results suggest that GDP and GTP convert transducin into two different functional states, i.e., the transducin X GTP complex binds to phosphodiesterase causing its stimulation, while the transducin X GDP complex is predominantly bound to rhodopsin.