Sulfhydryl group modification of photoreceptor G-protein prevents its light-induced binding to rhodopsin

The effect of sulfhydryl modification on the light-induced interaction between rhodopsin and the peripheral GTP-binding protein of the photoreceptor membrane (G-protein) has been investigated by time-resolved near-infrared light-scattering and polyacrylamide gel electrophoresis. It has been found that the modification of rhodopsin with the alkylating agent N-ethylmaleimide (NEM) does not affect its light-induced interaction with the G-protein. Modification of G-protein with NEM or other sulfhydryl agents prevents any light-induced binding to rhodopsin. Dark-association of G to the membrane as well as the light-induced complex with rhodopsin (once formed) is insensitive to NEM.     

Mechanism of action of monoclonal antibodies that block the light activation of the guanyl nucleotide-binding protein, transducin

Seven monoclonal antibodies to the alpha subunit (G alpha) of the frog photoreceptor guanyl nucleotide-binding protein (transducin or G-protein) have been characterized as to their effect on G-protein function, and this has been correlated in the accompanying paper (Deretic, D., and Hamm, H. E. (1987) J. Biol. Chem. 262, 10839-10847) with the antibody-binding sites on G alpha tryptic fragments. Antibodies 4A, 7A, 7B, 7C, and 7D are members of a class of antibodies that block G-protein activation by light and therefore also block activation of the cGMP phosphodiesterase. All these blocking antibodies also block the interaction of G-protein with rhodopsin as measured by the light-scattering "binding signal," and as measured by the stabilization of meta-rhodopsin II by bound G-protein (extra-meta-rhodopsin II). The antibodies (or Fab fragments) also solubilize G alpha beta gamma from the membrane in the dark under isosmotic conditions and thus interfere with G alpha interaction with the membrane. Antibody 4A also blocks the extra-meta-rhodopsin II generated by G-protein-rhodopsin interaction in detergent solubilized membranes. Thus, even in the absence of phospholipids, antibody 4A blocks G-protein-rhodopsin interaction. Therefore, we suggest that the antibodies recognize a region of G alpha involved with binding to rhodopsin. An alternative hypothesis is that this antigenic site is a region of interaction between the alpha and beta gamma subunits, disruption of this interaction leading to removal of both the alpha and beta gamma subunits from the membrane and blocking interaction with rhodopsin. This does not seem to be the case because the antibodies immunoprecipitate the alpha beta gamma complex, and not just the alpha subunit. Other antibodies, 4C and 4H, do not block phosphodiesterase activation, the light-scattering signal, extra-meta-rhodopsin II formation, or interaction with the membrane in the dark and therefore recognize other sites on G alpha.     

Light-induced conformational change in rhodopsin detected by modification of G-protein binding, GTP gamma S binding and cGMP phosphodiesterase activation

CNBr treatment of rod outer segments was performed in dark and in light conditions. With the subsequent modified rhodopsin and opsin the cGMP phosphodiesterase activation system was reconstituted. The recombination systems exhibited greatly reduced G-protein binding, GTP gamma S binding and cGMP phosphodiesterase activation. The reduction in activity of these three steps of the PDE activation cascade is most significant with modified opsin and is shown to be due to its inability to bind the G alpha subunit. The correlation between the localization of CNBr cleavage in dark and light conditions and these results is strongly indicative that a light-induced conformational change occurs in two extradiscal regions of rhodopsin.     

Biochemical aspects of the visual process. XXVIII. Classification of sulfhydryl groups in phodopsin and other photoreceptor membrane proteins.

Reaction of isolated bovine rod outer segment membrane with radioactive N-ethylmaleimide, both in the presence and absence of 1% dodecyl sulfate followed by dodecyl sulfate-polyacrylamide gel electrophoresis, shows that six sulfhydryl groups (96% of total sulfhydryl in this membrane) are located on the rhodopsin molecule. On the basis of their reactivity towards rho-chloromercuribenzoate and rho-chloromercuribenzene sulfonate in suspensions of outer segment membranes, the sulfhydryl groups of rhodopsin can be divided into three pairs. One pair is rapidly modified, both in light and darkness. This modification does not impair the recombination capacity of opsin with 11-cis retinaldehyde under regeneration of rhodopsin. A second pair is modified upon prolonged interaction with the rho-chloromercuriderivatives in darkness. Modification of this pair leaves the typical rhodopsin absorbance at 500 nm intact, but a proportional loss of recombination capacity does occur. The third pair is only modified after illumination and isprobably located in the vicinity of the chromophoric center. The differences between these results and those obtained by modification with dithiobis-(2-nitrobenzoic acid) or N-ethylmaleimide in suspension, where even upon prolonged exposure to light as well as in darkness only two sulfhydryl groups of rhodopsin are modified, is explained by the detergent-like character of the rho-chloromercuri-derivatives.     

Contribution of the guanosinetriphosphatase activity of G-protein to termination of light-activated guanosine cyclic 3',5'-phosphate hydrolysis in retinal rod outer segments

Light activation of GTP binding to G-protein and its eventual hydrolysis are hypothesized to lead to activation and inactivation of cGMP phosphodiesterase (PDE) in vertebrate rod disk membranes (RDM). However, the reported GTPase rate of 3 per minute is too slow to account for the observed rapid inactivation of PDE. Our investigations on GTPase activity showed that RDM isolated in the dark have considerable dark GTPase activity, which is enhanced by light. In dark and light, the enzyme exhibits biphasic substrate dependence with two Km's for GTP of 2-3 and 40-80 microM at 22 degrees C and less than 1 and 10-25 microM at 37 degrees C. The Km's were not influenced by light. On the basis of G-protein content of the RDM, the Vmax's for the two activities at 37 degrees C in light are 4-5 and 20-30 GTPs hydrolyzed per minute per G-protein. RDM washed free of soluble and peripheral proteins do not have measurable GTPase activity in the dark or light. Purified G-protein alone also did not turn over GTP, apparently because bleached rhodopsin is required for it to bind GTP. Reconstitution of washed membranes with purified G-protein restores both the low- and high-Km GTPase activities. Inactivation of G-protein as measured by PDE turnoff and dissociation signal recovery is found to be faster at higher than lower [GTP], consistent with the observation that the higher GTPase activity associated with the higher Km alos resides in the G-protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Arrestin and its splice variant Arr1-370A (p44). Mechanism and biological role of their interaction with rhodopsin

Deactivation of G-protein-coupled receptors relies on a timely blockade by arrestin. However, under dim light conditions, virtually all arrestin is in the rod inner segment, and the splice variant p(44) (Arr(1-370A)) is the stop protein responsible for receptor deactivation. Using size exclusion chromatography and biophysical assays for membrane-bound protein-protein interaction, membrane binding, and G-protein activation, we have investigated the interactions of Arr(1-370A) and proteolytically truncated Arr(3-367) with rhodopsin. We find that these short arrestins do not only interact with the phosphorylated active receptor but also with inactive phosphorylated rhodopsin or opsin in membranes or solution. Because of the latter interaction they are not soluble (like arrestin) but membrane-bound in the dark. Upon photoexcitation, Arr(3-367) and Arr(1-370A) interact with prephosphorylated rhodopsin faster than arrestin and start to quench G(t) activation on a subsecond time scale. The data indicate that in the course of rhodopsin deactivation, Arr(1-370A) is handed over from inactive to active phosphorylated rhodopsin. This mechanism could provide a new aspect of receptor shutoff in the single photon operating range of the rod cell.     

Rhodopsin-G-protein interactions monitored by resonance energy transfer

Resonance energy transfer measurements were implemented to monitor the specific interactions between G-protein and rhodopsin in phospholipid vesicles reconstituted with the purified proteins. Fluorescently labeled G-protein was extracted from bleached rod outer segments (ROS) reacted with several sulfhydryl reagents: N-(1-pyrenyl)maleimide (P), monobromobimane (B), 7-(diethylamino)-3-(4-maleimidylphenyl)-4-methylcoumarin (C), and N-(4-anilino-1-naphthyl)maleimide (A). Limited labeling of ROS, resulting in the modification of less than a single -SH residue per G-protein molecule and less than 0.2 residue per rhodopsin, did not impair the specific in situ interactions between rhodopsin and G-protein. This was demonstrated by preservation of their light-activated tight association and Gpp(NH)p binding and their fast dissociation with excess GTP. The distribution of fluorescent label among the three subunits of G-protein revealed a highly reactive -SH group in the gamma subunit accessible to labeling when G-protein was bound specifically to bleached rhodopsin. Recombination of purified fluorescent derivatives of G-protein with purified rhodopsin reconstituted in lipid vesicles restored the light-activated Gpp(NH)p binding to a level comparable to that measured with unlabeled G-protein. Similar observations were obtained with ROS depleted of peripheral proteins. Likewise, modification of up to two -SH groups per rhodopsin molecule with the fluorescent reagents did not affect the functional recombination of G-protein with rhodopsin in reconstituted lipid vesicles or in depleted ROS. Interactions between rhodopsin and G-protein were monitored by resonance energy transfer measurements, with the following fluorescent conjugates as donor/acceptor couples: P-rhodopsin/C-G-protein, P-rhodopsin/B-G-protein, and P-G-protein/C-rhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibody and peptide probes of interactions between the SH1-SH2 region of myosin subfragment 1 and actin's N-terminus.

The negatively charged residues in the N-terminus of actin and the 697-707 region on myosin subfragment 1 (S-1), containing the reactive cysteines SH1 and SH2, are known to be important for actin-activated myosin ATPase activity. The relationship between these two sites was first examined by monitoring the rates of SH1 and SH2 modification with N-ethylmaleimide in the presence of actin and, secondly, by testing for direct binding of SH1 peptides to the N-terminal segment on actin. While actin alone protected SH1 from N-ethylmaleimide modification, this effect was abolished by an antibody against the seven N-terminal amino acids on actin, F(ab)(1-7), and was greatly reduced when the charge of acidic residues at actin's N-terminus was altered by carbodiimide coupling of ethylenediamine. Neither F(ab)(1-7) nor ethylenediamine treatment reversed the effect of F-actin on SH2 reactivity in SH1-modified S-1. These results show a communication between the SH1 region on S-1 and actin's N-terminus in the acto-S-1 complex. To test whether such a communication involves the binding of the SH1 site on S-1 to the N-terminal segment of actin, the SH1 peptide IRICRKG-NH2(4+) was used. Cosedimentation experiments revealed the binding of three to six peptides per actin monomer. Peptide binding to actin was affected slightly, if at all, by F(ab)(1-7). The antibody also did not change the polymerization of G-actin by the peptides. The peptides caused a small reduction in the binding of S-1 to actin and did not change the binding of F(ab)(1-7).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of photoexcited rhodopsin in retinal rods: the roles of rhodopsin kinase and 48-kDa protein (arrestin)

The inactivation of excited rhodopsin in the presence of ATP, rhodopsin kinase, and/or arrestin has been studied from its effect on the two subsequent steps in the light-induced enzymatic cascade: metarhodopsin II catalyzed activation of G-protein and G-protein-dependent activation of cGMP phosphodiesterase. The inactivation of G-protein (from light-scattering measurements) and that of phosphodiesterase (from measurements of cGMP hydrolysis) have been studied and compared in reconstituted systems containing various combinations of the proteins involved (rhodopsin, G-protein, phosphodiesterase, kinase, and arrestin). Our results show that rhodopsin kinase alone can terminate the activation of G-protein and that arrestin speeds up the process at a relative concentration similar to that reported in the rod (half-maximal effect at 50 nM for 4.4 microM rhodopsin). Measurements of rhodopsin phosphorylation under identical conditions show that in the presence of arrestin total metarhodopsin II inactivation is achieved when only 0.5-1.4 phosphates are bound per bleached rhodopsin, whereas in the absence of arrestin it requires binding of 12-16 phosphates per bleached rhodopsin. Phosphodiesterase activity can similarly be turned off by kinase, and the process is similarly accelerated by arrestin.     

The G-protein of retinal rod outer segments (transducin). Mechanism of interaction with rhodopsin and nucleotides

The mechanism of interaction of the G-protein of retinal rods with rhodopsin and with nucleotides has been investigated using two independent techniques, light-scattering and direct binding measurements with labeled nucleotides. Binding of photoexcited rhodopsin (R*) and nucleotides are shown to be antagonist, and three conformations of the G-protein are described, each of which is proposed to be related to a different level of light-scattering, as follows: (a) the "dark" state, stable in the absence of photoexcited rhodopsin, in which the nucleotide site is poorly accessible and has a high affinity (dissociation constants, 0.1 microM for GDP and 0.01 microM for GppNHp); (b) the R*-bound state in which the nucleotide site is rapidly accessible with a lower affinity (dissociation constants, about 20 microM for GDP and GTP; 20-100 microM for GppNHp). Binding of R* to the G-protein therefore enables rapid binding or exchange of the nucleotide; this in turn reduces the affinity of the G-protein for R* (dissociation constants, 0.2 microM for G-protein with GDP bound and 2-10 microM for G-protein with GppNHp bound, compared to 1 nM in absence of bound nucleotide); and (c) the third state, the activator of the phosphodiesterase. In the presence of GTP, an additional irreversible and fast step, which is proposed to be the dissociation of alpha-GTP from beta gamma, is shown to occur; a steady state equilibrium is obtained, and the dissociation constant measured between GTP and this third state of the G-protein in the presence of R* is an apparent constant which depends on the rate of transconformation between the first two states and on the rate of GTP hydrolysis. The minimum value of this apparent dissociation constant for GTP (0.05-0.1 (microM) is obtained at high levels of illumination. Finally, some results (number of nucleotide sites and saturation of the rate of the light-scattering signal) suggest an oligomeric association of the G-protein.     

Effect of GTP on the rhodopsin-G-protein complex by transient formation of extra metarhodopsin II

The light-induced transient interaction between rhodopsin and G-protein in the presence of GTP has been measured by the formation of extra metarhodopsin II. Disc membranes were recombined with the hypotonic extract containing the G-protein. Without GTP, a flash induces stable rhodopsin-G-protein complexes which dissociate upon addition of GTP. In low GTP (less than 10 microM) transient rhodopsin X G-protein interaction is observed. Rhodopsin X G-protein dissociates the faster, the more GTP is present (rate of dissociation, 0.3/s at 5 microM GTP; T = 3.5 degrees C). The results corroborate that the uptake of GTP terminates the rhodopsin-G-protein complex and allow an estimation of the rhodopsin X G-protein lifetime.     

Regulation of arrestin binding by rhodopsin phosphorylation level

Arrestins ensure the timely termination of receptor signaling. The role of rhodopsin phosphorylation in visual arrestin binding was established more than 20 years ago, but the effects of the number of receptor-attached phosphates on this interaction remain controversial. Here we use purified rhodopsin fractions with carefully quantified content of individual phosphorylated rhodopsin species to elucidate the impact of phosphorylation level on arrestin interaction with three biologically relevant functional forms of rhodopsin: light-activated and dark phosphorhodopsin and phospho-opsin. We found that a single receptor-attached phosphate does not facilitate arrestin binding, two are necessary to induce high affinity interaction, and three phosphates fully activate arrestin. Higher phosphorylation levels do not increase the stability of arrestin complex with light-activated rhodopsin but enhance its binding to the dark phosphorhodopsin and phospho-opsin. The complex of arrestin with hyperphosphorylated light-activated rhodopsin is less sensitive to high salt and appears to release retinal faster. These data suggest that arrestin likely quenches rhodopsin signaling after the third phosphate is added by rhodopsin kinase. The complex of arrestin with heavily phosphorylated rhodopsin, which appears to form in certain disease states, has distinct characteristics that may contribute to the phenotype of these visual disorders.     

The reactivity and function of cysteine residues in rabbit liver aldolase B

Rabbit liver aldolase B (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) contains 8 SH groups/subunit and no disulfide bonds. In the native enzyme 3 SH groups/subunit are titrable with 5,5'-dithiobis(2-nitrobenzoic) acid (Nbs2), 2,2'-dithiodipyridine and N-ethylmaleimide, whereas p-mercuribenzoate is able to react with 4 thiol groups per subunit. Among the three thiol groups titrable with Nbs2, two react 'fast' with simple second-order kinetics, one reacts 'slow' and for this thiol group saturation kinetics is observed, suggesting a reversible binding of Nbs2 to the enzyme prior to covalent modification. It is shown that this binding most likely occurs via ionic interactions in the region close to the active site. The kinetic differentiation between the two 'fast' reacting groups is possible by kinetic analysis of the release of Nbs residues from the modified enzyme. Modification of all exposed SH groups of aldolase B results in 14-32% loss of enzymatic activity. The complete inactivation of liver aldolase by 1 mM p-mercuribenzoate reported previously (Waud, J.M., Feldman, E. and Schray, K.J. (1981) Arch. Biochem. Biophys. 206, 292-295) is shown to be caused by a nonspecific reaction of this reagent used in large excess. It is concluded that this isoenzyme differs from muscle aldolase in the reactivity of exposed SH groups, the mechanisms of the interaction with modifying agents and also in the effect of SH group modification on the enzymatic activity.     

Recoverin binds exclusively to an amphipathic peptide at the N terminus of rhodopsin kinase, inhibiting rhodopsin phosphorylation without affecting catalytic activity of the kinase

Recoverin is a calcium-dependent inhibitor of rhodopsin kinase. It prevents premature phosphorylation of rhodopsin until the opening of cGMP-gated ion channels causes a decrease in intracellular calcium levels, signaling completion of the light response. This calcium depletion causes release of recoverin from rhodopsin kinase, freeing the kinase to phosphorylate rhodopsin and to terminate the light response. Previous studies have shown that recoverin is able to bind to a region at the N terminus of rhodopsin kinase. In this study we map this interaction interface, showing that residues 1-15 of the kinase form the interaction site for recoverin binding. Mutation of hydrophobic residues in this region have the greatest effect on the interaction. The periodic nature of these residues suggests that they lie along one face of an amphipathic helix. We show that this region is essential for recoverin binding, as a catalytically active kinase lacking these residues is unable to bind recoverin. In addition, we show that neither the N-terminal deletion nor the presence of recoverin inhibits the overall catalytic activity of the kinase, as measured by light-independent autophosphorylation. Finally, we observe that a kinase mutant lacking the N-terminal recoverin binding site is unable to phosphorylate light-activated rhodopsin. Taken together, these data support a model in which recoverin prevents rhodopsin phosphorylation by sterically blocking a region of kinase essential for its interaction with rhodopsin, thereby preventing recognition of rhodopsin as a kinase substrate.     

cGMP influences guanine nucleotide binding to frog photoreceptor G-protein

A rapid light-induced decrease in cGMP is thought to play a role in regulating the permeability or light sensitivity of photoreceptor membranes. Photo-excited rhodopsin activates a guanine nucleotide-binding protein (G-protein) by catalyzing the exchange of bound GDP for GTP. This G-protein X GTP complex activates the phosphodiesterase resulting in a decrease in cGMP concentration. We have observed two processes in vitro which may be relevant for the regulation of G-protein activation. First, we have found that free GDP binds to G-protein with an affinity similar to that of GTP. These two nucleotides appear to compete for a common site. Since G-protein X GDP does not activate phosphodiesterase, light-induced changes in the GTP/GDP ratio known to occur on illumination may serve to reduce G-protein activation and hence reduce phosphodiesterase activation. Second, addition of cGMP in the presence of equimolar GTP and GDP causes GTP binding to G-protein to be enhanced compared to GDP binding. This effect increases as the cGMP concentration is increased from 0.05 to 2 mM. Thus, light-induced decreases in cGMP concentration may also act as a feedback control in reducing G-protein activation. One or both of these processes may be involved in the desensitization (light adaptation) of rod photoreceptors.     

A purified agonist-activated G-protein coupled receptor: truncated octopus Acid Metarhodopsin

G-protein coupled receptors (GPCRs) mediate responses to many types of extracellular signals. So far, bovine rhodopsin, the inactive form of a GPCR, is the only member of the family whose three dimensional structure has been determined. It would be desirable to determine the structure of the active form of a GPCR. In this paper, we report the large scale preparation of a stable, homogenous species, truncated octopus rhodopsin (t-rhodopsin) in which proteolysis has removed the proline-rich C-terminal; this species retains the spectral properties and the ability for light-induced G-protein activation of unproteolyzed octopus rhodopsin. Moreover, starting from this species we can prepare a pure, active form of pigment, octopus t-Acid Metarhodopsin which has an all-trans-retinal as its agonist. Photoisomerization of t-Acid Metarhodopsin leads back to the inactive form, t-rhodopsin with the inverse agonist 11-cis-retinal. Octopus t-Acid Metarhodopsin can activate an endogenous octopus G-protein in the dark and this activity is reduced by irradiation with orange light which photoregenerates t-Acid Metarhodopsin back to the initial species, t-rhodopsin.     

Functionally discrete mimics of light-activated rhodopsin identified through expression of soluble cytoplasmic domains

Numerous studies on the seven-helix receptor rhodopsin have implicated the cytoplasmic loops and carboxyl-terminal region in the binding and activation of proteins involved in visual transduction and desensitization. In our continuing studies on rhodopsin folding, assembly, and structure, we have attempted to reconstruct the interacting surface(s) for these proteins by inserting fragments corresponding to the cytoplasmic loops and/or the carboxyl-terminal tail of bovine opsin either singly, or in combination, onto a surface loop in thioredoxin. The purpose of the thioredoxin fusion is to provide a soluble scaffold for the cytoplasmic fragments thereby allowing them sufficient conformational freedom to fold to a structure that mimics the protein-binding sites on light-activated rhodopsin. All of the fusion proteins are expressed to relatively high levels in Escherichia coli and can be purified using a two- or three-step chromatography procedure. Biochemical studies show that some of the fusion proteins effectively mimic the activated conformation(s) of rhodopsin in stimulating G-protein or competing with the light-activated rhodopsin/G-protein interaction, in supporting phosphorylation of the carboxyl-terminal opsin fragment by rhodopsin kinase, and/or phosphopeptide-stimulated arrestin binding. These results suggest that specific segments of the cytoplasmic surface of rhodopsin can adopt functionally discrete conformations in the absence of the connecting transmembrane helices and retinal chromophore.     

Photoactivation of rhodopsin causes an increased hydrogen-deuterium exchange of buried peptide groups.

A key step in visual transduction is the light-induced conformational changes of rhodopsin that lead to binding and activation of the G-protein transducin. In order to explore the nature of these conformational changes, time-resolved Fourier transform infrared spectroscopy was used to measure the kinetics of hydrogen/deuterium exchange in rhodopsin upon photoexcitation. The extent of hydrogen/deuterium exchange of backbone peptide groups can be monitored by measuring the integrated intensity of the amide II and amide II' bands. When rhodopsin films are exposed to D2O in the dark for long periods, the amide II band retains at least 60% of its integrated intensity, reflecting a core of backbone peptide groups that are resistant to H/D exchange. Upon photoactivation, rhodopsin in the presence of D2O exhibits a new phase of H/D exchange which at 10 degrees C consists of fast (time constant approximately 30 min) and slow (approximately 11 h) components. These results indicate that photoactivation causes buried portions of the rhodopsin backbone structure to become more accessible.     

Functional reconstitution of rhodopsin into tubular lipid bilayers supported by nanoporous media

We report on a novel reconstitution method for G-protein-coupled receptors (GPCRs) that yields detergent-free, single, tubular membranes in porous anodic aluminum oxide (AAO) filters at concentrations sufficient for structural studies by solid-state NMR. The tubular membranes line the inner surface of pores that traverse the filters, permitting easy removal of detergents during sample preparation as well as delivery of ligands for functional studies. Reconstitution of bovine rhodopsin into AAO filters did not interfere with rhodopsin function. Photoactivation of rhodopsin in AAO pores, monitored by UV-vis spectrophotometry, was indistinguishable from rhodopsin in unsupported unilamellar liposomes. The rhodopsin in AAO pores is G-protein binding competent as shown by a [35S]GTPgammaS binding assay. The lipid-rhodopsin interaction was investigated by 2H NMR on sn-1- or sn-2-chain perdeuterated 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phospholine as a matrix lipid. Rhodopsin incorporation increased mosaic spread of bilayer orientations and contributed to spectral density of motions with correlation times in the range of nano- to microseconds, detected as a significant reduction in spin-spin relaxation times. The change in lipid chain order parameters due to interaction with rhodopsin was insignificant.