Effects of guanine nucleotides on cholera toxin catalyzed ADP-ribosylation in rat adipocyte plasma membranes

ADP-ribosylation of rat adipocyte plasma membrane proteins was investigated following incubation of membranes with [alpha-32P]NAD and cholera toxin in the presence and absence of various guanine nucleotides. In membranes incubated without guanine nucleotides, cholera toxin induced incorporation of 32P into three discrete proteins of 48, 45, and 41 kDa. In membranes containing 100 microM GTP or GDP, toxin-catalyzed incorporation of 32P into the 41-kDa protein was inhibited. GMP and Gpp(NH)p (100 microM) allowed moderate incorporation of 32P into the 41-kDa protein. Toxin-catalyzed labeling of all proteins was rapid, reaching maximal levels between 5 and 10 min. Toxin-catalyzed ADP-ribosylation of the 48- and 45-kDa proteins was stimulated by GTP, reaching maximal levels at 10(-5) M GTP. Inhibition of toxin-dependent labeling of the 41-kDa protein required GTP concentrations above 10(-7) M with complete inhibition occurring between 10(-5) and 10(-4) M GTP. Cholera toxin catalyzed ADP-ribosylation was increased up to 2-fold in membranes supplemented with adipocyte cytosol. These results indicate that cholera toxin catalyzes ADP-ribosylation of three distinct adipocyte plasma membrane proteins, each of which is regulated by the amount and type of added guanine nucleotides.     

Vitamin A deficiency reduces the concentration of visual pigment protein within blowfly photoreceptor membranes.

Visual pigment extracts prepared from rhabdomeric membranes of vitamin A deficient blowflies contain a 5-10 times lower concentration of rhodopsin than extracts from flies which were raised on a vitamin A rich diet. Spectrophotometry showed that digitonin-solubilized rhodopsin from blowfly photoreceptors R1-6 has an absorbance maximum at about 490 nm, but no unusually enhanced beta-band in the ultraviolet. The extracts did not contain detectable concentrations of other visual pigments nor was there any evidence for the presence of photostable vitamin A derivatives. Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that the concentration of opsin in the rhabdomeric membrane is significantly reduced in vitamin A deficient flies compared to normal flies. The results indicate that the synthesis of opsin or its incorporation into the photoreceptor membrane is regulated by the chromophore concentration in the receptor cell. Furthermore, our findings open up the possibility that differences in the spectral absorption and excitability of photoreceptors from normal and vitamin A deficient flies result from the differing opsin content of the rhabdomeres.     

Vitamin a deficiency reduces the concentration of visual pigment protein within blowfly photoreceptor membranes

Visual pigment extracts prepared from rhabdomeric membranes of vitamin A deficient blowflies contain a 5–10 times lower concentration of rhodopsin than extracts from flies which were raised on a vitamin A rich diet. Spectrophotometry showed that digitonin-solubilized rhodopsin from blowfly photoreceptors R_1–6 has an absorbance maximum at about 490 nm, but no unusually enhanced β-band in the ultraviolet. The extracts did not contain detectable concentrations of other visual pigments nor was there any evidence for the presence of photostable vitamin A derivatives.Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that the concentration of opsin in the rhabdomeric membrane is significantly reduced in vitamin A deficient flies compared to normal flies. The results indicate that the synthesis of opsin or its incorporation into the photoreceptor membrane is regulated by the chromophore concentration in the receptor cell. Furthermore, our findings open up the possibility that differences in the spectral absorption and excitability of photoreceptors from normal and vitamin A deficient flies result from the differing opsin content of the rhabdomeres.     

Mutation that selectively affects rhodopsin concentration in the peripheral photoreceptors of Drosophila melanogaster

A Drosophila mutant (ninaAP228) that is low in rhodopsin concentration but identical to the wild-type fly in photoreceptor morphology has been isolated. R1-6 photoreceptors of the mutant differ from those of wild type in that (a) the prolonged depolarizing afterpotential (PDA) is absent, (b) concentrations of rhodopsin and opsin are substantially reduced, and (c) intramembrane particle density in the membranes of the rhabdomeres is low. Each of these traits is mimicked by depriving wild- type flies of vitamin A. The ninaAP228 mutation differs from vitamin A deprivation in that in the mutant (a) the rhabdomeric membrane particle density is reduced only in the R1-6 photoreceptors and not in R7 or R8, (b) the PDA can be elicited from the R7 photoreceptors, and (c) photoconversion of R1-6 rhodopsin to metarhodopsin by ultraviolet (UV) light is considerably more efficient than in vitamin A-deprived flies. The absorption properties of the mutant rhodopsin in the R1-6 photoreceptors appear to be identical to those of wild type as judged from rhodopsin difference spectra. The results suggest that the mutation affects the opsin, rather than the chromophore, component of rhodopsin molecules in the R1-6 photoreceptors. The interaction between the chromophore and R1-6 opsin, however, appears to be normal.     

Reversible phosphorylation of opsin induced by irradiation of blowfly retinae

Summary Light-induced phosphorylation and dephosphorylation of the visual pigment protein, opsin, was investigated in isolated retinae of the blowfly making use of the fact that photon capture by rhodopsin leads to the formation of a thermostable metarhodopsin. Retinae were exposed, in the presence of exogenous³²P-orthophosphate, to an intense blue light which initiated the phosphorylation of opsin (half-time about 5 min at 25 °C). Subsequent exposure of the retina to red light converted all the metarhodopsin present into rhodopsin and triggered a relatively rapid dephosphorylation of rhodopsin (half-time less than 20 s). It is proposed that the phosphorylated forms of rhodopsin and metarhodopsin represent inactive states of the pigment, i.e. phosphorylated metarhodopsin does not initiate reactions leading to the excitation of the photoreceptor cell and phosphorylated rhodopsin cannot be converted into physiologically active metarhodopsin without first being dephosphorylated.Abbreviations R1–6 peripheral retinula cells of the blowfly ommatidium - PDA prolonged depolarizing afterpotential - R rhodopsin - M metarhodopsin - R-P _n phosphorylated rhodopsin - M-P _n phosphorylated metarhodopsin - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis     

Light-dependent GTP-binding proteins in squid photoreceptors.

Previous biochemical and electrophysiological evidence suggests that in invertebrate photoreceptors, a GTP-binding protein (G-protein) mediates the actions of photoactivated rhodopsin in the initial stages of transduction. We find that squid photoreceptors contain more than one protein (molecular masses 38, 42 and 46 kDa) whose ADP-ribosylation by bacterial exotoxins is light-sensitive. Several lines of evidence suggest that these proteins represent distinct alpha subunits of G-proteins. (1) Pertussis toxin and cholera toxin react with distinct subsets of these polypeptides. (2) Only the 42 kDa protein immunoreacts with the monoclonal antibody 4A, raised against the alpha subunit of the G-protein of vertebrate rods [Hamm & Bownds (1984) J. Gen. Physiol. 84. 265-280]. (3) In terms of ADP-ribosylation, the 42 kDa protein is the least labile to freezing. (4) Of the 38 kDa and 42 kDa proteins, the former is preferentially extracted with hypo-osmotic solutions, as demonstrated by the solubility of its ADP-ribosylated state and by the solubility of the light-dependent binding of guanosine 5'-[gamma-thio]triphosphate. The specific target enzymes for the observed G-proteins have not been established.     

Cholecystokinin activates Gi1-, Gi2-, Gi3- and several Gs-proteins in rat pancreatic acinar cells.

On separation of rat pancreatic plasma membrane proteins by two-dimensional gel electrophoresis, 15 GTP-binding protein (G-protein) alpha-subunits could be detected immunochemically using an alpha common antibody. These consisted of five 48 kDa proteins (pI 5.70, 5.80, 5.90, 6.10 and 6.25) and five 45 kDa proteins (pI 5.90, 6.05, 6.25, 6.30 and 6.70), presumably corresponding to low- and high-molecular mass forms of the Gs-protein, as well as three 40/41 kDa proteins (pI 5.50, 5.70 and 6.00) and two 39 kDa proteins (pI 5.50 and 6.00). All of these proteins except for the more acidic 39 kDa protein were ADP-ribosylated by cholera toxin (CT). In addition, the three 40/41 kDa proteins and the more alkaline 39 kDa protein were also ADP-ribosylated by pertussis toxin (PT). CT- and PT-induced ADP-ribosylation changed the pI values of G-protein alpha-subunits by 0.2 pI units to more acidic values. Preincubation of isolated pancreatic membranes with cholecystokinin octapeptide (CCK-OP), which stimulates phospholipase C in acinar cells, decreased CT-induced as well as PT-induced ADP-ribosylation of the three 40/41 kDa proteins, whereas CT-induced ADP-ribosylation of one 45 kDa (pI 5.80) and all 48 kDa proteins was enhanced in the presence of CCK. Carbachol, another stimulant of phospholipase C, had no effect. The three 40/41 kDa proteins and one 48 kDa protein could be labelled with the GTP analogue [alpha-32P]GTP-gamma-azidoanilide. CCK, but not carbachol, stimulated incorporation of the GTP analogue into all of these four proteins. Using different anti-peptide antisera specific for alpha-subunits of G-proteins we identified the three 40/41 kDa Gi-proteins as Gi1 (pI 6.00), Gi2 (pI 5.50) and Gi3 (pI 5.70). The Gi3-protein was found to be the major Gi-protein of pancreatic plasma membranes. One of the 39 kDa proteins (pI 6.0) was identified as Go. These results indicate that CCK receptors functionally interact with six Gs-proteins and with Gi1, Gi2 and Gi3-proteins. Since evidence suggests that a 40/41 kDa CT substrate is involved in the stimulation of phospholipase C in pancreatic acinar cells, it is likely that one, two or all three 40/41 kDa Gi-proteins are involved in the coupling of CCK receptors with phospholipase C.     

Kinetics, binding constant, and activation energy of the 48-kDa protein-rhodopsin complex by extra-metarhodopsin II

We have found that the 48-kDa protein (or S-antigen 48k) of the rod photoreceptor enhances the light-induced formation of the photoproduct metarhodopsin II (MII) from prephosphorylated rhodopsin. The effect is analogous to the known enhancement of MII (extra-MII) that results from selective interaction of MII with G-protein. We have determined some parameters of the MII-48k interaction by measuring the extra-MII absorption change induced by the 48-kDa protein. The amplitude saturation yields a dissociation constant for the MII-48k complex on the order of 50 nM. At the technical limit of these measurements, 13.7 degrees C and 12 microM 48-kDa protein, we find a rate of 2.3 s-1 for formation of the 48k-MII complex. Extrapolation of these values to cellular conditions yields an occupation time of phosphorylated MII by 48k less than 200 ms. This is short compared to estimated rates of phosphorylation. The temperature dependence of the MII-48k formation rate is very high (Q10 for 5 degrees C/15 degrees C = 9-10). The related Arrhenius activation energy (165 kJ mol-1) is correspondingly high and indicates a considerable transient chemical change during the binding process.     

Solubilization of somatostatin receptors in hamster pancreatic beta cells. Characterization as a glycoprotein interacting with a GTP-binding protein

Somatostatin receptors of plasma membranes from beta cells of hamster insulinoma were covalently labelled with 125I-[Leu8,D-Trp22,Tyr25]somatostatin-28 (125I-somatostatin-28) and solubilized with the non-denaturing detergent Triton X-100. Analysis by SDS/PAGE and autoradiography revealed three specific 125I-somatostatin-28 receptor complexes with similar molecular masses (228 kDa, 128 kDa and 45 kDa) to those previously identified [Cotroneo, P., Marie, J.-C. & Rosselin, G. (1988) Eur. J. Biochem. 174, 219-224]. The major labelled complex (128 kDa) was adsorbed to a wheat-germ-agglutinin agarose column and eluted by N-acetylglucosamine. Also, the binding of 125I-somatostatin-28 to plasma membranes was specifically inhibited by the GTP analog, guanosine-5'-O-(3-thiotriphosphate) (GTP[S]) in a dose-dependent manner. Furthermore, when somatostatin-28 receptors were solubilized by Triton X-100 as a reversible complex with 125I-somatostatin-28, GTP[S] specifically dissociated the bound ligand to a larger extent from the soluble receptors than from the plasma-membrane-embedded receptors, the radioactivity remaining bound after 15 min at 37 degrees C being 30% and 83% respectively. After pertussis-toxin-induced [32P]ADP-ribosylation of pancreatic membranes, a 41-kDa [32P]ADP-ribose-labelled inhibitory guanine nucleotide binding protein coeluted with the 128-kDa and 45-kDa receptor complexes. The labelling of both receptor proteins was sensitive to GTP[S]. The labelling of the 228-kDa band was inconsistent. These results support the conclusion that beta cell somatostatin receptors can be solubilized as proteins of 128 kDa and 45 kDa. The major labeled species corresponds to the 128-kDa band and is a glycoprotein. The pancreatic membrane contains a 41-kDa GTP-binding protein that can complex with somatostatin receptors.     

Effects of phosphorylation of inhibitory GTP-binding protein by cyclic AMP-dependent protein kinase on its ADP-ribosylation by pertussis toxin, islet-activating protein

Pretreatment of rat cardiac myocytes with the beta-adrenergic agonist, db-cAMP or forskolin decreased ADP-ribosylation of 40-41 kDa protein by islet-activating protein (IAP) in cell membranes. Addition of activated cyclic AMP-dependent protein kinase (protein kinase A) catalytic subunit and MgCl2 also decreased ADP-ribosylation of 40-41 kDa protein by IAP in cell membranes. The alpha- and beta-subunits of partially purified inhibitory GTP-binding protein (Gi) were both phosphorylated by protein kinase A. The amounts of phosphate incorporated into the subunits of Gi were 0.34 and 0.18 mol/mol protein. These show that phosphorylation of Gi by protein kinase A results in a decrease in its ADP-ribosylation by IAP.     

Regulation of squid visual phospholipase C by activated G-protein alpha.

Phospholipase C (PLC) is the key enzyme in the phototransduction cascade of invertebrate rhabdomeric photoreceptors. In addition to 130 kDa PLC, a 95 kDa protein recognized by antibody against the catalytic site of PLC was found in the squid retina. The PLC-like 95 kDa protein (95 kDa PLC) was produced from 130 kDa PLC by an intrinsic protease in the presence of calcium. The 130 kDa PLC was stimulated by the active form of Gq-class G-protein alpha (Gq alpha), but the 95 kDa PLC was not, although their PLC activity was similar. A 35 kDa fragment, the counterpart of 95 kDa PLC, was not recognized by antibodies against catalytic site or N-terminal site of the 130 kDa PLC, indicating that the cleavage site is on the C-terminal side beyond the catalytic site. In the presence of a large excess of the 35 kDa fragment, 95 kDa PLC was stimulated by Gq alpha to a similar extent as intact 130 kDa PLC. These results indicate that the C-terminal polypeptide of PLC is necessary for regulation of its enzyme activity by Gq alpha. The uncoupling of PLC from Gq alpha, caused by limited proteolysis, is therefore a candidate regulatory mechanism of the phototransduction cascade in rhabdomeric photoreceptors.     

Phosphorylation of the 48-kDa subunit of the glycine receptor by protein kinase C.

The postsynaptic glycine receptor purified from rat spinal cord is rapidly and specifically phosphorylated by protein kinase C. The target for phosphorylation is the strychnine-binding subunit of the receptor (molecular mass of approximately 48 kDa), which is phosphorylated on serine residues to a final stoichiometry of approximately 0.8 mol of phosphate/mol of subunit. The 48-kDa phosphoprotein was analyzed by proteolytic cleavage and peptide mapping in order to localize the site of phosphorylation within the receptor molecule. Examination of the 32P-labeled receptor fragments generated by digestion with N-chlorosuccinimide, cyanogen bromide, and endoproteinase lysine C and of the deduced amino acid sequence of the 48-kDa protein (Grenningloh, G., Rienitz, A., Schmitt, B., Methfessel, C., Zensen, M., Beyreuther, K., Gundelfinger, E. D., and Betz, H. (1987) Nature 328, 215-220) indicates that the phosphorylation site is located in a region corresponding to the major intracellular loop of the predicted structure of the glycine receptor subunit and suggests serine 391 as the phosphorylated residue. In fact, a synthetic peptide corresponding to residues 384-392 of the 48-kDa subunit was specifically phosphorylated by protein kinase C. Moreover, tryptic digests of this phosphopeptide and of the phosphorylated 48-kDa subunit of the glycine receptor migrated to the same position in two-dimensional peptide mapping. Furthermore, antibodies elicited against peptide 384-392 were shown to inhibit the protein kinase C-dependent phosphorylation of the 48-kDa polypeptide. Interestingly, the relative position of the phosphorylated domain is similar to those known or proposed to be phosphorylated in other ligand-gated ion channel receptor subunits, thus suggesting further the existence of a homologous regulatory region in these receptor proteins.     

Loop 2 of limulus myosin III is phosphorylated by protein kinase A and autophosphorylation

Little is known about the functions of class III unconventional myosins although, with an N-terminal kinase domain, they are potentially both signaling and motor proteins. Limulus myosin III is particularly interesting because it is a phosphoprotein abundant in photoreceptors that becomes more heavily phosphorylated at night by protein kinase A. This enhanced nighttime phosphorylation occurs in response to signals from an endogenous circadian clock and correlates with dramatic changes in photoreceptor structure and function. We seek to understand the role of Limulus myosin III and its phosphorylation in photoreceptors. Here we determined the sites that become phosphorylated in Limulus myosin III and investigated its kinase, actin binding, and myosin ATPase activities. We show that Limulus myosin III exhibits kinase activity and that a major site for both protein kinase A and autophosphorylation is located within loop 2 of the myosin domain, an important actin binding region. We also identify the phosphorylation of an additional protein kinase A and autophosphorylation site near loop 2, and a predicted phosphorylation site within loop 2. We show that the kinase domain of Limulus myosin III shares some pharmacological properties with protein kinase A, and that it is a potential opsin kinase. Finally, we demonstrate that Limulus myosin III binds actin but lacks ATPase activity. We conclude that Limulus myosin III is an actin-binding and signaling protein and speculate that interactions between actin and Limulus myosin III are regulated by both second messenger mediated phosphorylation and autophosphorylation of its myosin domain within and near loop 2.     

Subcellular localization of Gi alpha in human neutrophils

Subcellular fractions were prepared from human neutrophils by sucrose density gradient centrifugation and analyzed for Gi-like proteins by pertussis toxin-catalyzed [32P]ADP-ribosylation and by immunoblotting with rabbit antiserum AS/6 which recognizes purified transducin and Gi, but not Gs or Go alpha-subunits. In resting cells, approximately equal to 60% of pertussis toxin substrate retrieved from the sucrose density gradient localized to the plasma membrane-enriched fraction, approximately equal to 35% to the specific granule-enriched fraction, and approximately equal to 5% to cytosol. The azurophil granule-enriched fraction did not contain pertussis toxin substrate. In contrast to plasma membrane, the specific granule-enriched fraction demonstrated increased AS/6 immunoreactivity of a approximately equal to 41-kDa protein relative to a approximately equal to 40-kDa protein. Within the specific granule-enriched fraction, the peak of pertussis toxin substrate detected immunochemically or by [32P]ADP-ribosylation sedimented at a lighter density (rho = 1.6 g/ml) than did lactoferrin (rho = 1.19 g/ml), suggesting that the intracellular compartment bearing pertussis toxin substrate may not be the lactoferrin containing specific granule, per se. Furthermore, in neutrophils exposed to 10(-8) M N-formylmethionylleucylphenylalanine, a weak degranulating stimulus (7% lactoferrin degranulation), there was a 31-42% decline in pertussus toxin-catalyzed [32P]ADP-ribosylation of approximately equal to 40-41-kDa proteins in the specific granule-enriched fraction accompanied by a near-quantitative increase in labeling of plasma membrane. The pool of intracellular formyl peptide receptors localized to the specific granule-enriched fraction appeared functionally coupled to a cosedimenting G-protein in experiments demonstrating modulation of high affinity N-formylmethionylleucyl[3H]phenylalanine binding by guanosine 5'-(3-O-thio)triphosphate or pertussis toxin. The data indicate that neutrophils contain a surface translocatable pool of intracellular G-protein sedimenting in the specific granule-enriched fraction and support the view that mobilization of intracellular G-protein represents a mechanism by which cells can regulate receptor activity.     

Pattern of protein phosphorylation in aortic endothelial cells. Modulation by adenine nucleotides and bradykinin

In bovine aortic endothelial cells, ATP (10-100 microM) and bradykinin (0.1-1.0 microM) enhanced the phosphorylation of two major protein substrates with apparent molecular masses of 95 and 28 kDa. The action of ATP involved P2y purinoceptors. The kinetics were distinct for the two phosphopeptides. The phosphorylation of the 95-kDa protein was rapid (within 30 s) but transient (maintained for only 2 min). This time course agrees with that observed for the increase of the cytosolic Ca2+ level induced by ATP in these cells. Ionophore A23187 (greater than or equal to 100 nM) induced this phosphorylation for a longer period (5-10 min), whereas phorbol 12-myristate 13-acetate (PMA) was completely inactive. The enhancement of the 28-kDa protein phosphorylation was detectable after a 5-min lag and was maintained for at least 20 min. PMA (50 nM) stimulated weakly the phosphorylation of the 28-kDa protein, whereas A23187 (100-300 nM) was even more effective than ATP and bradykinin. The 95-kDa phosphoprotein seems to be related to a 100-kDa substrate of calmodulin-dependent protein kinase III recently identified as elongation factor-2. The 28-kDa protein, which was resolved as three variants in bidimensional gel electrophoresis, appears very similar to a slightly heavier phosphoprotein from thrombin-stimulated human platelets. In addition, bidimensional electrophoresis allowed the detection of at least 10 substrates (from 18 to 46 kDa) whose phosphorylation was enhanced equally well by ATP, bradykinin, and A23187 and only partially by PMA. In conclusion, protein phosphorylation induced by ATP and bradykinin in aortic endothelial cells seems to be catalyzed mostly by Ca2+-dependent kinases, distinct from protein kinase C.     

Interplay between hydroxylamine,metarhodopsin II and GTP-binding protein in bovine photoreceptor membranes

The decay reactions of metarhodopsin II and the dissociation of the complex between rhodopsin (in the metarhodopsin II state) and the GTP-binding protein (G-protein) (in its inactive, GDP-binding form) have been compared at various concentrations of hydroxylamine. The reactions of the chromophore were measured by absorption changes in the visible range, the complex dissociation by changes in the near-in-frared scattering. An additional monitor of the complex was given by the G-protein-dependent equilibrium between metarhodopsin I and metarhodopsin II. For all measurements, fragments of isolated bovine rod outer segments in suspension were used. In the absence of hydroxylamine, the rhodopsin-G-protein complex dissociated within 20–30 min at room temperature. The presence of hydroxylamine greatly accelerated (e.g., 5-fold at 1 mM NH₂OH) the dissociation. Under all conditions, the free, dissociated G-protein can reassociate to metarhodopsin II produced by subsequent bleaching. Dissociation of the metarhodopsin II-G-protein complex required the decay of photoproducts with a maximal absorbance of 380 nm, but was not affected by the simultaneous presence of metarhodopsin III or metarhodopsin III — like photoproducts with a maximal absorbance between 450 and 470 nm. Despite the acceleration of metarhodopsin II-G-protein dissociation by NH₂OH, metarhodopsin II-G-protein was relatively stabilized as compared to free metarhodopsin II. The ratio of the decay rates of free metarhodopsin II and metarhodopsin III-G-protein was increased as much as 10-fold in the presence of 25 mM NH₂OH. The results indicate a mutual interdependence of retinal, opsin and G-protein.     

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.     

Investigation of rhodopsin catalyzed G-protein GTP-binding using [35S] GTP gamma S--effects of regeneration and hydroxylamine

A simple reconstitution system for studying rhodopsin-catalyzed G-protein GTP-binding is described. Purified rhodopsin is recombined with a G-protein containing extract in the presence of [35S]GTP gamma S and after incubation the reaction is stopped by rapid cooling and filtration. The system has been used to study the effects of 11-cis-retinal and hydroxylamine on R*-catalyzed G-protein GTP-binding. Since both these compounds greatly reduced binding, our results provide further evidence that it is the rhodopsin intermediate metarhodopsin II which interacts with G-protein.     

Progesterone and cAMP-dependent protein kinase regulate in vivo the level of phosphorylation of two proteins (Mr 20,000 and Mr 32,000) in Xenopus oocytes

The [32P]phosphoproteins and [35S]thiophosphoproteins were analyzed by electrophoresis and autoradiography after microinjection of [gamma-32P]ATP or of [35S]ATP-gamma-S into living Xenopus oocytes. The level of 32P incorporation into a 20-kDA protein was decreased following progesterone treatment (between 1 and 2 hr). This 20-kDa protein was partially thiophosphorylated in vivo by [35S]ATP-gamma-S. Furthermore it was found that this phosphoprotein was partially purified by TCA (1%) extraction and heat treatment. Microinjection of the C-subunit of cAMP-dependent protein kinase (0.6 to 1.2 pmole) inhibited maturation and provoked an increase in the level of phosphorylation of the 20-kDa protein and of a 32-kDa protein, indicating that both proteins were in vivo substrates (directly or indirectly) for cAMP-dependent protein kinase. When inhibitor-1 of protein phosphatase-1 was microinjected (5 to 10 pmole per oocyte) meiotic maturation was inhibited and the level of phosphorylation of the 32-kDa protein was increased; the same result was obtained following ATP-gamma-S (1 mM) microinjection. Altogether these results suggest that a 20-kDa phosphoprotein, whose level of phosphorylation is decreased by progesterone, could be involved in the regulation of maturation by lowering the level phosphorylation of a 32-kDa phosphoprotein. An attractive hypothesis would be that the 20-kDa phosphoprotein is an inhibitor of protein phosphatase-1.     

The molecular basis of mechanisms underlying polarization vision

The underlying mechanisms of polarization sensitivity (PS) have long remained elusive. For rhabdomeric photoreceptors, questions remain over the high levels of PS measured experimentally. In ciliary photoreceptors, and specifically cones, little direct evidence supports any type of mechanism. In order to promote a greater interest in these fundamental aspects of polarization vision, we examined a varied collection of studies linking membrane biochemistry, protein-protein interactions, molecular ordering and membrane phase behaviour. While initially these studies may seem unrelated to polarization vision, a common narrative emerges. A surprising amount of evidence exists demonstrating the importance of protein-protein interactions in both rhabdomeric and ciliary photoreceptors, indicating the possible long-range ordering of the opsin protein for increased PS. Moreover, we extend this direction by considering how such protein paracrystalline organization arises in all cell types from controlled membrane phase behaviour and propose a universal pathway for PS to occur in both rhabdomeric and cone photoreceptors.