Mono ADP-ribosylation of transducin catalyzed by rod outer segment extract.

Transducin is the retinal rod outer segment (ROS)-specific G protein coupling the photoexcited rhodopsin to cyclic GMP-phosphodiesterase. The alpha subunit of transducin is known to be ADP-ribosylated by bacterial toxins. We investigated the possibility that transducin is modified in vitro by an endogenous ADP-ribosyltransferase activity. By using either ROS, cytosolic extract of ROS or purified transducin in the presence of [alpha-32P]nicotinamide adenine dinucleotide (NAD+), the alpha and beta subunits of transducin were found to be radiolabeled. The labeling was decreased by snake venom phosphodiesterase I (PDE I). The modification was shown to be mono ADP-ribosylation by analyses on thin layer chromatography of the PDE I-hydrolyzed products which revealed only 5'AMP residues. In addition we report that sodium nitroprusside activates the ADP-ribosylation of transducin.     

A monoclonal antibody against the rod outer segment guanyl nucleotide-binding protein, transducin, blocks the stimulatory and inhibitory G proteins of adenylate cyclase

GTP-binding proteins have been implicated as transducers of a variety of biological signaling processes. These proteins share considerable structural as well as functional homology. Due to these similarities, it was thought that a monoclonal antibody that inhibits the light activation of the rod outer segment GTP-binding protein, tranducin (Gt), might exert some functional effect upon the G proteins that regulate the adenylate cyclase system. Antibody 4A, raised against the alpha subunit of Gt, cross-reacted (by hybridization on nitrocellulose) with purified alpha subunits of other G proteins (Gi and Gs, regulatory guanyl nucleotide-binding proteins that mediate inhibition and stimulation of adenylate cyclase, respectively) as long as they were not denatured. This antibody, which interferes with rod outer segment cGMP phosphodiesterase activation by blocking interaction between rhodopsin and Gt, also interfered with actions of both the stimulatory and inhibitory G proteins of adenylate cyclase from rat cerebral cortex membranes. Effects of monoclonal antibody (mAb) 4A were dose-dependent and not reversed by washing. mAb 4A also blocked the Gi-mediated inhibition of adenylate cyclase in the cyc- variant of S49 lymphoma and in doing so raised the level of adenylate cyclase activity in both the cyc- variant and the S49 wild type. There was no effect of mAb 4A on adenylate cyclase activity of the resolved catalytic subunit. These results suggest that the well known sequence homologies among the G proteins involved in cellular signal transduction may extend to the sites that interact with other members of signal-transducing cascades (receptors and effector molecules). Therefore, antibody 4A may serve as a useful tool to probe the similarities and differences among the various systems.     

Effect of monoclonal antibody binding on alpha-beta gamma subunit interactions in the rod outer segment G protein, Gt

The guanyl nucleotide binding regulatory protein of retinal rod outer segments, called Gt, that couples the photon receptor rhodopsin with the light-activated cGMP phosphodiesterase, can be resolved into two functional components, alpha t and beta gamma t. The effect of monoclonal antibody binding to the alpha t subunit of Gt on subunit association has been investigated in the present study. It was previously shown that this monoclonal antibody, mAb 4A, blocks interactions with rhodopsin and its epitope was located within the region Arg310-Phe350 at the COOH terminus of the alpha t subunit. In this paper, we show that mAb 4A disrupts the Gt complex. Gt migrates in 5-20% linear sucrose density gradients as a monomer, with a sedimentation coefficient of 4.1 +/- 0.07 S, while in the presence of mAb 4A, the alpha t and beta gamma t subunits show sedimentation coefficients of 7.7 +/- 0.2 and 3.7 +/- 0.1 S, respectively. The beta gamma t subunit migrates with the same sedimentation rate as pure beta gamma t. Nonimmune rabbit IgG does not modify the sedimentation behavior of Gt. The Fab fragment of mAb 4A also dissociates the Gt complex, as suggested by the change of the sedimentation rate of alpha t. This effect of mAb 4A on Gt subunit association was also confirmed by immunoprecipitation studies in the presence of detergent. In the presence of detergent, subunit association is not affected, but the formation of Gt oligomers and, therefore, the nonspecific precipitation of beta gamma t subunit are reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular cGMP phosphodiesterase related to the rod outer segment phosphodiesterase isolated from bovine and monkey retinas

A phosphodiesterase (PDE) has been characterized in the interphotoreceptor matrix (IPM) of light-adapted fresh bovine retinas. It is obtained through a gentle rinsing of the retinal surface under conditions where the light-activated rod outer segment (ROS) enzyme remains attached. The enzyme has an apparent native molecular weight of 350 000 by gel filtration and appears as a doublet at Mr 47 000 and 45 000 on sodium dodecyl sulfate-polyacrylamide gels. It has an apparent Km value for cGMP of 33 microM and an apparent Km value for cAMP of 2200 microM. It is activated 3-6-fold by protamine and over 40-fold by trypsin. Protamine has no effect on the Km for cGMP while trypsin decreases the Km for cGMP by a factor of 2. The enzyme occurs in at least two forms as evidenced by two distinct peaks of activity after gel electrophoresis under nondenaturing conditions. A heat-stable inhibitor is tightly bound to the enzyme. The inhibitor obtained from the IPM PDE inhibits 98% of the activity of the trypsin-activated ROS PDE: conversely, the inhibitor obtained by boiling the ROS PDE completely inhibits the trypsin-activated IPM enzyme. A high-affinity monoclonal antibody to the active site of the ROS PDE, ROS 1 [Hurwitz, R., Bunt-Milan, A.H., & Beavo, J. (1984) J. Biol. Chem. 259, 8612-8618], quantitatively absorbs the IPM PDE. These observations indicate a clear relationship between these two PDEs even though their location, sizes, and specific functions in the retina appear to be distinct.     

Interaction of the gamma-subunit of retinal rod outer segment phosphodiesterase with transducin. Use of synthetic peptides as functional probes

There is considerable evidence which suggests that the gamma-subunit of cGMP phosphodiesterase (PDE gamma) is a multifunctional protein which may interact directly with both the catalytic subunits of PDE (PDE alpha beta) and the alpha-subunit of transducin (T alpha) (Whalen, M., and Bitensky, M. (1989) Biochem. J. 259, 13-19; Griswold-Prenner, I., Young, J. H., Yamane, H. K., and Fung, B. K.-K. (1988) Invest. Ophthalmol. & Visual Sci. 29, (Suppl.) 218). To determine the region of interaction between the multifunctional PDE gamma and T alpha, and to determine the significance of this interaction, peptides corresponding to various regions of PDE gamma were synthesized and tested for their ability to inhibit the GTPase activity of T alpha. One of these peptides, PDE gamma-3 (bovine amino acid residues 31-45), inhibited the GTPase activity of T alpha with an I50 of 450 microM. The peptide (PDE gamma-3) was found to inhibit the GTPase activity of T alpha by inducing the binding of transducin to the rod outer segment membrane and by altering the GTP/GDP exchange. Analogs of PDE gamma-3 were synthesized to determine the required structure of the PDE gamma-3 region needed for the interaction of PDE gamma with T alpha. The results of these studies indicated that the removal of the positively charged amino acids or any of the potential hydrogen-bonding amino acids increased the I50 for the inhibition of the GTPase activity of T alpha Substitution of the hydrophobic amino acids had no effect. These results indicate the hydrophilic interactions may be essential for the binding of PDE gamma to T alpha and for the inhibition of the GTPase activity of T alpha by PDE gamma. The observed effects of PDE gamma-3 on T alpha and on PDE suggest that PDE gamma is a multifunctional protein which may play more than one role in the deactivation of the retinal transduction cascade.     

Membrane stimulation of cGMP phosphodiesterase activation by transducin: comparison of phospholipid bilayers to rod outer segment membranes.

To clarify the role of phospholipids in G protein-effector interactions of vertebrate phototransduction, transducin activation of cGMP phosphodiesterase (PDE) has been reconstituted on the surface of well-defined phosphatidylcholine (PC) vesicles, using purified proteins from bovine rod outer segments (ROS). PC vesicles enhanced PDE stimulation by the GTP-gamma S-bound transducin alpha subunit (T alpha-GTP gamma S) as much as 17-fold over activation in the absence of membranes. In the presence of 3.5 microM accessible PC in the form of large (100 nm) unilamellar vesicles, 500 nM T alpha-GTP gamma S stimulated PDE activity to more than 70% of the maximum activity induced by trypsin. Activation required PC, PDE, and T alpha-GTP gamma S, but did not require prior incubation of any of the components, and occurred within 4 s of mixing. The PC vesicles were somewhat more efficient than urea-washed ROS membranes in enhancing PDE activation. Half-maximal activation occurred at accessible phospholipid concentrations of 3.8 microM for PC vesicles, and 13 microM for ROS membranes. Titrations of PDE with T alpha-GTP gamma S in the presence of membranes indicated a high-affinity (Kact less than 250 pM) activation of PDE by a small fraction (0.5-5%) of active T alpha-GTP gamma S, as did titrations of ROS with GTP gamma S. When activation by PC vesicles was compared to PDE binding to membranes, the results were consistent with activation enhancement resulting from formation of a T alpha-GTP gamma S-dependent PDE-membrane complex with half-maximal binding at phospholipid concentrations in the micromolar range. The value of the apparent dissociation constant, KPL, associated with the activation enhancement was estimated to be in the range of 2.5 nM (assuming an upper limit value of 1600 phospholipids/site) to 80 nM (for a lower limit value of 50 phospholipids/site). Another component of membrane binding was more than 100-fold weaker and was not correlated with activation by T alpha-GTP gamma S. Low ionic strength disrupted the ability of ROS membranes, but not PC vesicles, to bind and activate PDE. Removal of PDE's membrane-binding domain by limited trypsin digestion eliminated both the binding of PDE to vesicles and the ability of PDE to be activated by T alpha-GTP gamma S and membranes. These results suggest that ROS membrane stimulation of PDE activation by T alpha-GTP gamma S is due almost exclusively to the phospholipids in the disk membrane.     

Fluid lipid fraction in rod outer segment membrane.

Rod outer segment membrane is analyzed using the spin resonance label technique by means of two probes. The solubility of the first label,2,2,6,6-tetramethylpiperidin-1-oxyl, is correlated with the membrane fluidity which is measured using a stearic acid spin probe. The two values are compared to the solubility-fluidity relationship which characterizes a model system in which all lipids are in a fluid state. The analysis leads to the conclusion that only two thirds of the membrane lipids are fluid. This conclusion is reinforced by the observation that partial lipid removal leaves rigid lipids associated with the rhodopsin molecules.     

Rhodopsin in the rod outer segment plasma membrane

Isolated frog retinas were incubated in vitro with a 4-h pulse of [3H]leucine, then chased for 32 h with a nonradioactive amino acid mixture. At the end of the incubation, light and electron microscope autoradiograms were prepared from some of the retinas. The autoradiograms revealed: (a) intense radioactivity in the basal disks of the rod outer segments, (b) diffuse label evenly distributed throughout the rod outer segments, and (c) a high concentration of label in the entire rod outer segment plasma membrane. Incubation under identical conditions, but with puromycin added, significantly inhibited the labeling of all of these components. To identify the labeled proteins, purified outer segments from the remaining retinas were analyzed biochemically by SDS disc gel electrophoresis and gel filtration chromatography. SDS gel electrophoresis showed that about 90% of the total rod outer segment radioactivity chromatographed coincident with visual pigment, suggesting that the radiolabeled protein in the plasma membrane is visual pigment. Gel filtration chromatography demonstrated that the radiolabeled protein co-chromatographed with rhodopsin rather than opsin, and that the newly synthesized visual pigment is both the basal disks and the plasma membrane is present in the native configuration.     

Localization of rhodopsin in isolated, osmotically intact rod outer segment discs.

Localization of rhodopsin and its position in the membrane has been the subject of numerous studies. Most recently, immunocytochemical techniques have been employed to localize the opsin component of the molecule in in situ rod outer segments. Due to the problems inherent in localization procedures (penetration and mechanical interference) we have utilized isolated, osmotically intact rod outer segment discs in this study. Specific antibodies to chromatographically pure rhodopsin were prepared and enzymatically digested to their Fab components. The univalent Fab antibodies were conjugated to horseradish peroxidase and used to label the isolated rod outer segment discs. Discs treated with anti-opsin conjugate stained uniformly and heavily on their interdisc surfaces. Reaction product was also present on the intradisc surface in a thinner but still uniformly distributed layer. Controls treated with preimmune Fab - horseradish peroxidase conjugate showed no deposition of reaction product.     

Membrane fusion in the rod outer segment disk membrane

The rod outer segment disk membrane of bovine retina has been isolated in a predominantly fused state. The physical and chemical properties of the membrane in the fused state are profoundly different from the corresponding properties of the same membrane in the unfused state. Exposure to light induces the transition of the disk membrane from the fused to the unfused state. Evidence is presented which suggests that the fusion-defusion cycle of the disk membrane is a primary event of photoexcitation and nerve stimulation.     

Degradation of rod outer segment proteins by cathepsin D.

The degradation of proteins of the rod outer segment (ROS) fraction by partially purified cathepsin D [EC 3.4.23.5] from the retinal pigment epithelium was studied. The ROS fraction, prepared from bovine eyes by sucrose density gradient centrifugation, had little cathepsin D activity. Partially purified cathepsin D, obtained from crude extract of bovine retinal pigment epithelium using bovine serum albumin as a substrate, hydrolyzed the porteine of the ROS fraction. The rate of degradation of ROS proteins was proportional to both the enzyme concentration and the incubation time. With ROS proteins as substrate, the optimal pH of cathepsin D was about 3.5. The degradation of ROS proteins was inhibited by pepstatin.     

Calcium adenosinetriphosphatase of bovine retinal rod outer segment disks.

A Ca(2+)-pumping ATPase activity is present in bovine retinal rod outer segment purified disks. The ATPase has a high Ca2+ affinity (KM = 25 microM). Low Ca2+ (n-microM) concentrations stimulate an ATP-dependent Ca2+ uptake and the ATP hydrolysis in the absence of exogenous Mg2+. Electrophoretic analysis of disk proteins after treatment with (gamma-32P)ATP shows the existence of the enzyme-phosphate acid-stable, hydroxylamine-sensitive intermediate complex of molecular mass of about 135 kDa. The results would indicate the presence of an inwardly directed Ca(2+)-ATPase pump acting on the disk membrane, that could be involved in the regulation of cytosolic free Ca2+ levels inside ROS.     

Fluid lipid fraction in rod outer segment membrane

Rod outer segment membrane is analyzed using the spin label technique by means of two probes. The solubility of the first label, 2,2,6,6-tetramethylpiperidin-1-oxyl, is correlated with the membrane fluidity which is measured using a stearic acid spin probe. The two values are compared to the solubility-fluidity relationship which characterizes a model system in which all lipids are in a fluid state. The analysis leads to the conclusion that only two thirds of the membrane lipids are fluid. This conclusion is reinforced by the observation that partial lipid removal leaves rigid lipids associated with the rhodopsin molecules.     

Sub-second turnover of transducin GTPase in bovine rod outer segments. A light scattering study

A fast, regenerative light scattering signal from bovine ROS, the PA-signal, reflects the light-induced, transient activation of transducin. Its rate of recovery depends on the number of photolysed rhodopsin molecules, indicating that rhodopsin deactivation and not GTPase activity is rate limiting in our in vitro system. When rhodopsin deactivation is accelerated (in the presence of NH2OH), PA-signal recovery is also accelerated. A GTPase turnover number of more than 2 s-1 (at 37 degrees C) can be derived from these experiments. This is more than one order of magnitude faster than the GTPase rates so far described in the literature and is rapid enough for a physiological shut-off mechanism. The fast GTPase is attributed to a highly intact disk stack, which never releases transducin into the free aqueous space.     

Direct observation of the complex formation of GDP-bound transducin with the rhodopsin intermediate having a visible absorption maximum in rod outer segment membranes

Rhodopsin is a photoreceptive protein that is present in rod photoreceptor cells, inducing a GDP-GTP exchange reaction on the retinal G-protein transducin (Gt) upon light absorption. This exchange reaction proceeds through at least three steps, which include the binding of photoactivated rhodopsin to GDP-bound Gt, the dissociation of GDP from the rhodopsin-Gt complex, and the binding of GTP to the nucleotide-unbound Gt. These steps have been thought to occur after the formation of the rhodopsin intermediate, meta-II; however, the extra formation of meta-II, which reflects the formation of a complex with Gt, was inhibited in the presence of excess GDP. Here, we use a newly developed CCD spectrophotometer to show that a meta-II precursor, meta-Ib, which has an absorption maximum at visible region, can bind to Gt in its GDP-bound form in urea-washed bovine rod outer segment membranes. The affinity of meta-Ib for GDP-bound Gt is about two times less than that of meta-II for GDP-unbound Gt, indicating that the extra formation of meta-II is observed at equilibrium even in the presence of the meta-Ib-Gt complex. This is the first identification of a complex that includes the GDP-bound form of G protein. Our results strongly suggest that the protein conformational change of the rhodopsin intermediate after binding to Gt is important for the induction of the nucleotide release from the alpha-subunit of Gt.     

Proteomic analysis of the retinal rod outer segment disks

The initial events of vision at low light take place in vertebrate retinal rods. The rod outer segment consists of a stack of flattened disks surrounded by the plasma membrane. A list of the proteins that reside in disks has not been achieved yet. We present the first comprehensive proteomic analysis of purified rod disks, obtained by combining the results of two-dimensional gel electrophoresis separation of disk proteins to MALDI-TOF or nLC-ESI-MS/MS mass spectrometry techniques. Intact disks were isolated from bovine retinal rod outer segments by a method that minimizes contamination from inner segment. Out of a total of 187 excised spots, 148 proteins were unambiguously identified. An additional set of 61 proteins (partially overlapping with the previous ones) was generated by one-dimensional (1D) gel nLC-ESI-MS/MS method. Proteins involved in vision as well as in aerobic metabolism were found, among which are the five complexes of oxidative phosphorylation. Results from biochemical, Western blot, and confocal laser scanning microscopy immunochemistry experiments suggest that F 1F o-ATP synthase is located and catalytically active in ROS disk membranes. This study represents a step toward a global physiological characterization of the disk proteome and provides information necessary for future studies on energy supply for phototransduction.