Calcium-dependent activation and deactivation of rod outer segment phosphodiesterase is calmodulin-independent

ATP-dependent activation and deactivation of retinal rod outer segment phosphodiesterase is affected by calcium [Kawamura, S. and Bownds, M. D., J. Gen. Physiol. 77:571-591(1981)]. Our data demonstrate that although calmodulin has been found in rod outer segments [Liu, Y. P. and Schwartz, H., Biochim. Biophys. Acta 526:186-193(1978); Kohnken, R. E. et al, J. Biol. Chem. 256:12517-12522(1981)], this protein is not involved in calcium-dependent phosphodiesterase activation at light levels at which calcium clearly affects this enzyme's activity. Furthermore, calmodulin does not mediate the calcium-dependent deactivation of phosphodiesterase.     

Molecular species analysis of mitogen-stimulated 1,2-diglycerides in fibroblasts. Comparison of alpha-thrombin, epidermal growth factor, and platelet-derived growth factor

Recent studies have implicated the hydrolysis of phosphoinositides and phosphatidylcholine in agonist-stimulated events. The potent mitogen, alpha-thrombin, stimulates the generation of diglycerides in a biphasic and sustained manner in IIC9 fibroblasts (Wright, T. M., Rangan, L. A., Shin, H. S., and Raben, D. M. (1988) J. Biol. Chem. 263, 9374-9380). Using measurements of radiolabeled headgroup release and molecular species analysis, we previously determined that alpha-thrombin generates diglycerides through the hydrolysis of both the phosphoinositides and phosphatidylcholine at early times (15 s), and at later times (greater than or equal to 5 min) through the hydrolysis of primarily, if not exclusively, phosphatidylcholine (Pessin, M. S., and Raben, D. M. (1989) J. Biol. Chem. 264, 8729-8738). In contrast, IIC9 fibroblasts respond to the mitogenic treatments of (a) alpha-thrombin following chymotrypsin pretreatment or (b) epidermal growth factor by increasing their levels of diglycerides in a monophasic and sustained manner (Wright, T. M., Rangan, L. A., Shin, H. S., and Raben, D. M. (1988) J. Biol. Chem. 263, 9374-9380). In this report, we have analyzed the molecular species of the diglycerides generated by these two different treatments and have also examined the lipid response of IIC9 fibroblasts to platelet-derived growth factor. Based on both the molecular species analyses and the release of radiolabeled head-groups, all three of these different mitogenic treatments generate diglycerides primarily through the stimulation of phosphatidylcholine hydrolysis. However, while similar, the molecular species profiles of the diglycerides generated by these three treatments are not identical to the molecular species profile of total cellular phosphatidylcholine. In addition, the molecular species profiles of the diglycerides generated by these three mitogenic treatments greatly resemble each other, with significant differences between any two profiles occurring in at most one molecular species. This finding differs from that seen with alpha-thrombin stimulation alone, where the molecular species profile of the diglycerides generated following 5 min of alpha-thrombin stimulation is nearly identical to the molecular species profile of total cellular phosphatidylcholine. These data support the possibility of hormone-sensitive phosphatidylcholine pools or selective diglyceride metabolism.     

Relationship of cholesterol content to spatial distribution and age of disc membranes in retinal rod outer segments

The initial events of visual transduction occur on disc membranes which are sequestered within the photoreceptor outer segment. In rod cells, the discs are stacked in the outer segment. Discs are formed at the base of the rod outer segment (ROS) from evaginations of the plasma membrane. As new discs form, older discs move toward the apical tip of the rod, from which they are eventually shed and subsequently phagocytosed by the adjacent pigment epithelium. Thus, disc membranes within a given rod cell are not of uniform age. We have recently shown that disc membranes are not homogeneous with respect to cholesterol content (Boesze-Battaglia, K., Hennessey, T., and Albert, A. D. (1989) J. Biol. Chem. 264, 8151-8155). In the present study, freshly isolated bovine retinas were incubated with [3H]leucine for 4 h in order to allow sufficient time for the radiolabeled proteins to become incorporated into the basal-most (newest) discs. Osmotically intact discs were then isolated. After the addition of digitonin, the discs were fractionated based on cholesterol content, and radioactivity (indicative of newly synthesized protein) was measured. Discs which exhibited high cholesterol content also exhibited high radio-activity. These results demonstrate that the cholesterol heterogeneity of ROS disc membranes is related to the age, and thus the position, of the discs in the ROS.     

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.     

Phosphorylation by cyclin-dependent protein kinase 5 of the regulatory subunit of retinal cGMP phosphodiesterase. I. Identification of the kinase and its role in the turnoff of phosphodiesterase in vitro

Cyclic GMP phosphodiesterase (PDE) is an essential component in retinal phototransduction. PDE is regulated by Pgamma, the regulatory subunit of PDE, and GTP/Talpha, the GTP-bound alpha subunit of transducin. In previous studies (Tsuboi, S., Matsumoto, H. , Jackson, K. W., Tsujimoto, K., Williamas, T., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15016-15023; Tsuboi, S., Matsumoto, H., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15024-15029), we showed that Pgamma is phosphorylated by a previously unknown kinase (Pgamma kinase) in a GTP-dependent manner in photoreceptor outer segment membranes. We also showed that phosphorylated Pgamma loses its ability to interact with GTP/Talpha, but gains a 10-15 times higher ability to inhibit GTP/Talpha-activated PDE than that of nonphosphorylated Pgamma. Thus, we propose that the Pgamma phosphorylation is probably involved in the recovery phase of phototransduction through shut off of GTP/Talpha-activated PDE. Here we demonstrate that all known Pgammas preserve a consensus motif for cyclin-dependent protein kinase 5 (Cdk5), a protein kinase believed to be involved in neuronal cell development, and that Pgamma kinase is Cdk5 complexed with p35, a neuronal Cdk5 activator. Mutational analysis of Pgamma indicates that all known Pgammas contain a P-X-T-P-R sequence and that this sequence is required for the Pgamma phosphorylation by Pgamma kinase. In three different column chromatographies of a cytosolic fraction of frog photoreceptor outer segments, the Pgamma kinase activity exactly coelutes with Cdk5 and p35. The Pgamma kinase activity ( approximately 85%) is also immunoprecipitated by a Cdk5-specific antibody, and the immunoprecipitate phosphorylates Pgamma. Finally, recombinant Cdk5/p35, which were expressed using clones from a bovine retina cDNA library, phosphorylates Pgamma in frog outer segment membranes in a GTP-dependent manner. These observations suggest that Cdk5 is probably involved in the recovery phase of phototransduction through phosphorylation of Pgamma complexed with GTP/Talpha in mature vertebrate retinal photoreceptors.     

Immunocytochemical localization of opsin in outer segments and Golgi zones of frog photoreceptor cells. An electron microscope analysis of cross-linked albumin-embedded retinas

Adult vertebrate retinal cells (rod and cones) continuously synthesize membrane proteins and transport them to the organelle specialized for photon capture, the outer segment. The cell structures involved in the synthesis of opsin have been identified by means of immunocytochemistry at the electron microscope level. Two indirect detection systems were used: (a) rabbit antibodies to frog opsin were localized with ferritin conjugated F(ab')2 of sheep antibodies to rabbit F(ab')2 and (b) sheep antibodies to cattle opsin were coupled to biotin and visualized by means of avidin-ferritin conjugates (AvF). The reagents were applied directly to the surface of thin sections of frog retinal tissues embedded in glutaraldehyde cross-linked bovine serum albumin (BSA). Specific binding of anti-opsin antibodies indicates that opsin is localized in the disks of rod outer segments (ROS), as expected, and in the Golgi zone of the rod cell inner segments. In addition, we observed quantitatively different labeling patterns of outer segments of rods and cones with each of the sera employed. These reactions may indicate immunological homology of rod and cone photopigments. Because these quantitiative variations of labeling density extend along the entire length of the outer segment, they also serve to identify the cell which has shed its disks into adjacent pigment ipithelial cell phagosomes.     

Activation of phosphodiesterase in frog rod outer segment by an intermediate of rhodopsin photolysis. I.

Guanosine 3′,5′-cyclic monophosphate phosphodiesterase (EC 3.1.4.1) in frog rod outer segment prepared by a sucrose stepwise density gradient method was activated by light in the presence of GTP. Rhodopsin in rod outer segment was solubilized with sucrose laurylmonoester and then purified by concavanalin A-Sepharose column. Addition of photo-bleached preparation of the purified rhodopsin to the rod outer segment, which had been prepared by 43% (w/w) sucrose floatation, caused the activation of phosphodiesterase in the dark, while each component of the photo-product eluted from the column (all-trans retinal and opsin) did not. Regenerated rhodopsin prepared from 11-cis retinal and purified opsin activated phosphosdiesterase when it was bleached. From these facts it is suggested that an intermediate or a process of photolysis of rhodopsin causes activation of phosphodiesterase.     

Activation of phosphodiesterase by rhodopsin and its analogues

Activation of guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase (EC 3.1.4.35.) in frog rod outer segment membrane by rhodopsin and its analogues was investigated. The Schiff-base linkage between opsin and retinal in rhodopsin was not always necessary for the phosphodiesterase activation. The binding of beta-ionone ring of retinal to a hydrophobic region of opsin was not enough to induce the enzyme activation. A striking photo-activation of the enzyme was induced by photo-isomerization of rhodopsin analogues from cis to trans form. It seems probable that an "expanded" conformation of opsin around the retinylidene chromophore induced by the cis to trans isomerization may be the trigger for the activation of phosphodiesterase. On the other hand, the phosphodiesterase in frog rod outer segment was activated by warming of bathorhodopsin to -12 degrees C and then incubating it at the same temperature. Thus, metarhodopsin II or an earlier intermediate than metarhodopsin II should be a direct intermediate for the enzyme activation.     

Microinjected oligonucleotides complementary to the alpha-sarcin loop of 28 S RNA abolish protein synthesis in Xenopus oocytes

The integrity of the alpha-sarcin loop in 28 S ribosomal RNA is critical during protein synthesis. The toxins alpha-sarcin, ricin, Shiga toxin, and Shiga-like toxin inhibit protein synthesis in oocytes by attacking specific nucleotides within this loop (Ackerman, E.J., Saxena, S. K., and Ulbrich, N. (1988) J. Biol. Chem. 263, 17076-17083; Saxena, S.K., O'Brien, A.D., and Ackerman, E.J. (1989) J. Biol. Chem. 264, 596-601). We injected Xenopus oocytes with deoxyoligonucleotides complementary to the 17-nucleotide alpha-sarcin loop of Xenopus 28 S rRNA. Only injected oligonucleotides fully covering the alpha-sarcin loop or slightly beyond inhibited oocyte protein synthesis. Shorter alpha-sarcin domain deoxyoligonucleotides complementary to the alpha-sarcin and ricin sites but not spanning the entire loop were less effective inhibitors of protein synthesis. The alpha-sarcin domain oligonucleotides covering the entire loop were more effective inhibitors of protein synthesis than injected cycloheximide at equivalent concentrations. Control oligonucleotides complementary to nine other regions of Xenopus 28 S rRNA as well as universal M13 DNA sequencing primers had no effect on oocyte protein synthesis. Oligonucleotides complementary to the highly conserved alpha-sarcin domain therefore represent an alternative to catalytic toxins for causing cell death and may prove effective in immunotherapy.     

Mono(ADP-ribosyl)ation of Gi by eukaryotic cysteine-specific mono(ADP-ribosyl) transferase attenuates inhibition of adenylate cyclase by epinephrine

Eukaryotic cysteine-specific mono(ADP-ribosyl)transferase, named ADP-ribosyltransferase C (Tanuma, S., Kawashima, K. and Endo, H. (1988) J. Biol. Chem. 263, 5485-5489), attenuates inhibition of adenylate cyclase in human platelet membranes by epinephrine. This attenuation appeared to result from mono(ADP-ribosyl)ation by ADP-ribosyltransferase C of the inhibitory guanine nucleotide-binding protein (Gi) of adenylate cyclase. These results indicate a role of ADP-ribosyltransferase C in regulation of hormonal control of the adenylate cyclase system.     

Solubilization, purification, and reconstitution of the sodium-calcium exchanger from bovine retinal rod outer segments

We have previously described a method for the solubilization and reconstitution of the cGMP-gated cation channel from the membranes of bovine rod outer segments (Cook, N. J., Zeilinger, C., Koch, K.-W., and Kaupp, U. B. (1986) J. Biol. Chem. 261, 17033-17039). Here we report that not only cGMP but also sodium is capable of releasing entrapped calcium from liposomes reconstituted with total rod outer segment membrane proteins. Other alkali cations tested were unable to induce calcium efflux; therefore, we concluded that the sodium-induced calcium efflux was due to the sodium-calcium exchanger. Sodium was found to activate calcium efflux from these liposomes with an EC50 of approximately equal to 35 mM, comparable to values reported for the sodium-calcium exchanger in native rod outer segment membranes. We found that reconstitution of the sodium-calcium exchanger is quantitative and used this method to assay the exchange protein during purification using conventional protein chromatographic techniques. In this way, we were able to purify and identify as the rod outer segment sodium-calcium exchanger a glycoprotein of apparent Mr = 220,000 to greater than 90% homogeneity. The specific activity of the purified protein at room temperature was 8.2 mumol of Ca2+ exchanged min-1 mg-1 of protein at 50 mM Na+, corresponding to a turnover number of approximately equal to 30 Ca2+ (or 90 Na+) s-1 exchanger-1. The Mr = 220,000 protein reported here appears to be distinct from another protein ("rim protein") with an identical Mr known to exist in these membranes.     

Mutation of the two carboxyl-terminal tyrosines results in an insulin receptor with normal metabolic signaling but enhanced mitogenic signaling properties

Our previous studies have shown that the deletion of the insulin receptor carboxyl terminus impairs metabolic, but augments mitogenic, signaling (McClain, D. A., Maegawa, H., Levy, J., Huecksteadt, T., Dull, T. J., Lee, J., Ullrich, A., and Olefsky, J. M. (1988) J. Biol. Chem. 263, 8904-8911; Thies, R.S., Ulrich, A., and McClain, D. A. (1989) J. Biol. Chem. 264, 12820-12825). To explore further the regulatory role of the insulin receptor carboxyl terminus, a mutant insulin receptor was constructed in which the two tyrosines (Y1316 and Y1322) on the carboxyl terminus were replaced with phenylalanines. Rat 1 fibroblasts expressing high levels of this mutant receptor (Y/F2 cells) exhibited normal insulin binding and normal insulin internalization. The absence of the two tyrosines in the carboxyl terminus did not affect the phosphotransferase activity of the beta-subunit and insulin-stimulated glucose transport. However, the Y/F2 cells showed markedly enhanced sensitivity for insulin-stimulated DNA synthesis. Dose-response curves for both insulin-stimulated thymidine uptake and 5-bromo-2-deoxyuridine incorporation in the Y/F2 cell lines were shifted to the left (4-10-fold) compared with those observed in the cells expressing similar numbers of wild type receptors. Thus, the two tyrosines of the insulin receptor carboxyl terminus do not modulate the kinase function of the insulin receptor, although they are autophosphorylated in native receptors. Moreover, these tyrosines are not necessary for stimulation of glucose transport. On the other hand, these results suggest that the two carboxyl-terminal tyrosine residues exert an inhibitory effect on mitogenic signaling in native insulin receptors.     

In vivo incorporation of [2-3H]-myo-inositol into frog opsin

The in vivo incorporation of [2-3H]-myo-inositol into frog retinal rod outer segment membranes was examined. About 25% of the recovered radioactivity was found to be protein-associated. Following acid hydrolysis of this material and extraction with hexane, all the radioactivity remained in the aqueous phase, indicating that the label was not in fatty acids. Following ion exchange column chromatography of the hydrolysate, the major radioactive compound comigrated on TLC with an internal standard of [U-14C]-myo-inositol. SDS polyacrylamide gel electrophoresis of unextracted membranes indicated that the majority of the label was associated with opsin. These results indicate that [2-3H]-myo-inositol was incorporated in vivo into opsin, presumably with retention of its chemical identity.     

Rat liver polysome N alpha-acetyltransferase: isolation and characterization

Rat liver polysome N alpha-acetyltransferase has been purified to homogeneity by a four-step procedure that utilizes ammonium sulfate precipitation, gel filtration, hydroxylapatite chromatography, and Mono Q ion exchange chromatography. The enzyme is greatly stabilized by the inclusion of EDTA and 0.01% deoxycholate in the isolation buffers. The purified enzyme has a native molecular weight of 190,000 and a subunit molecular weight of 95,000, suggesting that it is a homodimer. The enzyme shows a pH optimum of 8.0 and is strongly inhibited by Cl-, I-, SCN-, and ClO4- and to a lesser degree by sulfate and acetate. It is unaffected by phosphate, citrate, and F- and by Na+ and K+; NH4+ is partially inhibitory. The enzyme is also sensitive to iodoacetic acid. It is generally more similar to yeast N alpha-acetyltransferase [Lee, F.-J. S., Lin, L.-W., & Smith, J. A. (1988) J. Biol. Chem. 263, 14948-14955] than to the hen oviduct enzyme, which contains a 7S RNA subunit [Kamitani, K., & Sakiyama, F. (1989) J. Biol. Chem. 264, 13194-13198], although the amino acid compositions are quite different.     

Immunogold localization of the regulatory subunit of a type II cAMP- dependent protein kinase tightly associated with mammalian sperm flagella

We have shown previously that the regulatory subunit (RII) of a type II cAMP-dependent protein kinase is an integral component of the mammalian sperm flagellum (Horowitz, J.A., H. Toeg, and G.A. Orr. 1984. J. Biol. Chem. 259:832-838; Horowitz, J.A., W. Wasco, M. Leiser, and G.A. Orr. 1988. J. Biol. Chem. 263:2098-2104). The subcellular localization of this flagellum-associated RII in bovine caudal epididymal sperm was analyzed at electron microscope resolution with gold-conjugated secondary antibody labeling techniques using anti-RII monoclonal antibodies. By immunoblot analysis, the flagellum-associated RII was shown to interact with mAb 622 which cross reacts with both neural and nonneural isoforms of RII. In contrast, a neural specific monoclonal antibody (mAb 526) failed to interact with flagellar RII. In the midpiece of the demembranated sperm tail, gold label after mAb 622 incubation was primarily associated with the outer mitochondrial membrane. Although almost all specific labeling in the midpiece can be assigned to the mitochondria, in the principal piece, there is some labeling of the fibrous sheath. Labeling of the outer dense fibers and the axoneme was sparse. Specific labeling was virtually absent in the sperm head. Sections of sperm tails incubated in the absence of primary antisera or with mAb 526 showed little labeling. A beta-tubulin monoclonal antibody localized only to the 9 + 2 axoneme. These results raise the possibility that a type II cAMP-dependent protein kinase located at the outer mitochondrial membrane plays a role in the direct cAMP stimulation of mitochondrial respiration during sperm activation.     

The plasmamembrane calmodulin-dependent calcium pump: a major regulator of nitric oxide synthase I

The plasma membrane calcium/calmodulin-dependent calcium ATPase (PMCA) (Shull, G.E., and J. Greeb. 1988. J. Biol. Chem. 263:8646-8657; Verma, A.K., A.G. Filoteo, D.R. Stanford, E.D. Wieben, J.T. Penniston, E.E. Strehler, R. Fischer, R. Heim, G. Vogel, S. Mathews, et al. 1988. J. Biol. Chem. 263:14152-14159; Carafoli, E. 1997. Basic Res. Cardiol. 92:59-61) has been proposed to be a regulator of calcium homeostasis and signal transduction networks of the cell. However, little is known about its precise mechanisms of action. Knock-out of (mainly neuronal) isoform 2 of the enzyme resulted in hearing loss and balance deficits due to severe inner ear defects, affecting formation and maintenance of otoconia (Kozel, P.J., R.A. Friedman, L.C. Erway, E.N. Yamoah, L.H. Liu, T. Riddle, J.J. Duffy, T. Doetschman, M.L. Miller, E.L. Cardell, and G.E. Shull. 1998. J. Biol. Chem. 273:18693-18696). Here we demonstrate that PMCA 4b is a negative regulator of nitric oxide synthase I (NOS-I, nNOS) in HEK293 embryonic kidney and neuro-2a neuroblastoma cell models. Binding of PMCA 4b to NOS-I was mediated by interaction of the COOH-terminal amino acids of PMCA 4b and the PDZ domain of NOS-I (PDZ: PSD 95/Dlg/ZO-1 protein domain). Increasing expression of wild-type PMCA 4b (but not PMCA mutants unable to bind PDZ domains or devoid of Ca2+-transporting activity) dramatically downregulated NO synthesis from wild-type NOS-I. A NOS-I mutant lacking the PDZ domain was not regulated by PMCA, demonstrating the specific nature of the PMCA-NOS-I interaction. Elucidation of PMCA as an interaction partner and major regulator of NOS-I provides evidence for a new dimension of integration between calcium and NO signaling pathways.     

Acylation of disc membrane rhodopsin may be nonenzymatic

Bovine retinal rod outer segments (ROS) support the incorporation of [3H]palmitate into rhodopsin. [14C] Palmitoyl-CoA serves as the donor with an apparent Km of 40 microM. Solubilization of ROS in the detergent, Emulphogene, results in increased incorporation of label into rhodopsin. A further increase is found when ConA-Sepharose-purified rhodopsin is used as the source of both "enzyme" and acceptor. Failure to separate enzyme from acceptor suggested the possibility of a nonenzymatic reaction. This was confirmed when boiled rhodopsin was found to support the reaction. However, the acylation of rhodopsin is not an artifact since analysis of purified native rhodopsin reveals the presence of covalently bound palmitate and we showed that whole bovine retinas incubated with [3H] palmitate incorporated the fatty acid into rhodopsin (O'Brien, P.J., and Zatz, M. (1984) J. Biol. Chem. 259, 5054-5057). Furthermore, in vivo experiments with rat retinas have revealed that opsin is acylated both in the rod inner and outer segments (St. Jules, R. S., and O'Brien, P.J. (1986) Exp. Eye Res. 43, 929-940). Incubation of labeled rhodopsin with mercaptoethanol resulted in release of the labeled palmitate indicating the presence of a thioester bond. This also illustrates the ease with which a thioester, such as palmitoyl cysteine or palmitoyl-CoA, can transfer the fatty acyl group to a free thiol, such as cysteine or mercaptoethanol.     

RNA polymerases B and C are more closely related to each other than to RNA polymerase A

Amino acid sequence comparison of the largest subunit of the three forms of yeast nuclear RNA polymerase disclosed six major conserved regions that are partly retained in the cognate subunits from bacteria, viral, and insect enzymes (Mémet, S., Gouy, M., Marck, C., Sentenac, A., and Buhler, J.-M. (1988) J. Biol. Chem. 263, 2830-2839). Within these conserved domains, the high sequence similarity of B220 and C160 subunits (52% identity) sets them apart from yeast enzyme A subunit A190. Parsimony analysis at the gene and protein levels suggests the existence of a transient ancestor to eukaryotic RNA polymerases B and C. These results are discussed in the light of the recent finding of class C genes containing RNA polymerase B promoter elements.