Calcium-dependent interaction of transducin with calmodulin Sepharose

Affinity chromatography on calmodulin Sepharose showed that transducin, the G protein of bovine retinal rod outer segments, interacts with the Ca²⁺-calmodulin complex. This may mean that in the dark, rod outer segment calmodulin is largely in the bound state. It was assumed that photoactivation of rods induces a change in the calmodulin concentration in the cytoplasm of rod outer segments and this may be one of the processes leading to light adaptation of the photoreceptor.     

A photoreceptor calcium binding protein is recognized by autoantibodies obtained from patients with cancer-associated retinopathy

Cancer-associated retinopathy (CAR), a paraneoplastic syndrome, is characterized by the degeneration of retinal photoreceptors under conditions where the tumor and its metastases have not invaded the eye. The retinopathy often is apparent before the diagnosis of cancer and may be associated with autoantibodies that react with specific sites in the retina. We have examined the sera from patients with CAR to further characterize the retinal antigen. Western blot analysis of human retinal proteins reveals a prominent band at 26 kD that is labeled by the CAR antisera. Antibodies to the 26-kD protein were affinity-purified from complex CAR antisera and used for EM-immunocytochemical localization of the protein to the nuclei, inner and outer segments of both rod and cone cells. Other antibodies obtained from the CAR sera did not label photoreceptors. Using the affinity-purified antibodies for detection, the 26-kD protein, designated p26, was purified to homogeneity from the outer segments of bovine rod photoreceptor cells by Phenyl-Sepharose and ion exchange chromatography. Partial amino acid sequence of p26 was determined by gas phase Edman degradation and revealed extensive homology with a cone-specific protein, visinin. Based upon structural relatedness, both the p26 rod protein and visinin are members of the calmodulin family and contain calcium binding domains of the E-F hand structure.     

Calcium-dependent interaction of transducin with calmodulin-sepharose

It has been shown by affinity chromatography on calmodulin-sepharose that transducin, a G protein of bovine retinal rod outer segments interacts with Ca(2+)-calmodulin. This result assumes that the main part of calmodulin in dark retinal rod outer segments is associated with transducin. It has been suggested that photoactivation of retinal rods induces changes in intracellular calmodulin concentration, which may be one of the steps involved in the light adaptation of photoreceptor.     

Comparison of the phosphodiesterase inhibitory subunit interactions of frog and bovine rod outer segments.

The rod outer segments of the bovine and frog retina possess a cyclic GMP phosphodiesterase (PDE) that is composed of two larger subunits, alpha and beta (P alpha beta), which contain the catalytic activity and a smaller gamma (P gamma) subunit which inhibits the catalytic activity. We studied the binding of P gamma to P alpha beta in both the bovine and frog rod outer segment membranes. Analysis of these data indicates that there are two classes of P gamma binding sites per P alpha beta in both species. The activation of PDE by the guanosine 5'-[gamma-thio]triphosphate form of the alpha subunit of transducin, T alpha.GTP gamma S, was also studied. These data indicate that the two classes of P gamma binding sites contribute to the formation of two classes of binding sites for T alpha.GTP gamma S. We demonstrate solubilization of a portion of the P gamma by T alpha.GTP gamma S in both species. There is also present, in both species, a second class of P gamma which is not solubilized even when it is dissociated from its inhibitory site on P alpha beta by T alpha.GTP gamma S. The amount of full PDE activity which results from release of the solubilizable P gamma is about 50% in the frog PDE but only approx. 17% in the bovine PDE. We also show that activation of frog rod outer segment PDE by trypsin treatment releases the PDE from the membranes. This type of release by trypsin has already been demonstrated in bovine rod outer segments [Wensel & Stryer (1986) Proteins: Struct. Funct. Genet. 1, 90-99].     

Correlation of rhodopsin biogenesis with ultrastructural morphogenesis in the chick retina

The developing chick retina from stages 39-45 has been examined by biochemical and electron microscope techniques. The levels of rhodopsin contained in the maturing chick retina were evaluated by detergent extraction and correlated with rod outer segment formation. It was found that the appearance of rhodopsin in significant levels preceded outer segment formation by at least 2 days, thus implying that rhodopsin is synthesized in the receptor cell inner segment and translocated to the outer limb when disk membrane biogenesis occurs. The level of rhodopsin continues to rise as the rod outer segment develops. Development of both rods and cones originates and proceeds most rapidly in the fundus or central region and proceeds toward the periphery. In general, rod outer segments were noted to develop far more rapidly than cone outer segments.     

Detection of annexin IV in bovine retinal rods

The fraction of proteins capable of binding to photoreceptor membranes in a Ca²⁺-dependent manner was isolated from bovine rod outer segments. One of these proteins with apparent molecular mass of 32 kD (p32) was purified to homogeneity and identified as annexin IV (endonexin) by MALDI-TOF mass-spectrometry. In immunoblot, annexin IV purified from bovine rod outer segments cross-reacted with antibodies against annexin IV from bovine liver. This is the first detection of annexin IV in vertebrate retina.     

Phosphorylation reactions in bovine rod outer segments studied by 32P-labelling of intact retina

The protein phosphorylation pattern in the intact bovine retina has been investigated by labelling with 32P-phosphate under incubation conditions that preserve the electrical photoresponse of the photoreceptor cells. The phosphorylation of rod outer segment proteins was analysed after isolation of outer segments from the labelled retina. The global influence of light, Ca2+ and the phosphodiesterase inhibitor, isobutylmethylxanthine, on protein phosphorylation in rod outer segments was analysed. A 12 kDa protein is the most prominent phosphorylated species in the intact bovine retina. Its phosphorylation is increased by light and/or Ca2+. Evidence is presented that this strongly phosphorylated protein is not located in the outer segment, and we suggest that it may be a synaptic protein. Retinal rod outer segment membrane proteins with apparent molecular weights of 245, 226, 125, 110, 50, 46, 38 and 20 all show light-stimulated phosphorylation. Lowering the extracellular Ca2+ levels results in a decrease of the phosphorylation level of some of these proteins, viz. at 125, 50, 38 and probably at 20 kDa. Such proteins, whose phosphorylation level is influenced both by light and by elevated Ca2+, are candidates for mediators of phototransduction. The phosphorylated species at 245, 226, 110, 50 and 20 kDa are enriched in rod outer segment plasma membrane preparations. These protein species could participate in the light-regulated modulation of the Na+-conductance of the plasma membrane.     

Light-induced dephosphorylation of a 33K protein in rod outer segments of rat retina

Phosphorylated proteins may play an important role in regulating the metabolism or function of rod photoreceptors. In mammalian retinas, a photoreceptor protein of 33 000 (33K) molecular weight is phosphorylated in a cyclic nucleotide dependent manner in vitro. Since light initiates the activation of a photoreceptor-specific phosphodiesterase and a rapid reduction in guanosine cyclic 3',5'-phosphate concentration, phosphorylation of the 33K protein may be modulated by light in situ. In order to test this possibility, dark-adapted rat retinas were incubated for 30 min in the dark in phosphate-free Kreb's buffer containing [32P]orthophosphate. Following incubation, rod outer segments were detached by shaking, and the 32P-labeled rod outer segment proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, detected by autoradiography, and quantitated by densitometric scanning. The incorporation of radioactivity (32P) into the 33K protein was higher than into any other rod outer segment protein, and the amount of 32P-labeled 33K protein in the detached rod outer segments remained unchanged during 10 additional min of darkness. The addition of isobutylmethylxanthine to the incubation medium enhanced the incorporation of 32P into 33K protein to about 400% of the original level. Exposure of freshly detached rod outer segments to room light for 90 s decreased the amount of labeled 33K protein to 45% of its original level. The dephosphorylation of labeled 33K protein continued, reaching 12% of the original dark value 10 min after the previously illuminated sample was returned to darkness. Light initiated the phosphorylation of rhodopsin, and rhodopsin phosphorylation continued during the postillumination period of darkness.     

Molecular mechanisms of photoreception. V. Isolation of cytoplasmic membranes of rod outer segments from the frog retina

Cytoplasmic membranes of rod outer segments from frog retina intact rods in retina were stained with fluorescent dye fluoresceinmonomercur acetate. The dye is covalently bound to proteins of cytoplasmic membrane and doesn't penetrate into the cells. Upon isolation of the purified outer segments with the labeled cytoplasmic membranes the cells were disrupted and fractionated in density sucrose gradient. Cytoplasmic membranes possess floating densities different from those of disk membranes and thus providing a mean for separating them from the latter. The main peptides of cytoplasmic membranes are 56, 53, 45, 30 and 28 kDa proteins.     

Metabolism of phosphatidylethanolamine in the frog retina

The synthesis and the turnover of phosphatidylethanolamine in frog retinal rod outer segments and microsomes were studied by monitoring the incorporation of five radioactive precursors: 32PO4, 33PO4 [3H]glycerol, [3H]serine, and [3H]ethanolamine. 1. Labeled serine was actively incorporated into phosphatidylethanolamine. The kinetics of the labeling patterns in both microsomes and rod outer segments was consistent with formation via decarboxylation of phosphatidylserine. 2. Ethanolamine was found to be an ineffective precursor of phosphatidylethanolamine, suggesting that the major pathway for phosphatidylethanolamine synthesis in the retina is via the decarboxylation reaction. 3. An active methylation of phosphatidylethanolamine to phosphatidylcholine was observed in both retinal microsomes and rod outer segments. 4. The kinetics of labeling of phosphatidylethanolamine in the rod outer segments was different for the various isotopic precursors, and was found to depend on the relative turnover times of the precursor pools. Glycerol was the only precursor that gave a true pulse of radioactivity. 5. The specific activity of phosphatidylethanolamine derived from labeled glycerol declined exponentially, demonstrating that the labeled lipid was diffusely distributed throughout the rod outer segments. The half-life of phosphatidylethanolamine in the rod outer segments was determined to be 18 days. Comparison of this value to the turnover time of rod outer segment integral proteins revealed that rod outer segment lipid is renewed at a faster rate than protein.     

Interaction of retinal guanylate cyclase with the alpha subunit of transducin: potential role in transducin localization

Vertebrate phototransduction is mediated by cGMP, which is generated by retGC (retinal guanylate cyclase) and degraded by cGMP phosphodiesterase. Light stimulates cGMP hydrolysis via the G-protein transducin, which directly binds to and activates phosphodiesterase. Bright light also causes relocalization of transducin from the OS (outer segments) of the rod cells to the inner compartments. In the present study, we show experimental evidence for a previously unknown interaction between G(alphat) (the transducin alpha subunit) and retGC. G(alphat) co-immunoprecipitates with retGC from the retina or from co-transfected COS-7 cells. The retGC-G(alphat) complex is also present in cones. The interaction also occurs in mice lacking RGS9 (regulator of G-protein signalling 9), a protein previously shown to associate with both G(alphat) and retGC. The G(alphat)-retGC interaction is mediated primarily by the kinase homology domain of retGC, which binds GDP-bound G(alphat) stronger than the GTP[S] (GTPgammaS; guanosine 5'-[gamma-thio]triphosphate) form. Neither G(alphat) nor G(betagamma) affect retGC-mediated cGMP synthesis, regardless of the presence of GCAP (guanylate cyclase activating protein) and Ca2+. The rate of light-dependent transducin redistribution from the OS to the inner segments is markedly accelerated in the retGC-1-knockout mice, while the migration of transducin to the OS after the onset of darkness is delayed. Supplementation of permeabilized photoreceptors with cGMP does not affect transducin translocation. Taken together, these results suggest that the protein-protein interaction between G(alphat) and retGC represents a novel mechanism regulating light-dependent translocation of transducin in rod photoreceptors.     

Surfaces of rod photoreceptor disk membranes: light-activated enzymes

The light-activated GTP-binding protein (GBP) in toad rod outer segments has been located on the cytoplasmic surface (CS) of rod disk membranes by correlating biochemical results with images of quick- frozen, freeze-fractured, and deep-etched rod outer segments. This has been accomplished by selectively removing and replacing the 8-12-nm particles that are found on the CS of disk membranes, exactly in parallel with the GBP. In contrast, the large particles are not correlated with another major disk enzyme, the light-activated cGMP phosphodiesterase. We have been unable to visualize this protein. The surface density of large particles, one particle per eleven rhodopsins in isolated rod outer segments and one particle per nine rhodopsins in intact retina, correlates well with previous biochemical estimates of GBP numbers based on enzyme activity. After the identification of the large particles, we tested the effects of light on the density of particles on the surface of disk membranes in intact retinas. Retinas quick-frozen at various intervals after a bright flash of light show a modest increase (approximately 30%) in particle density by 10 s after the flash but no increase before 1 s. The number of particles on the disk membrane returns to dark levels between 1 and 10 min after the flash. The 1-s latency in the change of particle binding would appear to rule out this process as a mechanism for initiating phototransduction in the rod.     

Guanylate cyclase in rod outer segments of the toad retina. Effect of light and Ca2+

Guanylate cyclase activity was measured in disrupted rod outer segments of the toad retina. The experiments showed that cGMP is synthesized from GTP at a rate of 3 +/- 1 nmol/min per mg protein. In darkness this value is largely independent of the Ca2+ concentration, while it is enhanced by flashes of light of increasing intensity upon lowering Ca from 10-5 to 10-8 M. In view of recent observations that shortly after a flash of light calcium activity inside the photoreceptor cell decreases, it seems likely that calcium plays a regulatory role in cGMP metabolism in visual excitation.     

Ultrastructural localization of rhodopsin in the vertebrate retina

Early work by Dewey and collaborators has shown the distribution of rhodopsin in the frog retina. We have repeated these experiments on cow and mouse eyes using antibodies specific to rhodopsin alone. Bovine rhodopsin in emulphogene was purified on an hydroxyapatite column. The purity of this reagent was established by spectrophotometric criteria, by sodium dodecyl sulfate (SDS) gel electrophoresis, and by isoelectric focusing. This rhodopsin was used as an immunoadsorbent to isolate specific antibodies from the antisera of rabbits immunized with bovine rod outer segments solubilized in 2% digitonin. The antibody so prepared was shown by immunoelectrophoresis to be in the IgG class and did not cross-react with lipid extracts of bovine rod outer segments. Papain-digested univalent antibodies (Fab) coupled with peroxidase were used to label rhodopsin in formaldehyde-fixed bovine and murine retinas. In addition to the disk membranes, the plasma membrane of the outer segment, the connecting cilium, and part of the rod inner segment membrane were labeled. We observed staining on both sides of the rod outer segment plasma membrane and the disk membrane. Discrepancies were observed between results of immunolabeling experiments and observations of membrane particles seen in freeze-cleaved specimens. Our experiments indicate that the distribution of membrane particles in freeze cleaving experiments reflects the distribution of membrane proteins. Immunolabeling, on the other hand, can introduce several different types of artifact, unless controlled with extreme care.     

EFFECT OF LIGHT ON GANGLIOSIDES FROM CALF RETINA AND PHOTORECEPTORS

—The gangliosides of the whole calf retina and the rod outer segments have been analysed. This has been done in two functional states: before and after stimulation by light. After exposure to light no statistically significant change in the gangliosides of the whole retina was observed, but a 40 per cent increase in concentration was found in the rod outer segments. This difference was apparent only when using the same batch of rod outer segments. The major ganglioside in the whole calf retina is G_D3 which accounts for 46 per cent of the total. Three other gangliosides G_D1a, G_D1b and G_T1 are quantitatively important, each being between 12 and 16 per cent. G_Q1, G_M1, and G_M3 are minor constituents. In contrast to the chicken retina, G_M2 was not detected. The ganglioside N-acetylneuraminic acid of the rod outer segments accounts for only 1 per cent of the gangliosides of the whole retina. The composition of the gangliosides in the rod outer segments is essentially the same as that of the whole retina. No difference in the relative proportion of the gangliosides of either the rod outer segments or the whole retina was observed after exposure to light.     

Properties of the membrane current of rod outer segments.

The membrane current of single rod outer segments in pieces of isolated toad retina was recorded with a glass suction electrode. Light evoked a slow net outward photocurrent consisting of a reduction in the steady inward dark current. In very dim light, the photocurrent broke up into discrete shot effects with a rounded shape and an amplitude of about 1 pA. These events were shown to result from photoisomerization of single rhodopsin molecules. The current in darkness showed fluctuations consisting of (a) discrete events apparently resulting from thermal isomerization of rhodopsin molecules, and (b) smaller amplitude shot effects shaped by two of the four rate processes of the light response.     

cGMP phosphodiesterase in rod and cone outer segments of the retina

Immunochemical, chromatographic, and sodium dodecyl sulfate gel electrophoresis studies suggest that immunologically related but distinct cyclic GMP phosphodiesterases are present in rod and cone outer segments of the retina. Immunocytochemical studies demonstrated that one monoclonal antibody (ROS-1) recognized a determinant present in both rod and cone outer segments, while another monoclonal antibody (ROS-2) only recognized rod outer segments. At least two peaks of phosphodiesterase activity could be separated by high-performance anion-exchange chromatography of retinal extracts. Both peaks were recognized by ROS-1. None of the first peak and only 80% of the second broad peak of activity were recognized by ROS-2. High-performance liquid chromatography profiles from human fovea and several other types of cone-enriched retina showed that most of the activity was contained in the first peak, suggesting that this activity was derived from cone outer segments. Conversely, the phosphodiesterase in rod-enriched preparations migrated predominately in the second peak. Sodium dodecyl sulfate-gel electrophoresis indicated that this first peak contained a single large immunoreactive polypeptide (alpha') that migrated with the same mobility as a phosphorylase b standard and was distinct from the more rapidly migrating large immunoreactive polypeptides (alpha and beta) present in a broad second peak. The second peak could be further separated into a first part that contained a doublet of two immunoreactive polypeptides (alpha and beta) that migrated faster than phosphorylase b and a later part that contained only the most rapidly migrating polypeptide (beta). All of the peaks could be activated by histone or transducin:GTP, implying that all contained a small 11-kDa inhibitory subunit (gamma) of the enzyme. Since the larger (alpha') and smaller (beta) immunoreactive polypeptides could be completely separated from the alpha polypeptide and from each other, yet still retain the ability to be activated by histone or transducin, the data suggest that only a single species of polypeptide-inhibitor complex (e.g. alpha' gamma, alpha gamma, or beta gamma) was required for histone or transducin:GTP activation.     

Visual transduction in vertebrate photoreceptors. Light activation of guanylate cyclase

Light activation of guanylate cyclase at different calcium concentrations was studied in the rod outer segments of the toad retina. The enzyme becomes sensitive to calcium ions after a flash of light, showing an enhancement of its activity when Ca2+ concentration is lowered from 10(-4) M to 10(-8) M. A possible pathway of guanylate cyclase activation by light was also investigated by means of the antibody 4A to transducin. When added in excess to transducin, the antibody inhibits light activation of phosphodiesterase as well as of cyclase, suggesting a possible coupling of the two enzymes.     

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.     

8-Br-cGMP和cGMP对蟾蜍视杆光敏感通道作用的比较研究

在蟾蜍视网膜的视杆外段上进行片膜钳记录.在同一片膜中,8-Br-cGMP开启的通道,似乎是cGMP所开启的同一光敏感通道.在相同的片膜电位下,由同一片膜诱发同样大小的膜电流,所灌流的8-Br-cGMP溶液的浓度要大大低于cGMP溶液的浓度.再者,8-Br-cGMP被磷酸二酯酸水解的速率,比cGMP的要慢得多.结果表明,在开启视杆外段的光敏感通道的过程中,cGMP分子中的3＇,5＇-环一磷酸可能起主要作用.