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PubMed1988

Insulin-dependent phosphorylation of GTP-binding proteins in phospholipid vesicles.

The involvement of GTP-binding proteins (G proteins) in insulin action has been investigated in an in vitro system. Insulin receptors that have been purified by wheat germ lectin chromatography and either tyrosine-agarose chromatography, sucrose density centrifugation, or insulin-Sepharose chromatography have been co-inserted into phospholipid vesicles with different purified G proteins. The results of these studies indicate that a specific insulin-promoted phosphorylation of two G proteins, Go and Gi, can occur in these phospholipid vesicles. Bovine retinal transducin is a poor substitute for Go and Gi, being only weakly phosphorylated by the insulin receptor, and bovine brain Gs is not a substrate. The phosphorylation of Gi and Go occurs primarily on the alpha-subunits. Under optimal conditions, about one alpha o- or alpha i-subunit is phosphorylated on a tyrosine residue for every two beta-subunits of the insulin receptor, suggesting a 1:1 interaction between these G proteins and the heterotetrameric (alpha 2 beta 2) insulin receptor molecular. The inactive (GDP-bound) form of the alpha-subunits appears to be the preferred substrate, with the phosphorylation being significantly reduced in alpha o and alpha i upon the binding of guanosine 5'-O-thiotriphosphate (GTP gamma S) and completely eliminated in the pure alpha-GTP gamma S complex of transducin. The Gi and Go proteins also cause an enhancement of the insulin-stimulated receptor autophosphorylation. This enhancement is a reflection of an increased incorporation of the insulin receptor into lipid vesicles which is induced by these G proteins. Taken together these results provide evidence for the interactions of G proteins with the insulin receptor in a lipid milieu.

Animals↗
PubMed1984

Rhodopsin-enhanced GTPase activity of the inhibitory GTP-binding protein of adenylate cyclase.

Work in several laboratories has shown that Gi, the inhibitory guanyl nucleotide-binding protein of the adenylate cyclase system, is similar in many ways to transducin, the guanyl nucleotide-binding protein of the retinal light-activated cGMP phosphodiesterase system. Separated subunits of purified transducin, T alpha (approximately 39 kDa) and T beta gamma (approximately 35 and approximately 10 kDa), do not exhibit GTPase activity; GTPase activity is observed when the subunits are combined in the presence of rhodopsin ( Fung , B. K.-K. (1983) J. Biol. Chem. 258, 10495-10502). Subunits of Gi, Gi alpha (approximately 41 kDa), and Gi beta gamma (approximately 35 and approximately 10 kDa) were prepared from rabbit liver membranes. It was found that Gi beta gamma could replace T beta gamma in reconstituting the rhodopsin-stimulated GTPase activity of T alpha. Gi alpha exhibited rhodopsin-stimulated GTPase activity when reconstituted with Gi beta gamma or T beta gamma. GTPase activity was a function of Gi alpha concentration when Gi beta gamma or T beta gamma was constant, and the GTPase activity of a given amount of Gi alpha was dependent on Gi beta gamma concentration. These studies demonstrate that the GTPase activity of Gi resides in Gi alpha and further establish that Gi alpha and Gi beta gamma are functionally analogous to T alpha and T beta gamma, respectively.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1993

Identification of guanine nucleotide binding proteins from Trypanosoma cruzi.

Guanine nucleotide binding proteins (GTP-binding proteins) function as transducers of signals in different cellular processes. We have identified several GTP-binding proteins in Trypanosoma cruzi by Western blot analyses. Six polypeptide bands, p20, p25, p28, p31, p37 and p38, were specifically detected in epimastigote crude extracts, using polyclonal antibodies directed against transducin (T) or the alpha-subunit of transducin (T alpha). Four of these bands, p28, p31, p37 and p38, were found in both the soluble and the particulate epimastigote fractions. On the other hand, two of the polypeptides, p20 and p25, were observed only in the particulate fraction, and were not solubilized using 0.2% Triton X-100 and 0.2% Nonidet P-40. A rat monoclonal antibody directed against the ras oncogene, immunorecognized a band with molecular mass of 20,000 daltons, in epimastigote homogenates. In view of their identical apparent molecular weight and solubilization properties, p20, recognized by anti-T or anti-T alpha antibodies, and the 20 KDa band, recognized by anti-ras antibodies, seem to correspond to the same polypeptide. [3H] GDP and [3H] GMP-PNP binding experiments revealed the presence of guanine nucleotide binding proteins in total epimastigote crude extracts, as well as, in the soluble, detergent soluble, and particulate fractions. A primary screening of a T. cruzi cDNA library with anti-T alpha antibodies, followed by secondary and tertiary screenings with anti-ras antibodies yielded six positive clones. One of these clones (Tc-ras1) contains a 600 bp insert which we believe encodes for the ras protein from T. cruzi. On a Northern blot, this cDNA hybridizes to a unique mRNA band of 2.0 Kilobases in epimastigotes.

Animals↗
PubMed1993

Immunochemical detection of GTP-binding protein in cephalopod photoreceptors by anti-peptide antibodies.

We prepared polyclonal antibodies (Pab) against the following peptides: partial sequences of bovine transducin alpha subunit including the ADP-ribosylation sites sensitive to cholera toxin (CTX) and pertussis toxin (PTX) and the N-terminus of Drosophila GTP-binding protein q alpha (DGqN); Pab CTX, Pab PTX and Pab DGqN, respectively. These antibodies were specific to the peptides used as antigen and no crossreactivity was observed. Pab CTX and Pab PTX reacted with bovine transducin alpha subunit and the reactivity was lost by preincubation with the specific antigen peptide. Proteins of 41-42 kDa in octopus and squid photoreceptor membranes were recognized by Pab DGqN but not by Pab CTX or Pab PTX. Anti-alpha antibody (GA/1) reacted with the same bands as Pab DGqN recognized. These results suggest that the major GTP-binding protein in cephalopod photoreceptors is a Gq-type, similar to Drosophila Gq.

Amino Acid Sequence↗
PubMed1994

Effects of carboxyl methylation of photoreceptor G protein gamma-subunit in visual transduction.

G protein gamma-subunits are isoprenylated and carboxyl-methylated at the C-terminal cysteine, which is indispensable for the function of photoreceptor G protein transducin (T alpha beta gamma). However, the physiological role of the methylation and its reversibility have been unclear. Here we isolated methylated and non-methylated forms of farnesylated T beta gamma, and demonstrated that the methylation remarkably facilitates not only the membrane association of T beta gamma but also the subunit interaction between T alpha and T beta gamma. Consequently, the functional coupling of transducin with light-activated receptor, metarhodopsin II, was stabilized by the methylation, resulting in acceleration of GTP gamma S (guanosine 5'-3-O-(thio) triphosphate) binding to T alpha. An examination of the reversibility of the methylation suggested that T gamma is kept fully methylated in rod outer segments. These observations indicate that the methylation of T gamma plays an important role in the most efficient photon-signal transduction process in rod cells.

Adenosine Diphosphate Ribose↗
PubMed1993

Specific interaction with rhodopsin is dependent on the gamma subunit type in a G protein.

Interaction with a receptor is the first step in the process of signal transduction by heterotrimeric (alpha beta gamma) G proteins. We have examined the role of the G protein gamma subunit in interaction between a receptor (rhodopsin) and a G protein, transducin (Gt). We have obtained recombinant beta gamma complexes containing the same beta subunit but three different gamma subunit types by expressing them in the baculovirus/insect cell system. We show that the different recombinant beta gamma complexes interact equally well with the alpha subunit of transducin (alpha t) but only the gamma subunit specific to rod photoreceptors (gamma 1) is able to support interaction of alpha t with rhodopsin. This indicates a direct role for the G protein gamma subunits, which are a family of proteins with diverse structures, in conferring specificity to receptor-G protein interaction.

Amino Acid Sequence↗
PubMed1997

Role of the pineal organ in the photoregulated hatching of the Atlantic halibut.

The timing of hatching in the Atlantic halibut (Hippoglossus hippoglossus) has been suggested to be regulated by environmental light conditions. However, the photosensory organ that perceives the triggering light has not been identified. In the present study, we investigated the morphogenesis of the pineal organ and the neurochemical differentiation of photoreceptors in the pineal organ and the retina of the Atlantic halibut during embryonic development. Immunocytochemical techniques were used for detection of integral protein components of the phototransduction process: opsins, arrestin (S-antigen) and alpha-transducin. We also studied the expression of serotonin (5-HT), a precursor of the neurohormone melatonin known to be synthesized by pineal photoreceptors. In the pineal anlage, opsin immunoreactive (ir) cells appear at 11 days post-fertilization (pf), arrestin, alpha-transducin and serotonin ir cells appear at 14 days pf; hatching took place 15 days pf. The retina contained no immunoreactive cells in embryos or in newly hatched larva. During this period, the pineal anlage is morphologically discernible only as a wedge-shaped region in the diencephalic roof, where elongated cells are aligned with their long axes converging toward a centrally located presumptive pineal lumen. The results show that the pineal photoreceptors contain serotonin and molecules involved in the phototransduction cascade before hatching. We suggest that the pineal organ has the capacity to perceive and mediate photic information before hatching in halibut embryos, and may thereby influence the timing of hatching.

Animals↗
PubMed1998

A CAT reporter construct containing 277bp GNAT2 promoter and 214bp IRBP enhancer is specifically expressed by cone photoreceptor cells in transgenic mice.

PURPOSE: The alpha-subunit of human cone transducin plays an important role in interacting with visual pigment and activating the cGMP-dependent phosphodiesterase (cGMP-PDE). The human GNAT2 gene (cone transducin alpha-subunit) has been cloned and characterized by Fong et al. In this report, we describe the use of transgenic mice to characterize the tissue specificity of the GNAT2 promoter. METHODS: A chimeric reporter gene construct which consists of a 277 bp 5'-flanking fragment of the GNAT2 gene at 5' end of the chloramphenicol acetyltransferase (CAT) gene and a 214 bp enhancer region from the human interphotoreceptor retinoid-binding protein (IRBP) gene at the 3' end of the CAT gene was used to generate transgenic mice. Transgenic mice were identified by Southern blot hybridization and polymerase chain reaction (PCR) analysis using tail DNA from experimental animals. Immunostaining was used to study the developmental expression of CAT and the endogenous GNAT2 gene. RESULTS: Analysis of four transgenic mouse lines revealed that three lines had low CAT activity in the retina. The CAT gene, along with the endogenous GNAT2 gene, was expressed at high levels in cone photoreceptor cells in the fourth transgenic mouse line as determined by CAT enzyme assays and immunostaining. CONCLUSION: The results show that the 277 bp 5'-flanking sequence from the human GNAT2 gene coupled with the 214 bp IRBP enhancer can direct a tissue-specific expression pattern of CAT reporter gene in mouse retina, which parallels the expression pattern of endogenous GNAT2.

Animals↗
PubMed1998

Glycosylation and palmitoylation are not required for the formation of the X-linked cone opsin visual pigments.

PURPOSE: This study was designed to test whether palmitoylation and glycosylation are required for the formation of the green opsin visual pigment. METHODS: Stable cell lines were established by transfecting EBNA-293 cells with a pMEP4ss recombinant plasmid containing wild-type bovine rhodopsin or wild-type or mutant (N32S) green opsin cDNA molecules that included a tag for the eight amino acid residues located at the C-terminus of rhodopsin. The opsins were induced by addition of CdCl2 into the medium and then reconstituted with 11-cis-retinal. The reconstituted opsins were purified by immunoaffinity chromatography, then analyzed by difference spectra, and by binding 35S-GTP in the presence of bovine transducin. Non-reconstituted opsins were analyzed by Western blotting and by pulse-labeling with 3H-palmitic acid followed by immunoprecipitation. RESULTS: Elimination of glycosylation by mutagenesis of the N-linked glycosylation site did not impair the ability of the resulting cone opsin to absorb light at the appropriate wavelength nor to activate transducin. Furthermore, as judged by pulse-labeling with 3H-palmitic acid and immunoprecipitation and by gas chromatography-mass spectroscopy, the wild type green opsin differs from rhodopsin by not being palmitoylated. CONCLUSIONS: Glycosylation and palmitoylation are not required for the formation of cone opsin visual pigments. For the previously described green opsin C203R mutation, disruption of folding and transport, rather than altered glycosylation is sufficient to explain the associated color vision deficiency.

Blotting, Western↗
PubMed2005

Photoreceptor synapses degenerate early in experimental choroidal neovascularization.

Severe visual loss in patients with age-related macular degeneration is associated with the development of choroidal neovascularization (CNV). The pathogenic mechanisms for CNV formation have been extensively investigated, but remarkably little research has addressed the mechanisms for dysfunction of the retina in CNV. Using laser-induced CNV in mice, we evaluated the mechanisms of retinal dysfunction. At 3 days, 1 week, 2 weeks, and 4 weeks after laser application, retinas under experimental CNV were characterized physiologically (ERG recordings, synaptic uptake of the exocytotic marker FM1-43, and light-induced translocation of transducin), histologically, and immunohistochemically. ERG amplitudes were reduced by 20% at 1 week after CNV. Depolarization-induced FM1-43 uptake in photoreceptor synapses was selectively reduced by 45% at 1 week after CNV. Although photoreceptor outer segments were shortened by 36%, light adaptation as measured by transducin translocation was mostly preserved. Early in CNV (3 days to 1 week), Muller cells demonstrated induction of c-fos and pERK expression. Also, the density of macrophage-like, F4/80 immunoreactive cells increased approximately 3-fold. Minimal photoreceptor death occurred during the first week, and was variable thereafter. At later times in CNV formation (> or =2 weeks), expression of photoreceptor synaptic markers was reduced in the outer plexiform layer, indicating loss of photoreceptor synaptic terminals. ERG amplitudes, synaptic uptake of FM1-43, and the induction of c-fos and pERK in Muller cells were altered within 1 week of experimental CNV, suggesting that during CNV formation, deficits in retinal function, in particular photoreceptor synaptic function, precede degeneration of photoreceptor terminals and photoreceptor cell death.

Adaptation, Ocular↗
PubMed2007

Photoreceptor proteins as cancer-retina antigens.

Melanocytes, melanoma and photoreceptor cells are of neuroectodermal origin and have a certain sensitivity to light. In this study, we present evidence for photoreceptor proteins that are responsible for visual transduction and its regulation function as a new class of cancer antigens in melanoma. Visual rhodopsin, transducin, cGMP-phosphodiesterase 6, cGMP-dependent channels, guanylyl cyclase, rhodopsin kinase, recoverin and arrestin are expressed in melanoma and can induce antibody responses in patients. Melanocytes also express mRNA of all photoreceptor genes besides transducin, but were devoid of the corresponding protein, which was tested for rhodopsin, cGMP-phosphodiesterase, guanylyl cyclase and recoverin. Furthermore, we show for the first time that some healthy tissues express mRNA of these genes, but never protein. Expression profiles and autoantibody responses were confirmed in the MT/ret and the HGF(tg)/Ink4a(-/-) transgenic mouse melanoma models. We propose a molecular transition of cancer-retina antigens from mRNA expression in melanocytes to protein expression in melanoma. Our work provides the basis for analyzing regulation of photoreceptor gene expression in normal and malignant cells as well as possible therapeutic tumor targeting using the newly defined class of cancer-retina antigens.

Animals↗
PubMed1986

Expression of p21 proteins in Escherichia coli and stereochemistry of the nucleotide-binding site.

v-Ha-ras encoded p21 protein (p21V), the cellular c-Ha-ras encoded protein (p21C) and its T24 mutant form p21T were produced in Escherichia coli under the control of the tac promoter. Large amounts of the authentic proteins in a soluble form can be extracted and purified without the use of denaturants or detergents. All three proteins are highly active in GDP binding, GTPase and, for p21V, autokinase activity. Inhibition of [3H]GDP binding to p21C by regio- and stereospecific phosphorothioate analogs of GDP and GTP was investigated to obtain a measure of the relative affinities of the three diphosphate and five triphosphate analogs of guanosine. p21 has a preference for the Sp isomers of GDP alpha S and GTP alpha S. It has low specificity for the Sp isomer of GTP beta S. Together with the data for GDP beta S and GTP gamma S these results are compared with those obtained for elongation factor (EF)Tu and transducin. This has enabled us to probe the structural relatedness of these proteins. We conclude that p21 seems to be more closely related to EF-Tu than to transducin.

Cell Line↗
PubMed2001

Inhibition of retinal guanylyl cyclase by the RGS9-1 N-terminus.

Cyclic GMP plays a key role in retinal phototransduction and its photoreceptor concentration is precisely controlled by the cooperative action of cGMP phosphodiesterase (PDE) and retinal guanylyl cyclase (retGC). However, studies of the relationship between these two systems have focused only on a Ca(2+)-mediated, indirect connection. Using a retinal "regulator of G-protein signaling" (RGS9-1) and its fragments, we show that the N-terminus of RGS9-1 inhibits retGC activity. We also indicate that the GGL domain and/or the RGS domain function as an internal suppressor against the N-terminus, suggesting that proteins bound to these domains regulate the inhibitory activity of the N-terminus. Direct interaction of retGC with RGS9-1 and its N-terminus is also proved by immunoprecipitation and an overlay technique. Since RGS9-1 also controls the lifetime of transducin-activated PDE through regulating GTPase activity of transducin, this study strongly suggests that RGS9-1 mediates the direct interaction between PDE and retGC systems, and that this ingenious mechanism plays an important role in tuning of cGMP concentration in photoreceptors.

Amino Acid Sequence↗
PubMed1999

Localization of a visual Gq protein in the photoreceptors of a polychaete, Perinereis brevicirris (Annelida).

Heterotrimeric GTP-binding proteins (G proteins) play an important role in phototransduction. The presence of G-protein subclasses has been reported in photoreceptive membranes, e.g., the Gi subgroup (transducin) in vertebrate rods, and the Gq subgroup in the eyes of the Arthropoda and the Mollusca. We examined the immunoreactivity and distribution of a Gq homologue in the cerebral ocelli of Perinereis brevicirris (Polychaeta, Annelida) using an anti-GqC antibody raised against a conserved sequence at the C-terminal of the alpha-subunit of Gq (Gq-alpha). The anti-GqC antibody labeled a 48-kDa band on the Western blot of proteins from the Perinereis ocelli. The anti-GtC antibody, which is raised against the C-terminal sequence of bovine transducin alpha-subunit (Gt-alpha), did not cross-react to the ocellar proteins of Perinereis. The rhabdomeric layers of the anterior and posterior ocelli were strongly labeled by anti-GqC on light-microscopic immunohistology. Immunoelectron microscopy showed that the Gq molecules were specifically localized in the photoreceptive membrane of the rhabdomeric microvilli. These results suggest that the Gq protein plays a role in the phototransduction of the Perinereis ocelli.

Amino Acid Sequence↗
PubMed1988

Two mechanisms of photoendocrine transduction in cultured chick pineal cells: pertussis toxin blocks the acute but not the phase-shifting effects of light on the melatonin rhythm.

We have recently described a system, using dispersed chick pineal cells in static culture, which displays a persistent, photosensitive, circadian rhythm of melatonin release. Light has two apparent effects on this melatonin rhythm: the first is an acute inhibition of melatonin output, the second is entrainment of the underlying pacemaker. These two effects could be mediated by the same or different mechanisms. Pertussis toxin, which acts to block the function of transducin and certain other G-proteins, blocked the acute effects of light on chick pineal cells, but not the ability of light pulses to induce phase-dependent phase shifts of the rhythm. There must, therefore, be at least two mechanistic pathways by which light affects chick pineal melatonin production. Transducin or other pertussis toxin-sensitive G-proteins would appear to be involved in the acute effects of light on the melatonin-synthesizing apparatus, but not in the effects of light on the circadian pacemaker which generates the melatonin rhythm. Some plausible pertussis toxin-sensitive mechanisms are discussed.

Animals↗
PubMed1988

Active sites of the cyclic GMP phosphodiesterase gamma-subunit of retinal rod outer segments.

Monoclonal antibodies were prepared to the gamma-subunit of the cGMP phosphodiesterase. One of them gamma p-1, suppresses the activation of phosphodiesterase through the alpha-subunit of transducin. The gamma-subunit fragment 24-45 rich in Arg and Lys residues is involved in gamma p-1 binding and is essential for the gamma-subunit interaction with transducin. Carboxypeptidase Y cleaves off seven amino acid residues from the C-terminus of the gamma-subunit resulting in phosphodiesterase activation. Thus, the C-terminal fragment of gamma-subunit participates in phosphodiesterase inhibition.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1991

Binding of inositol phosphates to arrestin.

Arrestin binds to phosphorylated rhodopsin in its light-activated form (metarhodopsin II), blocking thereby its interaction with the G-protein, transducin. In this study, we show that highly phosphorylated forms of inositol compete against the arrestin-rhodopsin interaction. Competition curves and direct binding assays with free arrestin consistently yield affinities in the micromolar range; for example, inositol 1,3,4,5-tetrakisphosphate (InP4) and inositol hexakisphosphate (InP6 bind to arrestin with dissociation constants of 12 microM and 5 microM, respectively. Only a small control amount of inositol phosphates is bound, when arrestin interacts with phosphorylated rhodopsin. This argues for a release of bound inositol phosphates by interaction with rhodopsin. Transducin, rhodopsin kinase, or cyclic GMP phosphodiesterase are not affected by inositol phosphates. These observations open a new way to purify arrestin and to inhibit its interaction with rhodopsin. Their physiological significance deserves further investigation.

Animals↗
PubMed1994

Photoreceptor and glial markers in human embryonic retina and in human embryonic retinal transplants to rat retina.

The purpose of this study was to compare the development of photoreceptor and glial cells in human embryonic retinal transplants with the development of normal human embryonic retina (13-20 weeks postconception). Human embryonic retinal cells (donor age 6-11 weeks postconception) were transplanted to the retinas of adult immunosuppressed rat hosts. Host animals were killed when the transplants were of 13-37 weeks total age (donor age+survival time after surgery). Immunohistochemistry was performed with antibodies specific for neuron-specific enolase (NSE), synaptophysin (SYN), cone-specific opsins, rhodopsin, rod alpha-transducin, S-antigen, vimentin, cellular retinaldehyde-binding protein (CRALBP) and glial fibrillary acidic protein (GFAP). With regards to photoreceptors, NSE and SYN immunoreactive cones were seen in transplants from 14-16 weeks of age, but cone opsin immunoreactivity was not seen until 25 weeks. Developing graft rods became S-antigen immunoreactive at 17-18 weeks. At 20 weeks, inner segments and some cell somas of graft rods stained faintly for alpha-transducin and rhodopsin. At 31 and 37 weeks, inner and outer rod segments were intensely labelled for the rod-specific antigens. The grafts exhibited areas of varying maturation with different staining intensities. Concerning the glial cells, vimentin immunoreactivity was seen in the earliest transplants studied (total age 14-16 weeks), but only in stages older than 19 weeks was the immunoreactivity of graft Müller cells comparable in intensity to those of the host retina. Host Müller cells were immunoreactive for GFAP near the lesion site at all times. At 20 weeks, some GFAP immunoreactive processes were seen inside the graft, apparently coming from the host retina. At 25 weeks, faintly stained Müller cells intrinsic to the graft were observed, indicating a gliosis within the graft. Graft Müller cells were first seen to express CRALBP immunoreactivity at 19-20 weeks and, at 25 weeks, intense immunoreactivity was seen in the transplant, mostly in regions near the host. In the transplants only the Müller cells were stained, whereas both Müller and retinal pigment epithelium cells were CRALBP immunoreactive in the host retina. The development of human embryonic retinal transplants appears to parallel approximately normal in utero development. Transplant cones, rods and Müller cells all express their cell-specific proteins. The photoreceptors develop both inner and outer segments and contain several essential proteins for processing light. The transplants can reach a degree of maturity comparable to newborn retina.

Animals↗