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PubMed1992

A third form of the G protein beta subunit. 2. Purification and biochemical properties.

Visual excitation in cones is thought to involve a cone-specific G protein (cone transducin) that transduces the light signal detected by the cone visual pigment into an increase in the enzymatic activity of a cGMP phosphodiesterase. In the preceding paper, we have shown that the G beta 3 isoform of G proteins is specifically localized in bovine cone photoreceptors and proposed that it might be a component of cone transducin. We reported here the purification from bovine retinal extract of a cone-specific T beta 3 gamma complex (where T is transducin), which is composed of a G beta 3 subunit and an immunochemically distinct G gamma subunit. Our purification of this complex is based on a two-stage procedure; the first stage consists of a series of column chromatographies that yield a mixture of purified T beta gamma substantially enriched in T beta 3 gamma, and the second stage involves the removal of all of the rod-specific T beta 1 gamma from the mixture using an affinity column of immobilized monoclonal antibodies directed against the rod T gamma subunit of transducin. Using this procedure, we were able to obtain sufficient amounts of T beta 1 gamma and T beta 3 gamma to begin a comparative study of their properties. We showed that T beta 3 gamma is distinguishable from T beta 1 gamma by isoelectric focusing under nondenaturing conditions. The G beta 3 polypeptide of T beta 3 gamma also migrates slightly slower than the G beta 1 polypeptide of T beta 1 gamma on denaturing polyacrylamide gels. Analysis of the interactions of T beta 3 gamma with other retinal proteins indicated that it has a lower affinity for the T alpha subunit of rod transducin but appears to complex with a phosducin-like protein. The differences in the intrinsic biochemical properties of T beta 3 gamma as compared to T beta 1 gamma may partially account for the lower light sensitivity of cones.

Amino Acid Sequence↗
PubMed1995

Depalmitoylation of rhodopsin with hydroxylamine.

Intrinsic membrane proteins that to date have been investigated with respect to the function of palmitoylation are the beta-adrenergic receptor, rhodopsin, the alpha 2A-adrenergic receptor, and the influenza virus spike glycoprotein. As described above, the studies have led to differing conclusions with respect to the influence of palmitoylation on physiological activity. The basis of the differences remains unclear, but it may relate at least in part to the membrane environment of the protein during these studies, that is, the presence of a native membrane, the membrane composition of the expression cell line (in the case of mutant proteins), or the absence of membrane (in the case of detergent-purified proteins). For example, in the case of rhodopsin, the composition of the ROS disk membrane differs from that of the rod plasma membrane, and presumably also from the plasma membranes of cell lines in which mutant rhodopsins are expressed. Variation in membrane composition is known to have marked effects on the ability of rhodopsin to mediate the photic activation of PDE. Thus, although Karnik et al. clearly demonstrated the absence of an absolute requirement for palmitate in activating transducin, the influence of detergent on tertiary protein structure may have masked the full effect of the elimination of palmitate on the transducin-activating property of rhodopsin. Alternatively, the differing results obtained in the studies of rhodopsin could be a consequence of differences in amino acid sequences of the proteins studied. The precise functional role of the palmitate groups of rhodopsin remains an important question for further research. It was suggested by Ovchinnikov et al. that the hydrolysis of covalently bound palmitate might occur during the process of rhodopsin bleaching, but more recent data argue against this hypothesis. Experiments using synthetic peptides (representing cytoplasmic loop regions of rhodopsin) to identify the sites of interaction of R* and transducin provide support for an alternative possibility, namely, that palmitoylation and the resulting cytoplasmic loop play a role in the coupling of rhodopsin with transducin. The finding that the binding of transducin to R* occurs independently of the presence of palmitate argues against an essential requirement of palmitoylation on the binding step itself. However, available data indicate an enhancement, by depalmitoylation, of light-dependent GTPase activity in ROS preparations, although not in assays of unpalmitoylated, purified mutant rhodopsins (see above).(ABSTRACT TRUNCATED AT 400 WORDS)

Acylation↗
PubMed1995

G protein regulation of the Na+/H+ antiporter in Xenopus laevis oocytes. Involvement of protein kinases A and C.

We have characterized the regulation of the endogenous Na+/H+ exchanger in Xenopus laevis oocytes by G proteins and protein kinases by measuring the ethylisopropylamiloride-sensitive Li+ uptake. Injection of oocytes with the stable GTP analog GTP gamma S stimulated Li+ uptake up to almost 4-fold, an effect blocked by coinjection with the GDP analog, guanyl-5'-yl thiophosphate. Injection into oocytes of beta gamma subunits of the heterotrimeric G protein transducin enhanced Li+ uptake by about 3-fold. This stimulation was blocked by transducin alpha subunits, which by themselves did not influence Li+ uptake. Using various activators and inhibitors of protein kinases, it is demonstrated that the X. laevis oocyte Na+/H+ antiporter can be stimulated by activation of both protein kinase A and C. Stimulation of Na+/H+ exchanger activity by GTP gamma S but not that induced by transducin beta gamma subunits was blocked by the protein kinase A inhibitor H-89. On the other hand, transducin beta gamma subunit-stimulated activity was prevented by the protein kinase C inhibitor, calphostin C. The non-selective protein kinase inhibitor H-7 blocked both GTP gamma S- and transducin beta gamma subunit-stimulated Na+/H+ exchanger activity. The results suggest that the Na+/H+ exchanger of X. laevis oocytes can be activated by G proteins and that this activation is not direct but mediated by protein kinase A- and/or protein kinase C-dependent pathways.

Animals↗
PubMed1986

Cyclic GMP cascade of vision.

Cyclic GMP is central to visual excitation in vertebrate retinal rod cells. Sodium channels in the plasma membrane of the outer segment are kept open in the dark by a high level of cGMP. Light closes these channels by activating an enzymatic cascade that leads to the rapid hydrolysis of cGMP. Photoexcited rhodopsin triggers transducin by catalyzing the exchange of GTP for bound GDP. The activated GTP-form of transducin then switches on the phosphodiesterase by overcoming an inhibitory constraint. The overall gain of this cascade is about 10(5). The cascade is turned off by the GTPase activity of transducin and by the action of rhodopsin kinase and arrestin. One of the challenges now is to delineate the interplay of cGMP, calcium ion, and phosphoinositides in excitation and adaptation. Transducin belongs to a family of signal-coupling proteins that includes the G proteins of the hormone-regulated adenylate cyclase cascade. The initial events in visual excitation in molluscs and arthropods are probably similar to those of vertebrates. The triggering of transducin by photoexcited rhodopsin is a recurring motif in visual transduction. The coming together of electrophysiology, biochemistry, and molecular genetics affords new opportunities in unraveling the molecular mechanism of visual transduction.

Animals↗
PubMed1992

A third form of the G protein beta subunit. 1. Immunochemical identification and localization to cone photoreceptors.

Vertebrate retinal cones play a major role in both photopic vision and color perception. Although the molecular mechanism of visual excitation in the cone is not as well understood as in the rod, it is generally thought to involve a cone-specific G protein (cone transducin) that couples the cone visual pigment to a cGMP phosphodiesterase. Like all other G proteins, cone transducin is most likely a heterotrimer consisting of G alpha, G beta, and G gamma subunits. A G alpha subunit of cone transducin has been localized to the outer segment of bovine cones, but its associated G beta and G gamma subunits are unknown. To identify the G beta subunit involved in the phototransduction process of cones, we have developed a panel of antipeptide antisera against the most diverse region of the amino acid sequences encoded by G beta 1, G beta 2, and G beta 3 cDNAs and used them to determine the distribution of the G beta isoforms in different retinal preparations. We found that the G beta 3 subunit is present in bovine retinal transducin and phosducin-T beta gamma complex preparations which were previously thought to contain only G beta 1. Analysis of its subcellular distribution indicated that G beta 3 is predominantly cytoplasmic. Immunocytochemical staining of bovine retinal sections with the anti-G beta 3 antiserum further revealed a specific localization of G beta 3 in cones but not in rods. In contrast, anti-G beta 1 antiserum stained only the rods. These results suggest that G beta 3 is the G beta subunit of cone transducin and confirms the proposition that rods and cones utilize distinct signaling proteins for phototransduction.

Amino Acid Sequence↗
PubMed1994

Expression of retina-specific genes by mouse retinoblastoma cells.

PURPOSE: Two cell lines derived from ocular tumors of a transgenic mouse expressing the SV40 large T antigen have been established as models of human retinoblastoma. One line, TM, originated from a metastasis, and the other, TE, originated from the primary tumor. The authors compared these two lines with the normal adult mouse eye by analysis of the expression of five photoreceptor cell-specific proteins: IRBP, opsin, rod- and cone-specific transducins, and S-antigen. The authors sought to determine which of these proteins was expressed qualitatively and to examine semi-quantitatively for changes in the levels of expression in the cell lines. METHOD: Western blot analysis was used to detect photoreceptor-specific intracellular or secreted proteins. Total RNA was prepared from cultured cells or from mouse adult whole eye. Specific messenger levels in total RNA were determined either by northern hybridization analysis or by a semi-quantitative polymerase chain reaction (PCR), coupled to complementary DNA (cDNA) substrates prepared from total RNA. RESULTS: IRBP was present in the retinoblastoma cell lines and secreted into the medium. Neither S-antigen nor opsin were detectable by immunoblotting. IRBP and cone transducin mRNA were present in both cell lines. In contrast, opsin, rod transducin, and S-Antigen mRNAs were not detectable by PCR. beta-actin was present in the mRNA populations of whole eye and retinoblastoma. SV40 large T antigen mRNA was present only in retinoblastoma cells. CONCLUSIONS: IRBP and cone transducin expression in mouse retinoblastoma cells is independent of signaling provided directly or indirectly through large T antigen or Rb105 regulatory cascades. The pattern of photoreceptor-specific gene expression is similar to that seen in human retinoblastoma cell lines. These murine-derived cell lines may be useful as a tool to study IRBP and cone transducin expression in vitro and to determine early retinoblast expression patterns in the mouse.

Animals↗
PubMed2002

Receptor-dependent G-protein activation in lipidic cubic phase.

The primary step in cellular signaling by G-protein-coupled receptors (GPCRs) is the interaction of the agonist-activated transmembrane receptor with an intracellular G-protein. Understanding the underlying molecular mechanisms requires the structural determination of receptor G-protein complexes that are not yet achieved. The crystal structure of the bovine photoreceptor rhodopsin, a prototypical GPCR, was solved recently and the structures of different states of engineered G-proteins were reported. Posttranslational hydrophobic modifications of G-proteins are in most cases removed for crystallization but play functional roles for interactions among G-protein subunits with receptors, as well as membranes. Bovine rhodopsin is reconstituted into lipidic cubic phases to assess their potential for crystallization of receptor G-protein complexes under conditions that may preserve the structural and functional roles of hydrophobic protein modifications. Three-dimensional bilayers of a bicontinuous lipidic cubic phase are successfully employed for crystallization of membrane and soluble proteins. UV-visible absorption and attenuated total reflection Fourier transform IR difference spectroscopy reveal that light activation of cubic phase reconstituted rhodopsin results in the generation of a metarhodopsin II-like state. Via diffusion along aqueous channels, transducin couples efficiently to this photoproduct as evidenced by the nucleotide-dependent increase of transducin fluorescence. Thus, rhodopsin transducin interactions do not crucially depend on the presence of sn1 and sn2 acyl chains, phospholipid head groups, or membrane planarity. Because lipidic cubic phases preserve the essential functional and structural properties of native rhodopsin and transducin, they appear suitable for the detergent-free crystallization of receptor G-protein complexes carrying a normal pattern of hydrophobic modifications.

Animals↗
PubMed1995

Mapping regions of G alpha q interacting with PLC beta 1 using multiple overlapping synthetic peptides.

The heterotrimeric G-protein alpha-chain G alpha q plays a critical role mediating receptor-linked activation of the beta isoforms of PLC which hydrolyse membrane inositol-containing phospholipids to generate the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. Despite knowledge of the three-dimensional structure of two G-protein alpha-chains (G alpha t and G alpha i1) as well as high regional amino acid conservation between members of the G-protein alpha-chain family, the precise molecular domains of G alpha q mediating activation of PLC beta 1 are unknown. To map sites responsible for effector interaction we employed 188 peptides each of 15 residues and corresponding to overlapping regions of the complete G alpha q sequence. These were tested for their ability to inhibit G alpha q-dependent activation of recombinant PLC beta 1 using an in vitro reconstitution assay. Peptides from two regions of G alpha q mediated up to 100% inhibition of GTP gamma S-stimulated PLC beta 1 activity, and representative peptides from each of these regions were half-maximally effective at 69.3 +/- 27.4 microM (n = 4) (G alpha q: 251-265) and 110.0 +/- 41.9 microM (n = 4) (G alpha q: 306-319). G alpha q regions described by inhibitory peptides are conserved selectively in other G-protein alpha-chains linked to PLC beta 1 activation (G alpha 11, G alpha 14) and correspond spatially to sites of effector interaction identified in G alpha s by scanning mutagenesis and in transducin using site-specific antibodies and peptides. Computer transducin using site-specific antibodies and peptides. Computer homology modelling of G alpha q based on the crystal structure of transducin indicates that regions interacting with PLC beta 1 form two parallel alpha-helices lying at the surface of the G alpha q structure. These observations provide the first description of two regions within G alpha q critically important for activating PLC beta 1, and moreover, indicate that effector binding domains identified in transducin and G alpha s are also conserved spatially in G alpha q.

Amino Acid Sequence↗
PubMed1992

Functional regions of the inhibitory subunit of retinal rod cGMP phosphodiesterase identified by site-specific mutagenesis and fluorescence spectroscopy.

In the dark, the activity of the cGMP phosphodiesterase (PDE) of retinal rod outer segments is held in check by its two inhibitory gamma subunits. Following illumination, gamma is rapidly removed from its inhibitory site by transducin, the G-protein of the visual system. In order to probe the functional roles of specific regions in the PDE gamma primary sequence, 10 variants of PDE gamma have been produced by site-specific mutagenesis and expression in bacteria and their properties compared to those of protein containing the wild-type bovine PDE gamma amino acid sequence. Three questions were asked about each mutant: What is its affinity for the alpha beta catalytic subunit of PDE? Does it inhibit catalytic activity? If so, can transducin relieve this inhibition? Binding to PDE alpha beta was determined directly using fluorescein-labeled gamma by measuring the increase in emission anisotropy that occurs when gamma binds to alpha beta. Inhibition of PDE alpha beta was measured by reconstitution of the gamma variants with gamma-free PDE generated by limited digestion with trypsin or endoproteinase Arg-C. Unlike trypsin, the latter enzyme did not remove PDE's ability to bind membranes and be activated by transducin, so that transducin activation of PDE containing specific gamma variants could be assayed directly. The results indicate that mutations in many regions of gamma affect its binding to alpha beta. A mutant missing the last five carboxy-terminal residues (83-87) was totally lacking in inhibitory activity. However, it still bound to PDE alpha beta tightly, although with a 100-fold lower dissociation constant (approximately 5 nM) than that of wild-type gamma (approximately 50 pM).(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2000

Light-induced conformational changes of rhodopsin probed by fluorescent alexa594 immobilized on the cytoplasmic surface.

A novel fluorescence method has been developed for detecting the light-induced conformational changes of rhodopsin and for monitoring the interaction between photolyzed rhodopsin and G-protein or arrestin. Rhodopsin in native membranes was selectively modified with fluorescent Alexa594-maleimide at the Cys(316) position, with a large excess of the reagent Cys(140) that was also derivatized. Modification with Alexa594 allowed the monitoring of fluorescence changes at a red excitation light wavelength of 605 nm, thus avoiding significant rhodopsin bleaching. Upon absorption of a photon by rhodopsin, the fluorescence intensity increased as much as 20% at acidic pH with an apparent pK(a) of approximately 6.8 at 4 degrees C, and was sensitive to the presence of hydroxylamine. These findings indicated that the increase in fluorescence is specific for metarhodopsin II. In the presence of transducin, a significant increase in fluorescence was observed. This increase of fluorescence emission intensity was reduced by addition of GTP, in agreement with the fact that transducin enhances the formation of metarhodopsin II. Under conditions that favored the formation of a metarhodopsin II-Alexa594 complex, transducin slightly decreased the fluorescence. In the presence of arrestin, under conditions that favored the formation of metarhodopsin I or II, a phosphorylated, photolyzed rhodopsin-Alexa594 complex only slightly decreased the fluorescence intensity, suggesting that the cytoplasmic surface structure of metarhodopsin II is different in the complex with arrestin and transducin. These results demonstrate the application of Alexa594-modified rhodopsin (Alexa594-rhodopsin) to continuously monitor the conformational changes in rhodopsin during light-induced transformations and its interactions with other proteins.

Amino Acid Sequence↗
PubMed1992

Regulation of deactivation of photoreceptor G protein by its target enzyme and cGMP.

The photoreceptor G protein, transducin, is one of the class of heterotrimeric G proteins that mediates between membrane receptors and intracellular enzymes or ion channels. Light-activated rhodopsin catalyses the exchange of GDP for GTP on multiple transducin molecules. Activated transducin then stimulates cyclic GMP phosphodiesterase by releasing an inhibitory action of the phosphodiesterase gamma-subunits. This leads to a decrease in cGMP levels in the rod, and closure of plasma membrane cationic channels gated by cGMP. In this and other systems, turn-off of the response requires the GTP bound to G protein to be hydrolysed by an intrinsic GTPase activity. Here we report that the interaction of transducin with cGMP phosphodiesterase, specifically with its gamma-subunits, accelerates GTPase activity by several fold. Thus the gamma-subunits of the phosphodiesterase serve a function analogous to the GTPase-activating proteins that regulate the class of small GTP-binding proteins. The acceleration can be partially suppressed by cGMP, most probably through the non-catalytic cGMP-binding sites of phosphodiesterase alpha and beta-subunits. This cGMP regulation may function in light-adaptation of the photo-response as a negative feedback that decreases the lifetime of activated cGMP phosphodiesterase as light causes decreases in cytoplasmic cGMP.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1997

G-protein deactivation is rate-limiting for shut-off of the phototransduction cascade.

Photoreceptors detect light through a seven-helix receptor (rhodopsin) and heterotrimeric G protein (transducin) coupled to a cyclic GMP phosphodiesterase. Similar pathways are used to amplify responses to hormones, taste and smell. The amplification of phototransduction is reduced by a fall in cytoplasmic Ca2+ , but it is not known how the deactivation of rhodopsin and transducin influence this response and hence the extent and duration of phosphodiesterase activity. Here we investigate this by recording the electrical response to flashes of light in truncated rod photoreceptors. By removing ATP to block the deactivation of rhodopsin by phosphorylation, we show that this reaction limits the amplitude of the response and begins within 3.2 s of a flash in a solution containing 1 microM Ca2+, falling to 0.9 s in a zero-Ca2+ solution. In contrast, the activation and amplitude of the response were unaffected when transducin deactivation by GTP hydrolysis was blocked by replacing GTP with its nonhydrolysable analogue GTP-gammaS, demonstrating that there is little GTP hydrolysis occurring over the period in which photoexcited rhodopsin is quenched. The rapid deactivation of rhodopsin is therefore a Ca2+-sensitive step controlling the amplitude of the light response, whereas transducin deactivation is slower and controls recovery.

Adenosine Triphosphate↗
PubMed1985

Effects of phospholipids and ADP-ribosylation on GTP hydrolysis by Escherichia coli-synthesized Ha-ras-encoded p21.

The Ha-ras protooncogene product p21, which may be involved in control of cellular growth, is a membrane protein that binds guanine nucleotides and hydrolyzes GTP. p21 GTPase activity is stimulated by lysophosphatidylcholine; a delay in activation was observed unless p21 was incubated with the phospholipid prior to assay. Maximal activation by the phospholipid was observed over a narrow concentration range; the presence in the assay mixture of lysophosphatidylcholine at concentrations above this optimum markedly inhibited p21 GTPase. GTP hydrolysis was also stimulated, but to a lesser degree, by phosphatidylcholine. Phosphatidylinositol and phosphatidylserine did not significantly enhance GTPase activity. The stimulatory effect of phospholipid was mimicked, in part, by nonionic detergents. p21 may be related to other GTPases, the regulatory guanine nucleotide-binding G proteins of the hormone-sensitive adenylate cyclase complex and transducin of the retinal light-activated phosphodiesterase system. The G proteins and transducin are heterotrimers; the alpha subunits possess GTPase activity and the beta gamma subunit complex along with agonist-receptor complex or light-activated rhodopsin enhance GTP hydrolysis. p21 GTPase activity was slightly stimulated by rhodopsin, but, in contrast to the GTPase activity of transducin, stimulation was not light-dependent. GTP hydrolysis was enhanced somewhat by beta gamma subunit complex in the absence, but not in the presence, of rhodopsin. Like the G proteins and transducin, activity of p21 was altered by ADP-ribosylation. Modification of p21 catalyzed by an NAD: arginine ADP-ribosyltransferase purified from turkey erythrocytes decreased both GTPase activity and guanine nucleotide binding activity.

Adenosine Diphosphate Ribose↗
PubMed1986

Phosphodiesterase activation by photoexcited rhodopsin is quenched when rhodopsin is phosphorylated and binds the intrinsic 48-kDa protein of rod outer segments.

Each photoexcited rhodopsin (R*) molecule catalyzes binding of GTP to many copies of the guanine nucleotide-binding protein transducin, which, in its GTP-binding form, then activates cGMP phosphodiesterase (PDEase). Subsequent deactivation of this light-activated enzyme cascade involves hydrolysis of the GTP bound to transducin, as well as decay of the activating capacity of R*. We report here that deactivation of PDEase in rod outer segment suspensions is highly enhanced by addition of ATP and purified 48-kDa protein, which is an intrinsic rod outer segment protein that is soluble in the dark but binds to photolyzed rhodopsin that has been phosphorylated by rhodopsin kinase and ATP [Kühn, H., Hall, S.W. & Wilden, U. (1984) FEBS Lett. 176, 473-478]. To analyze the mechanism by which ATP and 48-kDa protein deactivate PDEase, we used an ATP-free system consisting of thoroughly washed disk membranes, whose rhodopsin had been previously phosphorylated and chromophore-regenerated, and to which purified PDEase and transducin were reassociated. Such phosphorylated membranes exhibited a significantly lower (by a factor less than or equal to 5) light-induced PDEase-activating capacity than unphosphorylated controls. Addition of purified 48-kDa protein to phosphorylated membranes further suppressed their PDEase-activating capacity; suppression could be as high as 98% (as compared to unphosphorylated membranes), depending on the amount of 48-kDa protein and the flash intensity. Addition of ATP had little further effect. In contrast, PDEase activation or deactivation with unphosphorylated control membranes was not influenced by 48-kDa protein, even in the presence of ATP, provided rhodopsin kinase was absent. Our data suggest that 48-kDa protein binds to phosphorylated R* and thereby quenches its capacity to activate transducin and PDEase.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1988

Rod light adaptation may be mediated by acceleration of the phosphodiesterase-guanylate cyclase cycle.

We compare the retinal rod photocurrent before and after introduction of an hydrolysis-resistant analog of GTP into the outer segment by the whole-cell patch technique. Others have shown that GTP bound to transducin leads to the hydrolysis of cyclic GMP, causing the response to light--a decrease in dark current. The hydrolysis-resistant GTP analog prolongs the response to a bright flash, which leads us to suggest that prolonged transducin activation by bright light desensitizes the rod by a prolonged decrease in dark current. Recovery from the response to a bright flash does occur after introduction of the analog; that recovery requires acceleration of cyclase activity rather than inhibition of phosphodiesterase. The analog mimics light adaptation by desensitizing the rod and speeding the recovery from a dim flash. The analog plus light or light adaptation prolongs the activities of transducin and phosphodiesterase (oligonucleate 5'-nucleotidohydrolase, EC 3.1.4.1) to mediate desensitization by reducing the dark current. Hence, this faster recovery from a dim flash would be by increased activity of guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] rather than by inhibited phosphodiesterase. Accelerated activity of guanylate cyclase may speed recovery by response truncation. We conclude that transducin, activated by photolyzed rhodopsin, may lead to increased activity of both phosphodiesterase and guanylate cyclase to mediate the desensitization and the faster recovery of the light-adapted response.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1998

Characterization and solubilization of bitter-responsive receptors that couple to gustducin.

The tastes of many bitter and sweet compounds are thought to be transduced via guanine nucleotide binding protein (G-protein)-coupled receptors, although the biochemical nature of these receptors is poorly understood at present. Gustducin, a taste-specific G-protein closely related to the transducins, is a key component in transducing the responses to compounds that humans equate with bitter and sweet. Rod transducin, which is also expressed in taste receptor cells, can be activated by the bitter compound denatonium in the presence of bovine taste membranes. In this paper, we show that gustducin is expressed in bovine taste tissue and that both gustducin and transducin, in the presence of bovine taste membranes, can be activated specifically by several bitter compounds, including denatonium, quinine, and strychnine. We also demonstrate that the activation in response to denatonium of gustducin by presumptive bitter-responsive receptors present in taste membranes depends on an interaction with the C terminus of gustducin and requires G-protein betagamma subunits to provide the receptor-interacting heterotrimer. The taste receptor-gustducin interaction can be competitively inhibited by peptides derived from the sites of interaction of rhodopsin and transducin. Finally, as the initial step toward purifying taste receptors, we have solubilized this bitter-responsive taste receptor and maintained its biological activity.

Amino Acid Sequence↗
PubMed2005

Differential regulation of melatonin synthesis genes and phototransduction genes in embryonic chicken retina and cultured retinal precursor cells.

PURPOSE: Photoreceptor differentiation involves the activation of two specific sets of genes; those encoding the proteins of the phototransduction cascade and those encoding the enzymes of the melatonin synthesis pathway, arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole O-methyltransferase (HIOMT). The purpose of the present study was to examine the conditions of AANAT and HIOMT gene activation, relative to that of selected phototransduction markers (alpha-transducin and opsins), in both in vivo and in vitro differentiating photoreceptors of the chicken retina. METHODS: Neural retina RNA was obtained between embryonic day 7 (E7) and posthatch day 8 (P8) and analyzed on northern blots with cDNA probes to AANAT, HIOMT, visinin, alpha-transducin, rhodopsin, and the four cone opsins. Cell cultures were prepared from E7 chicken neural retina and incubated for two to four days in vitro, either in basal medium or in serum-supplemented medium or in medium containing an insulin-based supplement. RNA from the cultured cells was analyzed on northern blots as above. Real time RT-PCR was used to confirm in vitro changes in HIOMT and red opsin mRNA levels. The cultured cells were transfected with promoter-reporter plasmids for direct analysis of HIOMT promoter regulation by the dual luciferase method. RESULTS: The different mRNAs composing the photoreceptor phenotype appeared at E7 (visinin), E10 (alpha-transducin), E14 (HIOMT), E15 (rhodopsin, red opsin, and green opsin), E16 (AANAT), E17 (blue opsin), and E18 (violet opsin). In the early differentiating cones of the central retina, HIOMT mRNA appeared two days earlier than red opsin and green opsin mRNAs (E12 rather than E14). In cultured embryonic neural retina cells, basal medium was sufficient to activate alpha-transducin gene transcription, an insulin-based supplement was sufficient to activate HIOMT gene transcription, whereas serum was required for red opsin gene transcription after two days in vitro. All serum batches were able to activate red opsin gene transcription, whereas some of them failed to activate HIOMT gene transcription. Activation of the HIOMT gene promoter by an insulin-based supplement and by serum was confirmed after transfection of chicken embryonic neural retina cells with promoter-reporter plasmids. CONCLUSIONS: Activation of the melatonin synthesis genes in vivo takes place in a time window very close to that of early opsins. However, a 24-48 h lead of HIOMT gene expression over early opsins was clearly observed. Our in vitro experiments indicate that different exogenous signals are required to activate the different genes encoding photoreceptor specific functions. Significantly, marker genes for light sensitivity (red opsin) and for melatonin synthesis (HIOMT) appear to be activated in response to different signals.

Acetylserotonin O-Methyltransferase↗
PubMed1991

Identifying the recognition unit for G protein methylation.

Signal transducing G proteins, such as transducin, are prenylated and methylated at carboxyl-terminal cysteine residues. The methylation of transducin occurs by means of a membrane bound S-adenosyl methionine-dependent methyltransferase. This methyltransferase accepts the simple modified amino acid N-acetyl-S-farnesyl-L-cysteine (AFC) as a substrate. This means that the enzyme does not require peptide sequences of transducin in a putative substrate. Moreover, small structural changes in the AFC structural unit all lead to molecules incapable of being substrates. For example, neither N-acetyl-S-farnesylhomocysteine (AFHC) nor the saturated form of AFC are substrates. Interestingly, substitution of the N-acetyl moiety of AFC with a hydrogen atom leads to S-farnesylthiopropionic acid (FTP), which is an excellent substrate for the methyltransferase. The methyltransferase shows great specificity for the the FTP pharmacophore. So far, alterations in this structure have not led to active substrates. For example, removal of a methylene group of FTP, producing S-farnesylthioacetic acid (FTA), abolished substrate activity. FTA is a potent competitive inhibitor of the enzyme. FTP is thus the ultimately simplified substrate for the methyltransferase and does not contain any remnants of the peptide structure of transducin.

Animals↗