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PubMed1990

dgq: a drosophila gene encoding a visual system-specific G alpha molecule.

We describe the isolation and preliminary characterization of a new G alpha gene (dgq) in Drosophila. The dgq gene is differentially spliced, yielding two putative proteins, both of which contain guanine nucleotide binding and hydrolysis domains and share 50% identity with transducins and other G proteins. These proteins represent a new class of G alpha subunits because they lack both high amino acid identity with other G alpha proteins and the pertussis toxin ADP ribosylation site. The dgq mRNA is detected by RNA-RNA Northern hybridization in wild-type heads but not in wild-type bodies or in the mutant eyes absent heads. Tissue in situ hybridization detects dgq expression only in the retina and ocellus of the adult head, making it a prime candidate for encoding the Drosophila transducin analog, the G protein required for phototransduction.

Adenosine Diphosphate Ribose↗
PubMed2004

The role of opsin expression and apoptosis in determination of cone types in human retina.

In primates, short wavelength sensitive cones (S cones) and medium- or long-wavelength-sensitive cones (L/M cones) are two separate populations. Each cone type has a different developmental timecourse, contributes to different intra-retinal circuits, and transmits different types of information to the brain. However, in fetal human retina a significant population of cones express both S and L/M opsin (S+L/M cones), raising questions about whether S+L/M cones die or change opsin expression during development. We have utilized fetal, postnatal and adult human retinae to study the immunohistochemical distribution and morphology of S+L/M cones during development. Because S cones appear to be at higher density in fetal compared to adult retinae, we used antibodies to S opsin and alpha-transducin to estimate the proportion of S-cones, and TUNEL labelling to detect apoptotic death in the L/M, S or S+L/M population during development. S cones were present in central retina from fetal week (Fwk)11 and covered the retina by Fwk20. L/M cones appeared in the foveal cone mosaic 3-4 weeks after S-opsin was first detected, and covered the retina by birth. S+L/M cones were detected in all retinae older than Fwk14. They were most numerous at the retinal eccentricity where L/M opsin was just appearing; i.e. at the 'front' of L/M opsin expression. In this region, five morphological types of cones were present. (1) Heavily labelled S cones had thick cell bodies, a thick basal axon and pedicle, and a nucleus at any level of the outer nuclear layer (ONL). (2) Heavily labelled L/M cones were wine goblet shaped with a small round cell body, a large nucleus at the outer ONL edge, and a thin axon with a prominent synaptic pedicle. (3) Goblet-shaped S+L/M cones. (4) Goblet-shaped cones lightly labelled for S-opsin. (5) Cones that were not immunoreactive to either opsin. Only type 1 S cones were present peripheral to the L/M expression front, and their labelling intensity, morphology and distribution indicates that these are the 'true blue' cones of the adult mosaic. Only type 2 L/M cones were present in the foveal cone mosaic. Types 3 and 4 were most numerous within 500-750 microm of the L/M expression front, but type 3 S+L/M cones were also scattered throughout more central regions in fetal, infant and adult retinae. S+L/M cones comprised 5-10% of opsin immunoreactive cones at the L/M front in fetal and early postnatal retinas but 0.01-0.03% throughout P8mo and adult retinae. We found no evidence of significant levels of apoptosis in L/M cones at the expression front, suggesting that this decrease was not due to cell death. The findings suggest that goblet-shaped cones destined to express L or M opsin may initially and transiently express S opsin. Near the optic disc, at Fwk17 S cone density was around 2000 cells mm(-2), which dropped 50% by Fwk20 and stabilized at around 500 cells mm(-2) by birth. Double labelling with alpha-transducin showed that throughout this period 8-10% of all cones expressed S opsin. TUNEL labelling found no significant apoptosis in the S cone population. The decrease in S cone density near the optic disc occurs in the absence of apoptosis, and is likely due to other developmental events acting on the photoreceptor layer, including displacement of cones towards the fovea.

Adult↗
PubMed2004

Rhodopsin photoproducts in 2D crystals.

The published electron microscope and X-ray structures of rhodopsin have made available a detailed picture of the inactive dark state of rhodopsin. Yet, the photointermediates of rhodopsin that ultimately lead to the activated receptor species still await a similar analysis. Such an analysis first requires the generation and characterization of the photoproducts that can be obtained in crystals of rhodopsin. We therefore studied with Fourier-transform infrared (FTIR) difference spectroscopy the photoproducts in 2D crystals of bovine rhodopsin in a p22(1)2(1) crystal form. The spectra obtained by cryotrapping revealed that in this crystal form the still inactive early intermediates batho, lumi, and meta I are similar to those obtained from rhodopsin in native disk membranes, although the transition from lumi to meta I is shifted to a higher temperature. However, at room temperature, the formation of the active state, meta II, is blocked in the crystalline environment. Instead, an intermediate state is formed that bears some features of meta II but lacks the specific conformational changes required for activity. Despite being unable to activate its cognate G protein, transducin, to a significant extent, this intermediate state is capable of interacting with functional transducin-derived peptides to a limited extent. Therefore, while unable to support formation of rhodopsin's active state meta II, 2D p22(1)2(1) crystals proved to be very suitable for determining 3D structures of its still inactive precursors, batho, lumi, and meta I. In future studies, FTIR spectroscopy may serve as a sensitive assay to screen crystals grown under altered conditions for potential formation of the active state, meta II.

Animals↗
PubMed2006

Metarhodopsin-II stabilization by crosslinked Gtalpha C-terminal peptides and implications for the mechanism of GPCR-G protein coupling.

Metarhodopsin-II is the light-excited form of rhodopsin that triggers G protein function. Metarhodopsin-II is stabilized when the N-terminus of the carboxyl (340-350) tail peptide of the alpha-subunit of transducin (Gtalpha) is crosslinked to rhodopsin cysteine 140 or the 340-350 peptide C-terminus of Gtalpha is crosslinked to rhodopsin cysteine 316. When the N-terminus of the peptide is coupled to C316 the MI<-->MII equilibrium is not affected. The evidence suggests that the N-terminus of the 340-350 region of Gtalpha is located near C140 when transducin stabilizes metarhodopsin-II and alternative explanations are suggested for the effectiveness of the 340-350 Gtalpha tail peptide when bound to C316.

Animals↗
PubMed2000

Molecular genetics of Oguchi disease, fundus albipunctatus, and other forms of stationary night blindness: LVII Edward Jackson Memorial Lecture.

PURPOSE: To compare the clinical findings of the various forms of stationary night blindness caused by mutations in identified genes encoding proteins of photoreceptors or the retinal pigment epithelium. METHODS: Review of the visual acuities, visual fields, fundi, dark-adaptation curves, and electroretinograms from patients with stationary night blindness caused by mutations in the genes RHO, GNAT1, PDE6B, RHOK, SAG, RDH5, and CACNA1F, respectively encoding rhodopsin, the alpha subunit of rod transducin, the beta subunit of rod cGMP-phosphodiesterase, rhodopsin kinase, arrestin, 11-cis retinol dehydrogenase, and a retinal L-type calcium channel. RESULTS: In the evaluated forms of stationary night blindness, the time course of dark adaptation and the characteristics of the electroretinogram indicate that rod photoreceptors are present and that they function, although abnormally. In night blindness resulting from defects in rhodopsin, the alpha subunit of rod transducin, or the beta subunit of rod cGMP phosphodiesterase, rod photoreceptors respond only to light intensities far brighter than normal, and the sensitivity of rods to light is similar to that of normal individuals who are not dark adapted. In fundus albipunctatus and in Oguchi disease, the rod photoreceptors can achieve normal sensitivity to dim light but only after 2 or more hours of dark adaptation, compared with approximately 0.5 hours for normal individuals. In each of these forms of stationary night blindness, the poor rod sensitivity and the time course of dark adaptation correlate with the known or presumed physiologic abnormalities caused by the identified gene defects. Patients with some forms of stationary night blindness, such as fundus albipunctatus and Oguchi disease, may develop degeneration of the retina leading to severe loss of vision in later life. CONCLUSIONS: The identification of the mutant genes causing forms of stationary night blindness refines the classification of these diseases and enhances our understanding of the underlying physiologic defects. Ophthalmologists must be aware that although these diseases are traditionally categorized as "stationary," some of them lead to reduced visual acuity or constricted visual fields, especially in older patients. Efforts to develop therapies for these diseases should concentrate on these more severe forms.

Amino Acid Sequence↗
PubMed1988

Different types of ADP-ribose protein bonds formed by botulinum C2 toxin, botulinum ADP-ribosyltransferase C3 and pertussis toxin.

We attempted to characterize ADP-ribose-amino acid bonds formed by various bacterial toxins. The ADP-ribose-arginine bond formed by botulinum C2 toxin in actin was cleaved with a half-life of about 2 h by treatment with hydroxylamine (0.5 M). In contrast, the ADP-ribose-cysteine bond formed by pertussis toxin in transducin and the ADP-ribose-amino acid linkage formed by botulinum ADP-ribosyltransferase C3 in platelet cytosolic proteins were not affected by hydroxylamine. HgCl2 cleaved the ADP-ribose-amino acid bond formed by pertussis toxin in transducin but not those formed by botulinum C2 toxin or botulinum ADP-ribosyltransferase C3 in actin and platelet cytosolic proteins, respectively. NaOH (0.5 M) cleaved the ADP-ribose-amino acid bonds formed by botulinum C2 toxin and pertussis toxin but not the one formed by botulinum ADP-ribosyltransferase C3. The data indicate that the ADP-ribose bond formed by botulinum ADP-ribosyltransferase C3 differs from those formed by the known bacterial ADP-ribosylating toxins.

ADP Ribose Transferases↗
PubMed2003

Piecing together the timetable for visual transduction with transgenic animals.

Transgenic mice bearing null or functional mutations are being used to define the roles of specific elements in phototransduction and also to time the molecular interactions. Genetic manipulation of the collision frequency between rhodopsin and transducin molecules identified this parameter as rate-limiting for the photoresponse onset. Genetic interference with rhodopsin phosphorylation and arrestin binding, transducin shut-off and calcium feedback has revealed their respective roles in shaping the response waveform. The timetable for all of these molecular events determines the amplitude, kinetics and reproducibility of the photoresponse.

Amino Acid Sequence↗
PubMed2003

Light-mediated activation of Rac-1 in photoreceptor outer segments.

Small GTP binding proteins regulate diverse biological processes including gene expression, cytoskeleton reorganization, and protein and vesicular transport. While small GTPases have been investigated in a wide variety of cells, few studies have addressed their role in photoreceptors. In vertebrate retinal rods, the light stimulus is transmitted from rhodopsin via the pathway mediated by the heterotrimeric G protein transducin. To increase their sensitivity to light, photoreceptors accumulate remarkably high concentrations of rhodopsin and transducin in specialized cellular compartments, the outer segments (OS). Transport of these proteins from the inner segments is regulated by the small GTPases Rab6 and Rab8, which do not enter OS. Here, we asked if small G proteins have other functions in photoreceptors. We show that OS contain the small GTPase Rac-1, a member of the Rho family. In contrast to other cells, Rac-1 in OS is exclusively associated with the membranes and resides in lipid rafts. Most importantly, Rac-1 is activated by light. This activation is specifically blocked by a synthetic peptide corresponding to the Asn-Pro-X-X-Tyr motif found in rhodopsin, and Rac-1 coprecipitates with rhodopsin on Concanavalin A Sepharose. These data provide the first direct evidence for the existence of a novel pathway activated by rhodopsin.

Animals↗
PubMedDate not supplied

Cyclic AMP has no effect on the generation, recovery, or background adaptation of light responses in functionally intact rod outer segments: with implications about the function of phosducin.

In retinal rods, light exposure decreases the total outer segment content of both cGMP and cAMP by about 50%. The functional role of the light-evoked change in cAMP is not known. It is postulated to trigger changes in the phosphorylation state of phosducin, a phosphoprotein that is phosphorylated in the dark by cAMP-dependent protein kinase (PKA) and dephosphorylated by basal phosphatase activity when PKA is inhibited by the light-evoked drop in cAMP. In biochemical studies, dephosphorylated phosducin binds to free beta gamma dimer of transducin (Tbeta gamma) and prevents the regeneration of heterotrimeric transducin by blocking the re-association of the beta gamma and alpha subunits. Phosducin's interaction with Tbeta gamma is blocked when it is phosphorylated on a single residue by PKA. To evaluate the effect of the light-evoked fall in cAMP, functionally intact isolated lizard rod outer segments were dialyzed in whole-cell voltage clamp with a standard internal solution and electrical light responses were recorded with and without adding cAMP to the dialysis solution. Since the total outer segment content of cAMP in darkness is approximately 5 microM, internal dialysis with solution containing a much higher concentration (100 microM) of cAMP (or 8-bromo-cAMP) will overcome the effects of a light-evoked decrease in its concentration by keeping cAMP-dependent processes fully activated. Neither cyclic nucleotide had any influence on the generation, light sensitivity, recovery, or background adaptation of the flash response. These results also argue against the participation of phosducin in the sequence of events that are responsible for these aspects of rod function. This does not exclude the possibility of phosducin being involved in adaptation caused by higher light levels than used in the present study, that is, bleaching adaptation, or in light-dependent processes other than phototransduction.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

Structure and function in rhodopsin. 7. Point mutations associated with autosomal dominant retinitis pigmentosa.

Autosomal dominant retinitis pigmentosa (ADRP) is a hereditary form of retinitis pigmentosa which accounts for about 15% of all types of the latter disease. Recently, close to 50 mutations, mostly point mutations, have been identified in the rhodopsin gene in ADRP patients. We have introduced these mutations in the synthetic bovine rhodopsin gene and herein report on the expression of the mutant genes in COS-1 cells and studies in vitro of the properties of the expressed opsins. The mutant phenotypes fall into three classes: Class I mutants are expressed in COS-1 cells at wild-type levels, form the normal rhodopsin chromophore with 11-cis-retinal, and are transported to the cell surface. However, on illumination, they activate transducin inefficiently. Class II mutants remain in the endoplasmic reticulum and do not bind 11-cis-retinal to form the chromophore. Class III mutants are expressed at low levels and form rhodopsin chromophore only poorly. They also remain in the endoplasmic reticulum and, as expected, show high mannose glycosylation. Nearly all of the mutants studied show abnormal sensitivity to light compared to the wild type, and they activate transducin less efficiently. We conclude that the majority of the ADRP mutants have folding defects.

Amino Acid Sequence↗
PubMed1994

Tubulin-G protein association stabilizes GTP binding and activates GTPase: cytoskeletal participation in neuronal signal transduction.

It has been suggested that dimeric tubulin can participate in the signal transduction process through its association with the GTP-binding (G) proteins Gs and Gi1. Using the photoaffinity GTP analog, azidoanilido-GTP, it has been shown that the transfer of nucleotide from tubulin to G alpha s and G alpha i1 is the key step of this activation. The binding sites between tubulin and Gs or G alpha i1 appear to involve microtubule polymerization domains, since G protein alpha subunits were demonstrated to inhibit microtubule assembly [Wang, N., & Rasenick, M. M. (1991) Biochemistry 30, 10957-10965]. In order to understand tubulin-G protein interaction and the nucleotide transfer process in detail, tubulin was labeled with [alpha-32P]GTP or [35S]GTP gamma S and was incubated with recombinant G alpha i1 at increasing molar ratios. Rapid filtration through nitrocellulose was used to determine nucleotide binding in the protein complex. A substantial amount of bound nucleotide was lost from tubulin during the filtration assay. However, the addition of G alpha i1 to [alpha-32P]-GTP-tubulin protected the nucleotide binding in a dose-dependent manner, suggesting a stabilization of GTP binding in the tubulin-G alpha i1 complex. G beta gamma mitigated this effect, and this was not dependent upon the presence of G alpha, suggesting a direct interaction between beta gamma and tubulin. The retinal G protein, transducin, which displayed a much lower affinity for tubulin, did not elicit similar stabilization of GTP binding, and transducin beta gamma did not release GTP from tubulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

A mutant rhodopsin photoproduct with a protonated Schiff base displays an active-state conformation: a Fourier-transform infrared spectroscopy study.

In the rhodopsin mutant E113A/A117E the position of the protonated Schiff base counterion, Glu113, is moved by one helix turn from position 113 to 117. The photoreaction of this mutant pigment was studied by Fourier-transform infrared (FTIR) difference spectroscopy. At acidic pH, formation of a 474-nm absorbing photoproduct previously characterized biochemically as a species that activates transducin caused infrared absorption changes typical of metarhodopsin II (MII) formation in native rhodopsin. Specific spectral alterations revealed a localized perturbation near the protonated Schiff base in the dark state. In addition, an infrared band assigned to the C = O stretching vibration of Glu113 in MII of rhodopsin was abolished in the mutant. Absorption changes caused by Asp83 and Glu122 C = O stretching vibrations characteristic of rhodopsin MII formation were not affected. At alkaline pH, mutant E113A/A117E formed predominantly a 382-nm absorbing photoproduct. It displayed infrared-difference absorption bands significantly different from those of native MII over a large spectral range. These results support the conclusion that the 474-nm photoproduct of mutant E113A/A117E, despite a protonated Schiff base linkage, displays a predominantly MII-like conformation capable of catalyzing guanine-nucleotide exchange by transducin.

Carboxylic Acids↗
PubMed1986

Stimulation of the thiol-dependent ADP-ribosyltransferase and NAD glycohydrolase activities of Bordetella pertussis toxin by adenine nucleotides, phospholipids, and detergents.

Pertussis toxin catalyzed ADP-ribosylation of the guanyl nucleotide binding protein transducin was stimulated by adenine nucleotide and either phospholipids or detergents. To determine the sites of action of these agents, their effects were examined on the transducin-independent NAD glycohydrolase activity. Toxin-catalyzed NAD hydrolysis was increased synergistically by ATP and detergents or phospholipids; the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) was more effective than the nonionic detergent Triton X-100 greater than lysophosphatidylcholine greater than phosphatidylcholine. The A0.5 for ATP in the presence of CHAPS was 2.6 microM; significantly higher concentrations of ATP were required for maximal activation in the presence of cholate or lysophosphatidylcholine. In CHAPS, NAD hydrolysis was enhanced by ATP greater than ADP greater than AMP greater than adenosine; ATP was more effective than MgATP or the nonhydrolyzable analogue adenyl-5'-yl imidodiphosphate. GTP and guanyl-5'-yl imidodiphosphate were less active than the corresponding adenine nucleotides. Activity in the presence of CHAPS and ATP was almost completely dependent on dithiothreitol; the A0.5 for dithiothreitol was significantly decreased by CHAPS alone and, to a greater extent, by CHAPS and ATP. To determine the site of action of ATP, CHAPS, and dithiothreitol, the enzymatic (S1) and binding components (B oligomer) were resolved by chromatography. The purified S1 subunit catalyzed the dithiothreitol-dependent hydrolysis of NAD; activity was enhanced by CHAPS but not ATP. The studies are consistent with the conclusion that adenine nucleotides, dithiothreitol, and CHAPS act on the toxin itself rather than on the substrate; adenine nucleotides appear to be involved in the activation of toxin but not the isolated catalytic unit.

Adenine Nucleotides↗
PubMed2001

Phosphorylation of the regulator of G protein signaling RGS9-1 by protein kinase A is a potential mechanism of light- and Ca2+-mediated regulation of G protein function in photoreceptors.

In vertebrate photoreceptors, photoexcited rhodopsin interacts with the G protein transducin, causing it to bind GTP and stimulate the enzyme cGMP phosphodiesterase. The rapid termination of the active state of this pathway is dependent upon a photoreceptor-specific regulator of G protein signaling RGS9-1 that serves as a GTPase activating protein (GAP) for transducin. Here, we show that, in preparations of photoreceptor outer segments (OS), RGS9-1 is readily phosphorylated by an endogenous Ser/Thr protein kinase. Protein kinase C and MAP kinase inhibitors reduced labeling by about 30%, while CDK5 and CaMK II inhibitors had no effect. cAMP-dependent protein kinase (PKA) inhibitor H89 reduced RGS9-1 labeling by more than 90%, while dibutyryl-cAMP stimulated it 3-fold, implicating PKA as the major kinase responsible for RGS9-1 phosphorylation in OS. RGS9-1 belongs to an RGS subfamily also including RGS6, RGS7, and RGS11, which exist as heterodimers with the G protein beta subunit Gbeta5. Phosphorylated RGS9-1 remains associated with Gbeta5L, a photoreceptor-specific splice form, which itself was not phosphorylated. RGS9-1 immunoprecipitated from OS was in vitro phosphorylated by exogenous PKA. The PKA catalytic subunit could also phosphorylate recombinant RGS9-1, and mutational analysis localized phosphorylation sites to Ser(427) and Ser(428). Substitution of these residues for Glu, to mimic phosphorylation, resulted in a reduction of the GAP activity of RGS9-1. In OS, RGS9-1 phosphorylation required the presence of free Ca(2+) ions and was inhibited by light, suggesting that RGS9-1 phosphorylation could be one of the mechanisms mediating a stronger photoresponse in dark-adapted cells.

Amino Acid Sequence↗
PubMed2006

Minimal determinants for binding activated G alpha from the structure of a G alpha(i1)-peptide dimer.

G-proteins cycle between an inactive GDP-bound state and an active GTP-bound state, serving as molecular switches that coordinate cellular signaling. We recently used phage display to identify a series of peptides that bind G alpha subunits in a nucleotide-dependent manner [Johnston, C. A., Willard, F. S., Jezyk, M. R., Fredericks, Z., Bodor, E. T., Jones, M. B., Blaesius, R., Watts, V. J., Harden, T. K., Sondek, J., Ramer, J. K., and Siderovski, D. P. (2005) Structure 13, 1069-1080]. Here we describe the structural features and functions of KB-1753, a peptide that binds selectively to GDP x AlF4(-)- and GTPgammaS-bound states of G alpha(i) subunits. KB-1753 blocks interaction of G alpha(transducin) with its effector, cGMP phosphodiesterase, and inhibits transducin-mediated activation of cGMP degradation. Additionally, KB-1753 interferes with RGS protein binding and resultant GAP activity. A fluorescent KB-1753 variant was found to act as a sensor for activated G alpha in vitro. The crystal structure of KB-1753 bound to G alpha(i1) x GDP x AlF4(-) reveals binding to a conserved hydrophobic groove between switch II and alpha3 helices and, along with supporting biochemical data and previous structural analyses, supports the notion that this is the site of effector interactions for G alpha(i) subunits.

Aluminum Compounds↗
PubMed1993

Projection structure of rhodopsin.

Light absorption by the visual pigment rhodopsin triggers, through G-protein coupling, a cascade of events in the outer segment of the rod cell of the vertebrate retina that results in membrane hyperpolarization and nerve excitation. Rhodopsin, which contains 348 amino acids, has seven helices that cross the disk membrane and its amino terminus is extracellular. A wealth of biochemical data is available for rhodopsin: 11-cis retinal is bound to lysine 296 in helix VII; glutamic acid 113 on helix III is the counterion to the protonated Schiff's base; a disulphide bridge, cystine 110-187, connects helix III to the second extracellular loop e2 (refs 13, 14); the carboxy terminus has two palmitoylated cysteines forming a cytoplasmic loop i4 (ref. 15); three intracellular loops i2, i3 and i4 mediate activation of the heterotrimeric G protein transducin; glutamic acid 135 and arginine 136 at the cytoplasmic end of helix III affect binding of transducin. But to provide a framework to interpret these data, not only for rhodopsin but for other G-protein-coupled receptors, requires the structure to be determined. Here we present a projection map of rhodopsin showing the configuration of the helices.

Animals↗
PubMed1991

Ischemia of rat brain decreases pertussis toxin-catalyzed [32P]ADP ribosylation of GTP-binding proteins (Gi1 and G0) in membranes.

As an approach to understanding the molecular basis of the pathophysiology of cerebral ischemia, we examined qualitative and quantitative changes in pertussis toxin substrates, Gi1 and G0, in the membrane of rat cerebral cortex after decapitation. Within 1 min after decapitation, the extent of pertussis toxin-catalyzed [32P]ADP ribosylation of the G proteins in the cerebral cortex membrane was significantly decreased and the magnitude of the decrease became slightly larger upon further incubation of the decapitated brain. Addition of guanine nucleotides, GTP and GDP, or the purified beta gamma subunits of transducin to the membranes of control and ischemic cerebral cortex stimulated [32P]ADP ribosylation of the G proteins. The stimulation of [32P]ADP ribosylation in the control situation by guanine nucleotides was almost to the same extent as that in ischemia. However, the stimulation by transducin beta gamma subunits was different; the control stimulation was greater than that in ischemia. In immunoblots probed with antibodies against Gi1 alpha, G0 alpha, and T beta, the immunoreactivity of the corresponding proteins in ischemia was similar to that in control, suggesting that the amounts of G proteins were not changed in ischemia. These results suggest that ischemia accelerates the dissociation of alpha-GDP-beta gamma to alpha-GDP and free beta gamma and causes the denaturation of the dissociated alpha-GDP, thereby decreasing [32P]ADP ribosylation.

Adenosine Diphosphate Ribose↗
PubMed1988

Differential effects of fluoride on adenylate cyclase activity and guanine nucleotide regulation of agonist high-affinity receptor binding.

Fluoride ion, presumably an Al3+-F- complex, has been proposed to activate the guanine nucleotide regulatory protein (G-protein) of the visual system, transducin, by associating with GDP at the nucleotide-binding site and thus mimicking the effects of non-hydrolysable GTP analogues [Bigay, Deterre, Pfister & Chabre (1985) FEBS Lett. 191, 181-85]. We have examined this proposed model by using the adenylate cyclase complexes of frog erythrocytes, S49 lymphoma cells and human platelets. Preincubation of plasma membranes from frog erythrocytes and S49 cells with 20 mM-fluoride for 20 min at 30 degrees C strongly stimulated adenylate cyclase activity. In contrast, the preactivated membranes were still able to bind beta-adrenergic agonist with high affinity, as determined by radioligand-binding techniques. Moreover, high-affinity agonist binding in fluoride-treated membranes was fully sensitive to guanine nucleotide, which decreased beta-adrenergic-receptor affinity for agonist. Very similar results were obtained for [3H]prostaglandin E1 binding to S49 membranes pretreated with fluoride. Incubation of human platelet membranes with increasing concentrations of fluoride (1-50 mM) resulted in biphasic regulation of adenylate cyclase activity, with inhibition observed at concentrations greater than 10 mM. Preincubation of platelet membranes with 20 mM-fluoride did not affect agonist high-affinity binding to alpha 2-adrenergic receptors, nor receptor regulation by guanine nucleotide. These results suggest that the model developed from the study of transducin may not be generally applicable to the G-proteins of the adenylate cyclase system.

Adenylyl Cyclases↗