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P Deterre

Publications and source records attributed to P Deterre.

At least 19 recordsLinked to original sources
PubMed1994

Activation of phosphodiesterase by transducin in bovine rod outer segments: characteristics of the successive binding of two transducins.

In bovine retinal rods, transducin loaded with GTP or GTP gamma S (T*) activates a cGMP phosphodiesterase (PDE) by forming a tightly membrane-bound complex with it [Catty, P., et al. (1992) J. Biol. Chem. 267, 19489-19493]. Up to two T*s are able to bind to PDE [Clerc, A., & Bennett, N. (1992) J. Biol. Chem. 267, 6620-6627]. We analyze here PDE activation by two successive bindings of T*. In the mathematical model used, we took into account that the membrane concentration determines the amount of PDE able to interact efficiently with T* through the attachment of PDE itself to the membrane. We therefore fitted the data obtained over a wide range of membrane and PDE concentrations. We found that the binding of the first T* to PDE elicits 80-100% of the maximal activity of PDE, whereas the binding of the second T* to PDE elicits little or no additional activation of PDE. This finding profoundly differs from previous conclusions. The carefully controlled conditions of our experiments permit one to understand these discrepancies. In the physiological situation, PDE would be nearly maximally activated through its interaction with only one T*. The efficient binding of the second T* to those complexes would then ensure a rapid deactivation of T* through the enhancement of the rate of GTP hydrolysis in T* bound to PDE [Pagès, F., et al. (1992) J. Biol. Chem. 267, 22018-22021; Pagès, F., et al. (1993) J. Biol. Chem. 268, 26358-26364].

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1994

Cell calcium signaling via GM1 cell surface gangliosides in the human Jurkat T cell line.

The cell surface ganglioside GM1 is the specific receptor for the B subunit of cholera toxin. We show here that in the human Jurkat T cell line an increase in intracellular free Ca2+ concentration can be elicited by using B subunits to ligate GM1 molecules. This Ca2+ signaling effect is clearly mediated through GM1 because it can be observed after direct insertion of exogenous GM1 in a Jurkat cell variant deficient in GM1 expression. The observed Ca2+ response clearly involves both the release of Ca2+ from intracellular stores and a Ca2+ influx from extracellular spaces. It is sustained in the presence of 1 mM extracellular Ca2+, whereas it becomes transient in Ca(2+)-free medium. We show that the GM1-mediated stimulation partially empties the CD3-dependent and inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ pool suggesting a dependence of the Ca2+ response from activation of phospholipase C (PLC) metabolism. Accordingly, tyrosine phosphorylation of PLC gamma-1 can be evidenced but only in Jurkat cells highly expressing GM1. GM1 stimulation results in an IL-2 production comparable to that obtained after CD3 activation. Finally, the GM1-linked cell Ca2+ activation pathway is also observed in a Jurkat cell clone lacking Ag-specific receptor expression suggesting that the presence of functional CD3/TCR molecules is not essential for GM1-induced cell Ca2+ response. Altogether, these data show that cell surface gangliosides GM1 may act as a signaling molecule in Jurkat T cells possibly by a new pathway, a finding of importance when considering a possible function for ubiquitous membrane carbohydrate structures in T cell recognition systems.

CD3 Complex↗
PubMed1993

Enhancement by phosphodiesterase subunits of the rate of GTP hydrolysis by transducin in bovine retinal rods. Essential role of the phosphodiesterase catalytic core.

Phosphodiesterase (PDE) in bovine retinal rod outer segments is activated when it forms a membrane-bound complex with the alpha-subunit of transducin loaded with GTP (T alpha*). At maximal activation, this complex contains two T alpha* and all the subunits of native PDE (PDE alpha, PDE beta, and two inhibitory PDE gamma). We observed previously (Pagès, F., Deterre, P., and Pfister, C. (1992) J. Biol. Chem. 267, 22018-22021) that the rate of GTP hydrolysis by transducin in a rod outer segment suspension is enhanced when T alpha* is bound to native PDE (PDE alpha beta gamma 2). In this article, we compare the effects of PDE species with different PDE gamma contents. We show that T alpha* hydrolyzes its GTP faster not only when bound to PDE alpha beta gamma 2, but also when bound to PDE alpha beta gamma or PDE alpha beta. Moreover, trypsin-treated PDE (PDE gamma-deprived soluble PDE) also induces an acceleration of GTP hydrolysis. On the contrary, addition of isolated PDE gamma alone does not accelerate GTP hydrolysis. The interaction between T alpha* and PDE gamma, which is essential for the activation of PDE by T alpha*, is apparently not responsible of the feedback of PDE on T alpha*. The interaction of primary importance for the acceleration of GTP hydrolysis would be that existing between T alpha* and PDE alpha beta.

Animals↗
PubMed1993

The lymphocyte surface antigen CD38 acts as a nicotinamide adenine dinucleotide glycohydrolase in human T lymphocytes.

The extracellular domain of the lymphocyte surface antigen CD38 has been recently shown to share a high sequence homology with a nicotinamide adenine dinucleotide (NAD+)-specific hydrolyzing enzyme cloned from the ovotestis of the gastropod Aplysia (E. States, D.J., Walseth, T.F., Lee, H. C., Trends Biochem. Sci. 1992. 17:495). In agreement with this finding, we present here evidence that CD38-overexpressing T cells, such as human thymocytes and cells from the human HPB-ALL T cell line, exhibit a NAD(+)-hydrolyzing enzymatic activity present on the outer surface of the cell membrane. In contrast, T lymphocytes with relatively low levels of CD38 marker, such as the human Jurkat cell line, display a lower activity. This suggests a relationship between ecto-NAD+ glycohydrolase activity and CD38 expression, as confirmed here when comparing wild-type Jurkat cells and a Jurkat cell variant overexpressing the CD38 molecule. Moreover, CD38 immunoprecipitates from thymocytes behave as an authentic NAD+ glycohydrolase enzyme: it transforms NAD+ stoichiometrically into nicotinamide plus adenosine 5'-diphosphoribose. Altogether these results strongly support the assumption that CD38 is actually a lymphocyte-specific NAD(+)-hydrolyzing enzyme, a finding that give new prospects to understand the in vivo function of this cell membrane protein.

ADP-ribosyl Cyclase↗
PubMed1992

Enhanced GTPase activity of transducin when bound to cGMP phosphodiesterase in bovine retinal rods.

The generation of the physiological response of a retinal rod cell to an incident photon involves activation of a cGMP phosphodiesterase (PDE) by a GTP-binding protein, transducin (T). This activation has been shown to occur by formation of a membrane-bound T alpha GTP-PDE complex (Clerc, A., and Bennett, N. (1992) J. Biol. Chem. 267, 6620-6627; Catty, P., Pfister, C., Bruckert, F., and Deterre, P. (1992) J. Biol. Chem 267, 19489-19493). The recovery of the response involves turning-off of T by its intrinsic GTPase activity. We show here that the formation of the membrane-bound T alpha GTP-PDE complex correlates with an enhanced rate of GTP hydrolysis. In vivo, this would provide an appropriate mechanism for fast turn-off of cGMP hydrolysis.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1992

The cGMP phosphodiesterase-transducin complex of retinal rods. Membrane binding and subunits interactions.

cGMP-specific phosphodiesterase (PDE) of vertebrate retinal rod outer segments (ROS) is composed of two catalytic subunits (PDE alpha and PDE beta) and two identical inhibitory subunits (PDE gamma). Native PDE alpha beta gamma 2 is peripherally bound to the membranes of ROS discs. We studied quantitatively its partition between soluble and membrane-bound fractions in ROS homogenates. In the presence of its activator, the alpha-subunit of transducin loaded with a triphosphate guanine nucleotide (T alpha*), PDE displayed a greatly enhanced membrane binding. Neither the purified PDE gamma.T alpha* complex, nor the PDE alpha beta and PDE alpha beta gamma forms of active PDE, showed a membrane binding comparable to that of PDE alpha beta gamma 2 in the presence of T alpha*. The T alpha*-activated PDE is therefore an undissociated complex tightly bound to the ROS membranes. Using limited proteolysis, we showed that the membrane anchoring of the whole complex implies not only PDE (mainly by the C terminus of PDE beta) but also both termini of T alpha*. The membrane binding of the purified PDE alpha beta species was also enhanced in the presence of T alpha*; a direct link would therefore exist between the activator and the catalytic subunits. From this work emerges a plausible structural model of the T alpha*-activated PDE, with its internal interactions and its sites of anchoring into the ROS membrane.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1992

Role of G protein beta gamma subunits in the regulation of the plasma membrane Ca2+ pump.

In Zajdela hepatoma cells (ZHC) the plasma membrane Ca2+ pump displayed no sensitivity to glucagon (19-29) (mini-glucagon), whereas in hepatocyte this metabolite of glucagon evoked a biphasic regulation of the Ca2+ pump system via a cholera toxin-sensitive G protein. Analysis of G protein subunits in ZHC membranes indicated the presence of cholera toxin-sensitive Gs alpha and G beta gamma proteins, whose functionality was manifested by GTP and NaF stimulation of adenylylcyclase activity, and pertussis toxin-catalyzed ADP-ribosylation of Gi alpha, respectively. However, immunoblotting experiments suggested a lower content in beta gamma subunits in ZHC as compared with hepatocyte plasma membranes. Complementation of ZHC or hepatocyte plasma membranes with purified beta gamma subunits from transducin (T beta gamma) caused inhibition of the basal activity of the Ca2+ pump at 10 and 300 ng/ml, respectively, and revealed (in ZHC) or increased (in hepatocytes) sensitivity of the system to mini-glucagon. After cholera toxin treatment of ZHC, T beta gamma no longer reconstituted the response of the Ca2+ pump to mini-glucagon, suggesting that the mechanism of beta gamma action is dependent on an association with the alpha subunit of a cholera toxin-sensitive G protein. It is concluded that G beta gamma subunits control both the basal activity of the plasma membrane Ca2+ pump and its inhibition by mini-glucagon.

Adenosine Diphosphate Ribose↗
PubMed1991

Activation and solubilization of the retinal cGMP-specific phosphodiesterase by limited proteolysis. Role of the C-terminal domain of the beta-subunit.

The cGMP-specific phosphodiesterase (PDE) of vertebrate retinal rod outer segments (ROS) is a peripheral enzyme activated in vivo by transducin. In vitro artificial activation can be achieved using trypsin. This was described as resulting from degradation of the inhibitory gamma subunit (2 copies/PDE molecule), leaving intact the alpha beta catalytic core. It was, however, observed that trypsin could induce the release of PDE (or solubilization) from the ROS membranes before its activation [Wensel, T. G. & Stryer, L. (1986) Proteins Struct. Funct. Genet. 1, 90-99]. Studying the time course of this solubilization, we were able to purify a trypsin-solubilized PDE still completely inhibited (i.e. with its two gamma subunits bound). The tryptic solubilization of PDE is therefore complete before any functional degradation of the gamma subunits occurs. It was recently suggested that this solubilization could coincide with the cleavage of a C-terminal fragment of the alpha subunit, which can be labeled by methylation of a terminal cysteine residue [Ong, O. C., Ota, I. M., Clarke, S. & Fung, B. K. K. (1989) Proc. Natl Acad. Sci. USA 86, 9238-9242]. We present the following evidence indicating that the C-terminus of the PDE beta subunit is mainly responsible for PDE anchorage to the ROS membrane. (a) The trypsin-solubilized PDE alpha beta gamma 2 has intact blocked N-termini. (b) It is still methylated on PDE alpha. (c) The C-terminus of PDE beta can also be labeled by methylation and its tryptic cleavage coincides well with the PDE solubilization. (d) Sequential cleavage of the alpha and beta polypeptides can also be detected by high-resolution gel electrophoresis: the first cleavage appears on the beta subunit and is completed when cleavage of the alpha subunit begins. The time course for cleavage of the gamma subunits appears to be slower than for the beta subunit and comparable to that of the alpha subunit. Upon longer trypsinization, a 70-kDa polypeptide appears which seems to be a degradation product of PDE beta. Gel-filtration analysis, however, shows that this 70-kDa fragment does not dissociate from the catalytic core.

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

cGMP phosphodiesterase of retinal rods is regulated by two inhibitory subunits.

The cGMP phosphodiesterase (PDE) of cattle retinal rod outer segments comprises three types of subunits: the two heavy catalytic ones, PDE alpha and PDE beta, each around 85 kDa, and the light inhibitory one, PDE gamma or I (11 kDa). The relative stoichiometry is usually assumed to be 1:1:1. PDE activation in the visual transduction cascade results from removal of the inhibitor by the alpha subunit of transducin (T alpha). The stoichiometric complex T alpha-I, separated from activated PDE, has been isolated and characterized. Analyzing now the activated PDE, we find that it still contains some inhibitor and is resolvable into two species, one with 50% of the inhibitor content of the native enzyme and the other totally devoid of it. The same two species are observed upon activation of PDE by very short tryptic proteolysis, which specifically degrades the inhibitor. This leads us to conclude that the composition of the native enzyme is PDE alpha beta-I2. The two inhibitory subunits are differentially bound, sequentially removable, and exchangeable between the native complex PDE alpha beta-I2 and the fully active PDE alpha beta. The possibility of this exchange precludes as yet an unambiguous estimate of the actual activity of the intermediate complex PDE alpha beta-I. The differential binding and the exchangeability of the inhibitors raises the possibility of a fast, diffusion controlled, switch-off mechanism of PDE activity after a flash, which would shortcut the inactivation resulting from the slow GTPase rate of transducin.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1987

Antibodies directed against transducin beta subunits interfere with the regulation of adenylate cyclase activity in brain membranes.

In an attempt to study the mechanisms of action of membrane-bound adenylate cyclase, we have applied to rat brain synaptosomal membranes antibodies raised against purified bovine transducin (T) beta gamma subunits. The antibodies recognized one 36-kDa protein in Western blots of the membranes. Adenylate cyclase activation by GTP non-hydrolyzable analogues was greatly decreased in immune, as compared to preimmune, antibody-treated membranes, whereas the enzyme basal activity was unaffected by both types of antibodies. The inhibition of forskolin-stimulated adenylate cyclase by guanine 5'-(beta, gamma-imino)triphosphate (Gpp-(NH)p) was decreased in membranes preincubated with immune, but not preimmune, antibodies. Anti-T beta antibodies moderately decreased the extent of subsequent adenylate cyclase activation by forskolin, while not affecting activation by Al3+/F-. The enzyme activation by Gpp(NH)p in untreated membranes remained the same upon further incubation in the presence of either type of antibodies. Such results were consistent with the decreased exchange of guanine nucleotides which occurred in membrane treated with immune, but not preimmune antibodies, upon addition of GTP. The blockade of the regulation of adenylate cyclase by Gpp(NH)p observed in membranes pretreated by anti-T beta antibodies thus appears to be caused by the impairment of the guanine nucleotide exchange occurring on Gs alpha subunits. The G beta subunits in the adenylate cyclase complex seem to be instrumental in the guanine nucleotide exchange on G alpha subunits, just as T beta subunits are in the transducin complex.

Adenylyl Cyclases↗
PubMed1987

Fluoride complexes of aluminium or beryllium act on G-proteins as reversibly bound analogues of the gamma phosphate of GTP.

Fluoride activation of G proteins requires the presence of aluminium or beryllium and it has been suggested that AIF4- acts as an analogue of the gamma-phosphate of GTP in the nucleotide site. We have investigated the action of AIF4- or of BeF3- on transducin (T), the G protein of the retinal rods, either indirectly through the activation of cGMP phosphodiesterase, or more directly through their effects on the conformation of transducin itself. In the presence of AIF4- or BeF3-, purified T alpha subunit of transducin activates purified cyclic GMP phosphodiesterase (PDE) in the absence of photoactivated rhodopsin. Activation is totally reversed by elution of fluoride or partially reversed by addition of excess T beta gamma. Activation requires that GDP or a suitable analogue be bound to T alpha: T alpha-GDP and T alpha-GDP alpha S are activable by fluorides, but not T alpha-GDP beta S, nor T alpha that has released its nucleotide upon binding to photoexcited rhodopsin. Analysis of previous works on other G proteins and with other nucleotide analogues confirm that in all cases fluoride activation requires that a GDP unsubstituted at its beta phosphate be bound in T alpha. By contrast with alumino-fluoride complexes, which can adopt various coordination geometries, all beryllium fluoride complexes are tetracoordinated, with a Be-F bond length of 1.55 A, and strictly isomorphous to a phosphate group. Our study confirms that fluoride activation of transducin results from a reversible binding of the metal-fluoride complex in the nucleotide site of T alpha, next to the beta phosphate of GDP, as an analogue of the gamma phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1987

The retinal phototransduction process: enzymatic cascade and regulation.

Among cellular systems performing the transduction of an external stimulus, phototransduction in vertebrate rod cells is a unique case which allows convergent approaches to electrophysiological, biophysical and biochemical analyses. The framework of the molecular processes involved in the corresponding enzymatic cascade is now elucidated and can be considered as a model for G protein mediated transductions. We present here the main features of this cascade, its amplification and regulation properties. The mode of stimulation by the aluminofluoride ion is particularly addressed.

Membrane Proteins↗
PubMed1986

Activation of retinal rod cyclic GMP-phosphodiesterase by transducin: characterization of the complex formed by phosphodiesterase inhibitor and transducin alpha-subunit.

The GTP-binding subunit of transducin (T alpha) activates the cGMP phosphodiesterase (PDE) of bovine retinal rods by relieving the constraint imposed by the inhibitory subunit PDE gamma. We have isolated and characterized the complex T alpha.GTP gamma S-PDE gamma formed when T alpha is activated by the nonhydrolyzable analog GTP gamma S. Sedimentation and light-scattering techniques demonstrate that, in contrast to free T alpha.GTP gamma S, which is soluble, the T alpha.GTP gamma S-PDE gamma complex, as well as T alpha.GTP-PDE gamma, is membrane bound at cytosolic ionic strength. It is eluted from the membrane at low ionic strength as a monomeric and 1:1 stoichiometric complex. The relative affinities of PDE gamma for PDE alpha beta and for T alpha.GTP are discussed.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1986

Serotonin- and dopamine-sensitive adenylate cyclase in molluscan nervous system. Biochemical and electrophysiological analysis of the pharmacological properties and the GTP-dependence.

Helix aspersa neuronal cell membranes contain distinct serotonin (5-HT) and dopamine (DA) sensitive adenylate cyclases. We have taken advantage of the fact that in this system, both in vitro (enzymatic assays) and in vivo (electrophysiological measurements) experiments can be used to explore the GTP dependence and the pharmacological properties of this neurotransmitter-sensitive enzyme system. The first property was studied using non-hydrolysable GTP analogs (guanosine 5'-O-(3-thio-triphosphate) or GTP gamma S, and guanosine 5'-imido diphosphate or Gpp(NH)p). In vitro, these two components stimulate the enzyme activity but with different potencies (Kapparent = 10(-8) to 5 X 10(-8) M for GTP gamma S, and 10(-5) M for Gpp(NH)p). Intracellular injections of GTP gamma S, but not of Gpp(NH)p, produced an electrophysiological response similar to the one elicited by 5-HT and DA. These results imply that, even in the presence of the high endogenous GTP concentration normally present in the cell (10(-3) M), GTP gamma S may bind to the GTP-binding protein. Such an interpretation is consistent with the in vitro competition experiments between GTP and GTP gamma S for adenylate cyclase activation. The pharmacology of 5-HT and DA receptors involved in adenylate cyclase stimulation and electrophysiological responses was studied. Serotoninergic antagonists and neuroleptics inhibited the 5-HT-sensitive adenylate cyclase in a stereospecific manner. However, their inhibition was not simply competitive. Our results suggest that they irreversibly bind a component localized on the cytoplasmic side of the membrane. Unexpectedly, the DA receptor coupled with adenylate cyclase was insensitive to any of the several antagonists tested.

Adenylyl Cyclases↗
PubMed1985

Fluoroaluminates activate transducin-GDP by mimicking the gamma-phosphate of GTP in its binding site.

Fluoride activation of the cGMP cascade of vision requires the presence of aluminum, and is shown to be mediated by the binding of one A1F-4 to the GDP/GTP-binding subunit of transducin. The presence of GDP in the site is required: A1F-4 is ineffective when the site is empty or when GDP beta S is substituted for GDP. This sensitivity to the sulfur of GDP beta S suggests that A1F-4 is in contact with the GDP. Striking structural similarities between A1F-4 and PO3-4 lead us to propose that A1F-4 mimics the role of the gamma-phosphate of GTP.

Aluminum↗