The G-alpha protein GNA3 of Hypocrea jecorina (Anamorph Trichoderma reesei) regulates cellulase gene expression in the presence of light.

Although the enzymes enabling Hypocrea jecorina (anamorph Trichoderma reesei) to degrade the insoluble substrate cellulose have been investigated in some detail, little is still known about the mechanism by which cellulose signals its presence to the fungus. In order to investigate the possible role of a G-protein/cyclic AMP signaling pathway, the gene encoding GNA3, which belongs to the adenylate cyclase-activating class III of G-alpha subunits, was cloned. gna3 is clustered in tandem with the mitogen-activated protein kinase gene tmk3 and the glycogen phosphorylase gene gph1. The gna3 transcript is upregulated in the presence of light and is almost absent in the dark. A strain bearing a constitutively activated version of GNA3 (gna3QL) exhibits strongly increased cellulase transcription in the presence of the inducer cellulose and in the presence of light, whereas a gna3 antisense strain showed delayed cellulase transcription under this condition. However, the gna3QL mutant strain was unable to form cellulases in the absence of cellulose. The necessity of light for stimulation of cellulase transcription by GNA3 could not be overcome in a mutant which expressed gna3 under control of the constitutive gpd1 promoter also in darkness. We conclude that the previously reported stimulation of cellulase gene transcription by light, but not the direct transmission of the cellulose signal, involves the function and activation of GNA3. The upregulation of gna3 by light is influenced by the light modulator ENVOY, but GNA3 itself has no effect on transcription of the light regulator genes blr1, blr2, and env1. Our data for the first time imply an involvement of a G-alpha subunit in a light-dependent signaling event in fungi.

Purification and in vitro functional analysis of R7 subfamily RGS proteins in complex with Gbeta5.

The study of purified regulator of G-protein signaling (RGS) proteins in steady-state GTPase assays using reconstituted proteoliposomes is a powerful approach to characterizing the RGS protein-mediated acceleration of intrinsic Galpha subunit GTPase activity in the context of various G-protein and G-protein-coupled receptor (GPCR) combinations. This approach has been applied successfully to the R7 subfamily of RGS proteins, RGS6, -7, -9, and -11, which form heterodimers with Gbeta5 subunits via the G-protein gamma-like domain of R7 proteins. This article describes the purification of heterodimers from Sf9 insect cells following the expression of recombinant R7 protein and histidine-tagged Gbeta5 using affinity and ion-exchange chromatography. The ability of the heterodimers to accelerate the intrinsic GTPase activity of Galpha subunits was assessed in steady-state GTPase assays performed on proteoliposomes consisting of phospholipids, purified G proteins, and purified GPCRs.

Quantitative analysis of mammalian GIRK2 channel regulation by G proteins, the signaling lipid PIP2 and Na+ in a reconstituted system.

GIRK channels control spike frequency in atrial pacemaker cells and inhibitory potentials in neurons. By directly responding to G proteins, PIP2 and Na(+), GIRK is under the control of multiple signaling pathways. In this study, the mammalian GIRK2 channel has been purified and reconstituted in planar lipid membranes and effects of Gα, Gßγ, PIP2 and Na(+) analyzed. Gßγ and PIP2 must be present simultaneously to activate GIRK2. Na(+) is not essential but modulates the effect of Gßγ and PIP2 over physiological concentrations. Gαi1(GTPγS) has no effect, whereas Gαi1(GDP) closes the channel through removal of Gßγ. In the presence of Gßγ, GIRK2 opens as a function of PIP2 mole fraction with Hill coefficient 2.5 and an affinity that poises GIRK2 to respond to natural variations of PIP2 concentration. The dual requirement for Gßγ and PIP2 can help to explain why GIRK2 is activated by Gi/o, but not Gq coupled GPCRs.

ß-Arrestin-1 directly interacts with Gαs and regulates its function.

ß-Arrestins function to mediate G protein-coupled receptor (GPCR) desensitization and internalization and to initiate G protein independent signaling of GPCRs. Elucidating how ß-arrestin and G protein signal pathways coordinate with each other is important to fully understand GPCR signaling. Here we report that ß-arrestin-1 directly interacts with Gα(s). Purified ß-arrestin-1 binds to Gα(s) in a rapid association and dissociation manner. ß-Arrestin-1 promotes the binding and the release of GTPγS from Gα(s) in vitro. ß-Arrestin-1 L33K mutant shows reduced interaction with Gα(s) and has no detectable effects on Gα(s) function. Our study thus reveals a direct crosstalk of ß-arrestin-1 with Gα(s).

Dissecting the role of the S1P/S1PR axis in health and disease.

Sphingosine-1-phosphate (S1P) is a pleiotropic sphingophospholipid generated from the phosphorylation of sphingosine by sphingosine kinases (SPHKs). S1P has been experimentally demonstrated to modulate an array of cellular processes such as cell proliferation, cell survival, cell invasion, vascular maturation, and angiogenesis by binding with any of the five known G-protein-coupled sphingosine 1 phosphate receptors (S1P1-5) on the cell surface in an autocrine as well as a paracrine manner. Recent studies have shown that the S1P receptors (S1PRs) and SPHKs are the key targets for modulating the pathophysiological consequences of various debilitating diseases, such as cancer, sepsis, rheumatoid arthritis, ulcerative colitis, and other related illnesses. In this article, we recapitulate these novel discoveries relative to the S1P/S1PR axis, necessary for the proper maintenance of health, as well as the induction of tumorigenic, angiogenic, and inflammatory stimuli that are vital for the development of various diseases, and the novel therapeutic tools to modulate these responses in oral biology and medicine.

GDP-GTP exchange processes of G{alpha}i1 protein are accelerated/decelerated depending on the type and the concentration of added detergents.

Although detergents have been widely used in G-protein studies to increase solubility and stability of the protein, we noticed that detergents modulate the nucleotide-binding properties of G-proteins. Hence, we analysed the effects of detergents on guanine nucleotide exchange reactions of Galpha(i1). Lubrol PX, a non-ionic detergent, which has been widely used in nucleotide dissociation/binding assays, was found to accelerate both GDP dissociation and GTPgammaS binding from/to Galpha in parallel at above its critical micelle concentration (cmc). Sodium cholate, an anionic detergent, which have been used to extract G-proteins from animal tissues, decelerated and accelerated GDP dissociation below and above its cmc, respectively. Surprisingly, micellar cholate decelerated GTPgammaS binding, and the binding rate constant was decreased by three orders of magnitude in the presence of 2% cholate. These results demonstrate that the guanine nucleotide exchange reactions of Galpha(i1) are drastically modulated by detergents differently depending on the type and the state (monomeric or micellar) of the detergents and that dissociation of GDP from Galpha(i1) does not necessarily lead to immediate binding of GTP to Galpha(i1) in some cases. These effects of detergents on G-proteins must be taken into account in G-protein experiments.