A pea plasma membrane protein exhibiting blue light-induced phosphorylation retains photosensitivity following triton solubilization.

Phosphorylation of a polypeptide of approximately 120 kD in pea (Pisum sativum L.) plasma membranes in response to blue light has been shown to be involved in phototropic curvature, but the relationship of this protein to the kinase and photoreceptor acting upon it is uncertain. Using two-phase aqueous partitioning to isolate right-side-out plasma membrane vesicles, we have obtained evidence suggesting that the photoreceptor, kinase, and substrate are localized to the plasma membrane fraction. Latent phosphorylation accessible through Triton X-100 or freeze/thaw treatments of purified plasma membrane vesicles indicates that at least the kinase moiety is present on the internal face of the plasma membrane. Effects of solubilization of vesicles on fluence-response characteristics and on phosphorylation levels provide evidence that the receptor, kinase, and protein substrate are present together in individual mixed detergent micelles, either as a stable complex or as domains of a single polypeptide. In vivo blue-light irradiation results in a small but significant decrease in mobility of the 120-kD phosphorylated protein on sodium dodecylsulfate gel electrophoresis. This mobility shift is evident on Coomassie-stained gels and on western blots probed with polyclonal antibodies raised against the 120-kD protein. Among the plasma membrane proteins bound to the reactive nucleotide analog fluorosulfonylbenzoyladenine (FSBA), a distinct protein band at 120 kD can be detected on blots probed with anti-FSBA antibodies. This band exhibits an in vivo light-dependent mobility shift identical to that observed for the protein band and antibodies specific for the 120-kD protein, implying that the 120-kD protein has an integral nucleotide binding site and consistent with the possibility that the substrate protein is also a kinase.

Sorting mechanisms regulating membrane protein traffic in the apical transcytotic pathway of polarized MDCK cells.

The transcytotic pathway followed by the polymeric IgA receptor (pIgR) carrying its bound ligand (dIgA) from the basolateral to the apical surface of polarized MDCK cells has been mapped using morphological tracers. At 20 degreesC dIgA-pIgR internalize to interconnected groups of vacuoles and tubules that comprise the endosomal compartment and in which they codistribute with internalized transferrin receptors (TR) and epidermal growth factor receptors (EGFR). Upon transfer to 37 degreesC the endosome vacuoles develop long tubules that give rise to a distinctive population of 100-nm-diam cup-shaped vesicles containing pIgR. At the same time, the endosome gives rise to multivesicular endosomes (MVB) enriched in EGFR and to 60-nm-diam basolateral vesicles. The cup-shaped vesicles carry the dIgA/pIgR complexes to the apical surface where they exocytose. Using video microscopy and correlative electron microscopy to study cells grown thin and flat we show that endosome vacuoles tubulate in response to dIgA/pIgR but that the tubules contain TR as well as pIgR. However, we show that TR are removed from these dIgA-induced tubules via clathrin-coated buds and, as a result, the cup-shaped vesicles to which the tubules give rise become enriched in dIgA/pIgR. Taken together with the published information available on pIgR trafficking signals, our observations suggest that the steady-state concentrations of TR and unoccupied pIgR on the basolateral surface of polarized MDCK cells are maintained by a signal-dependent, clathrin-based sorting mechanism that operates along the length of the transcytotic pathway. We propose that the differential sorting of occupied receptors within the MDCK endosome is achieved by this clathrin-based mechanism continuously retrieving receptors like TR from the pathways that deliver pIgR to the apical surface and EGFR to the lysosome.

Enhanced heat shock protein 70 expression alters proteasomal degradation of IkappaB kinase in experimental acute respiratory distress syndrome.

OBJECTIVES: Acute respiratory distress syndrome is a common and highly lethal inflammatory lung syndrome. We previously have shown that an adenoviral vector expressing the heat shock protein (Hsp)70 (AdHSP) protects against experimental sepsis-induced acute respiratory distress syndrome in part by limiting neutrophil accumulation in the lung. Neutrophil accumulation and activation is modulated, in part, by the nuclear factor-kappaB (NF-kappaB) signal transduction pathway. NF-kappaB activation requires dissociation/degradation of a bound inhibitor, IkappaBalpha. IkappaBalpha degradation requires phosphorylation by IkappaB kinase, ubiquitination by the SCFbeta-TrCP (Skp1/Cullin1/Fbox beta-transducing repeat-containing protein) ubiquitin ligase, and degradation by the 26S proteasome. We tested the hypothesis that Hsp70 attenuates NF-kappaB activation at multiple points in the IkappaBalpha degradative pathway. DESIGN: Laboratory investigation. SETTING: University medical center research laboratory. SUBJECTS: Adolescent (200 g) Sprague-Dawley rats and murine lung epithelial-12 cells in culture. INTERVENTIONS: Lung injury was induced in rats via cecal ligation and double puncture. Thereafter, animals were treated with intratracheal injection of 1) phosphate buffer saline, 2) AdHSP, or 3) an adenovirus expressing green fluorescent protein. Murine lung epithelial-12 cells were stimulated with tumor necrosis factor-alpha and transfected. NF-kappaB was examined using molecular biological tools. MEASUREMENTS AND MAIN RESULTS: Intratracheal administration of AdHSP to rats with cecal ligation and double puncture limited nuclear translocation of NF-kappaB and attenuated phosphorylation of IkappaBalpha. AdHSP treatment reduced, but did not eliminate, phosphorylation of the beta-subunit of IkappaB kinase. In vitro kinase activity assays and gel filtration chromatography revealed that treatment of sepsis-induced lung injury with AdHSP induced fragmentation of the IkappaB kinase signalosome. This stabilized intermediary complexes containing IkappaB kinase components, IkappaBalpha, and NF-kappaB. Cellular studies indicate that although ubiquitination of IkappaBalpha was maintained, proteasomal degradation was impaired by an indirect mechanism. CONCLUSIONS: Treatment of sepsis-induced lung injury with AdHSP limits NF-kappaB activation. This results from stabilization of intermediary NF-kappaB/IkappaBalpha/IkappaB kinase complexes in a way that impairs proteasomal degradation of IkappaBalpha. This novel mechanism by which Hsp70 attenuates an intracellular process may be of therapeutic value.

ParA2, a Vibrio cholerae chromosome partitioning protein, forms left-handed helical filaments on DNA.

Most bacterial chromosomes contain homologs of plasmid partitioning (par) loci. These loci encode ATPases called ParA that are thought to contribute to the mechanical force required for chromosome and plasmid segregation. In Vibrio cholerae, the chromosome II (chrII) par locus is essential for chrII segregation. Here, we found that purified ParA2 had ATPase activities comparable to other ParA homologs, but, unlike many other ParA homologs, did not form high molecular weight complexes in the presence of ATP alone. Instead, formation of high molecular weight ParA2 polymers required DNA. Electron microscopy and three-dimensional reconstruction revealed that ParA2 formed bipolar helical filaments on double-stranded DNA in a sequence-independent manner. These filaments had a distinct change in pitch when ParA2 was polymerized in the presence of ATP versus in the absence of a nucleotide cofactor. Fitting a crystal structure of a ParA protein into our filament reconstruction showed how a dimer of ParA2 binds the DNA. The filaments formed with ATP are left-handed, but surprisingly these filaments exert no topological changes on the right-handed B-DNA to which they are bound. The stoichiometry of binding is one dimer for every eight base pairs, and this determines the geometry of the ParA2 filaments with 4.4 dimers per 120 A pitch left-handed turn. Our findings will be critical for understanding how ParA proteins function in plasmid and chromosome segregation.

Enhanced sensitivity detection of protein immobilization by fluorescent interference on oxidized silicon.

We present a silicon chip-based approach for the enhanced sensitivity detection of surface-immobilized fluorescent molecules. Green fluorescent protein (GFP) is bound to the silicon substrate by a disuccinimidyl terephtalate-aminosilane immobilization procedure. The immobilized organic layers are characterized by surface analysis techniques, like ellipsometry, atomic force microscopy (AFM) and X-ray induced photoelectron spectroscopy. We obtain a 20-fold enhancement of the fluorescent signal, using constructive interference effects in a fused silica dielectric layer, deposited before immobilization onto the silicon. Our method opens perspectives to increase by an order of magnitude the fluorescent response of surface immobilized DNA- or protein-based layers for a variety of biosensor applications.

Homologous DNA pairing domain peptides of RecA protein: intrinsic propensity to form beta-structures and filaments.

The 20 amino acid residue peptides derived from RecA loop L2 have been shown to be the pairing domain of RecA. The peptides bind to ss- and dsDNA, unstack ssDNA, and pair the ssDNA to its homologous target in a duplex DNA. As shown by circular dichroism, upon binding to DNA the disordered peptides adopt a beta-structure conformation. Here we show that the conformational change of the peptide from random coil to beta-structure is important in binding ss- and dsDNA. The beta-structure in the DNA pairing peptides can be induced by many environmental conditions such as high pH, high concentration, and non-micellar sodium dodecyl sulfate (6 mM). This behavior indicates an intrinsic property of these peptides to form a beta-structure. A beta-structure model for the loop L2 of RecA protein when bound to DNA is thus proposed. The fact that aromatic residues at the central position 203 strongly modulate the peptide binding to DNA and subsequent biochemical activities can be accounted for by the direct effect of the aromatic amino acids on the peptide conformational change. The DNA-pairing domain of RecA visualized by electron microscopy self-assembles into a filamentous structure like RecA. The relevance of such a peptide filamentous structure to the structure of RecA when bound to DNA is discussed.

A template-assembled synthetic protein surface mimetic of the von Willebrand factor A1 domain inhibits botrocetin-induced platelet aggregation.

Platelet adhesion, the initial step of platelet activation, is mediated by the interaction of von Willebrand factor (VWF) with its platelet receptor, the GPIb-IX complex. The binding of VWF to GPIb-IX is induced either by increased shear stress or by exogenous modulators, such as botrocetin. At a molecular level, this interaction takes place between the A1 domain of VWF and the GPIb alpha chain of the GPIb-IX complex. We report here the design and functional characteristics of a VWF template-assembled synthetic protein (TASP), a chimeric four-helix-bundle TASP scaffold mimicking the surface of the A1 domain. Twelve residues located on helices alpha 3 and alpha 4 in the native A1 domain were grafted onto a surface formed by two neighboring helices of the TASP. VWF TASP was found to inhibit specifically botrocetin-induced platelet aggregation and to bind both botrocetin and GPIb alpha. However, in contrast to the native A1 domain, VWF TASP did not bind simultaneously to both ligands. Modeling studies revealed that the relative orientation of the alpha helices in VWF TASP led to a clash of bound botrocetin and GPIb alpha. These results demonstrate that a chimeric four-helix-bundle TASP as a scaffold offers a suitable surface for presenting crucial residues of the VWF A1 domain; the potential of the TASP approach for de novo protein design and mimicry is thereby illustrated.

Hsp110 is a bona fide chaperone using ATP to unfold stable misfolded polypeptides and reciprocally collaborate with hsp70 to solubilize protein aggregates.

Structurally and sequence-wise, the Hsp110s belong to a subfamily of the Hsp70 chaperones. Like the classical Hsp70s, members of the Hsp110 subfamily can bind misfolding polypeptides and hydrolyze ATP. However, they apparently act as a mere subordinate nucleotide exchange factors, regulating the ability of Hsp70 to hydrolyze ATP and convert stable protein aggregates into native proteins. Using stably misfolded and aggregated polypeptides as substrates in optimized in vitro chaperone assays, we show that the human cytosolic Hsp110s (HSPH1 and HSPH2) are bona fide chaperones on their own that collaborate with Hsp40 (DNAJA1 and DNAJB1) to hydrolyze ATP and unfold and thus convert stable misfolded polypeptides into natively refolded proteins. Moreover, equimolar Hsp70 (HSPA1A) and Hsp110 (HSPH1) formed a powerful molecular machinery that optimally reactivated stable luciferase aggregates in an ATP- and DNAJA1-dependent manner, in a disaggregation mechanism whereby the two paralogous chaperones alternatively activate the release of bound unfolded polypeptide substrates from one another, leading to native protein refolding.

Two-step chromatographic purification of human plasma alpha(1)-acid glycoprotein: its application to the purification of rare phenotype samples of the protein and their study by chromatography on immobilized metal chelate affinity adsorbent.

Alpha1-Acid glycoprotein (AAG) or orosomucoid was purified to homogeneity from human plasma by a separate two-step method using chromatography on immobilized Cibacron Blue F3G-A to cross-linked agarose and chromatography on hydroxyapatite. The conditions for the pre-purification of AAG by chromatography on immobilized Cibacron Blue F3G-A were first optimized using different buffer systems with different pH values. The overall yield of the combined techniques was 80% and ca. 12 mg of AAG were purified from an initial total amount of ca. 15 mg in a ca. 40 ml sample of human plasma. This method was applied to the purification of AAG samples corresponding to the three main phenotypes of the protein (FI*S/A, F1/A and S/A), from individual human plasma previously phenotyped for AAG. A study by isoelectric focusing with carrier ampholytes showed that the microheterogeneity of the purified F1*S/A, F1/A and S/A AAG samples was similar to that of AAG in the corresponding plasma, thus suggesting that no apparent desialylation of the glycoprotein occurred during the purification steps. This method was also applied to the purification of AAG samples corresponding to rare phenotypes of the protein (F1/A*AD, S/A*X0 and F1/A*C1) and the interactions of these variants with immobilized copper(II) ions were then studied at pH 7, by chromatography on an iminodiacetate Sepharose-Cu(II) gel. It was found that the different variants encoded by the first of the two genes coding for AAG in humans (i.e. the F1 and S variants) interacted non-specifically with the immobilized ligand, whereas those encoded by the second gene of AAG (i.e. the A, AD, X0 and C1 variants) strongly bound to immobilized Cu(II) ions. These results suggested that chromatography on an immobilized affinity Cu(II) adsorbent could be helpful to distinguish between the respective products of the two highly polymorphic genes which code for human AAG.

Neutron structure of type-III antifreeze protein allows the reconstruction of AFP-ice interface.

Antifreeze proteins (AFPs) inhibit ice growth at sub-zero temperatures. The prototypical type-III AFPs have been extensively studied, notably by X-ray crystallography, solid-state and solution NMR, and mutagenesis, leading to the identification of a compound ice-binding surface (IBS) composed of two adjacent ice-binding sections, each which binds to particular lattice planes of ice crystals, poisoning their growth. This surface, including many hydrophobic and some hydrophilic residues, has been extensively used to model the interaction of AFP with ice. Experimentally observed water molecules facing the IBS have been used in an attempt to validate these models. However, these trials have been hindered by the limited capability of X-ray crystallography to reliably identify all water molecules of the hydration layer. Due to the strong diffraction signal from both the oxygen and deuterium atoms, neutron diffraction provides a more effective way to determine the water molecule positions (as D(2) O). Here we report the successful structure determination at 293 K of fully perdeuterated type-III AFP by joint X-ray and neutron diffraction providing a very detailed description of the protein and its solvent structure. X-ray data were collected to a resolution of 1.05 Å, and neutron Laue data to a resolution of 1.85 Å with a "radically small" crystal volume of 0.13 mm(3). The identification of a tetrahedral water cluster in nuclear scattering density maps has allowed the reconstruction of the IBS-bound ice crystal primary prismatic face. Analysis of the interactions between the IBS and the bound ice crystal primary prismatic face indicates the role of the hydrophobic residues, which are found to bind inside the holes of the ice surface, thus explaining the specificity of AFPs for ice versus water.

Synergistic action of GA-binding protein and glucocorticoid receptor in transcription from the mouse mammary tumor virus promoter.

B lymphocytes are among the first cells to be infected by mouse mammary tumor virus (MMTV), and they play a crucial role in its life cycle. To study transcriptional regulation of MMTV in B cells, we have analyzed two areas of the long terminal repeat (LTR) next to the glucocorticoid receptor binding site, fp1 (at position -139 to -146 from the cap site) and fp2 (at -157 to -164). Both showed B-cell-specific protection in DNase I in vitro footprinting assays and contain binding sites for Ets transcription factors, a large family of proteins involved in cell proliferation and differentiation and oncogenic transformation. In gel retardation assays, fp1 and fp2 bound the heterodimeric Ets factor GA-binding protein (GABP) present in B-cell nuclear extracts, which was identified by various criteria: formation of dimers and tetramers, sensitivity to pro-oxidant conditions, inhibition of binding by specific antisera, and comigration of complexes with those formed by recombinant GABP. Mutations which prevented complex formation in vitro abolished glucocorticoid-stimulated transcription from an MMTV LTR linked to a reporter gene in transiently transfected B-cell lines, whereas they did not affect the basal level. Exogenously expressed GABP resulted in an increased level of hormone response of the LTR reporter plasmid and produced a synergistic effect with the coexpressed glucocorticoid receptor, indicating cooperation between the two. This is the first example of GABP cooperation with a steroid receptor, providing the opportunity for studying the integration of their intracellular signaling pathways.

"Peptabody": a new type of high avidity binding protein.

A new type of high avidity binding molecule, termed "peptabody" was created by harnessing the effect of multivalent interaction. A short peptide ligand was fused via a semi-rigid hinge region with the coiled-coil assembly domain of the cartilage oligomeric matrix protein, resulting in a pentameric multivalent binding molecule. In the first peptabody (Pab-S) described here, a peptide (S) specific for the mouse B-cell lymphoma BCL1 surface Ig idiotype, was selected from a phage display library. A fusion gene was constructed encoding peptide S, followed by the 24 aa hinge region from camel IgG and a modified 55 aa cartilage oligomeric matrix protein pentamerization domain. The Pab-S fusion protein was expressed in Escherichia coli in a soluble form at high levels and purified in a single step by metal-affinity chromatography. Pab-S specifically bound the BCL1 surface idiotype with an avidity of about 1 nM, which corresponds to a 2 x 10(5)-fold increase compared with the affinity of the synthetic peptide S itself. Biochemical characterization showed that Pab-S is a stable homopentamer of about 85 kDa, with interchain disulfide bonds. Pab-S can be dissociated under denaturing and reducing conditions and reassociated as a pentamer with full-binding activity. This intrinsic feature provides an easy way to combine Pab molecules with two different peptide specificities, thus producing heteropentamers with bispecific and/or chelating properties.

Microtubule-associated protein 1B binds glyceraldehyde-3-phosphate dehydrogenase.

Microtubule-associated protein 1B, MAP1B, is a major cytoskeletal protein during brain development and one of the largest brain MAPs associated with microtubules and microfilaments. Here, we identified several proteins that bind to MAP1B via immunoprecipitation with a MAP1B-specific antibody, by one and two-dimensional gel electrophoresis and subsequent mass spectrometry identification of precipitated proteins. In addition to tubulin and actin, a variety of proteins were identified. Among these proteins were glyceraldehyde-3-phosphate dehydrogenase (GAPDH), heat shock protein 8, dihydropyrimidinase related proteins 2 and 3, protein-L-isoaspartate O-methyltransferase, beta-spectrin, and clathrin protein MKIAA0034, linking either directly or indirectly to MAP1B. In particular, GAPDH, a key glycolytic enzyme, was bound in large quantity to the heavy chain of MAP1B in adult brain tissue. In vitro binding studies confirmed a direct binding of GAPDH to MAP1B. In PC12 cells, GAPDH was found in cytoplasm and nuclei and partially co-localized with MAP1B. It disappeared from the cytoplasm under oxidative stress or after a disruption of cytoskeletal elements after colcemid or cytochalasin exposure. GAPDH may be essential in the local energy provision of cytoskeletal structures and MAP1B may help to keep this key enzyme close to the cytoskeleton.

The Bacillus subtilis bacteriophage SPP1 G39P delivers and activates the G40P DNA helicase upon interacting with the G38P-bound replication origin.

Initiation of Bacillus subtilis bacteriophage SPP1 replication requires the phage-encoded genes 38, 39 and 40 products (G38P, G39P and G40P). G39P, which does not bind DNA, interacts with the replisome organiser, G38P, in the absence of ATP and with the ATP-activated hexameric replication fork helicase, G40P. G38P, which specifically interacts with the phage replication origin (oriL) DNA, does not seem to form a stable complex with G40P in solution. G39P when complexed with G40P-ATP inactivates the single-stranded DNA binding, ATPase and unwinding activities of G40P, and such effects are reversed by increasing amounts of G38P. Unwinding of a forked substrate by G40P-ATP is increased about tenfold by the addition of G38P and G39P to the reaction mixture. The specific protein-protein interactions between oriL-bound G38P and the G39P-G40P-ATPgammaS complex are necessary for helicase delivery to the SPP1 replication origin. Formation of G38P-G39P heterodimers releases G40P-ATPgammaS from the unstable oriL-G38P-G39P-G40P-ATPgammaS intermediate. G40P-ATPgammaS binds to the origin region, the uncomplexed G38P fraction remains bound to oriL, and the G38P-G39P heterodimer is lost from the complex. We demonstrate that G39P is a component of an oligomeric nucleoprotein complex which plays an important role in the initiation of SPP1 replication.

Regulation of frizzled receptor activity at the plasma membrane : an interplay of the receptor, the trimeric G protein Go, and RGS protein and a scaffolding protein Kermit

G-protein-signaling pathways convey extracellular signals inside the cells and regulate distinct physiological responses. This type of signaling pathways consists of three major components: G-protein-coupled receptors (GPCRs), heterotrimeric G proteins (G-proteins) and downstream effectors. Upon ligand binding, GPCRs activate heterotrimeric G proteins to initiate the signaling cascade. Dysfunction of GPCR signaling correlates with numerous diseases such as diabetes, nervous and immune system deficiency, and cancer. As the signaling switcher, G-proteins (Gs, Gq/11, G12/13, and Gi/o) have been an appealing topic of research for decades. A heterotrimeric G-protein is composed of three subunits, the guanine nucleotide associated a-subunit, ß and y subunits. In general, the duration of signaling is determined by the lifetime of activated (GTP bound) Ga subunits. Identification of novel communication partners of Ga subunits appears to be an attractive way to understand the machinery of GPCR signaling. In our lab, we mainly focus on Gao, which is abundantly expressed in the nervous system. Here we present two novel interacting partners of Drosophila Gao: Dhit and Kermit, identified through yeast two-hybrid screening and genetic screening respectively. Dhit is characterized by a small size with a conserved RGS domain and an N-terminal cysteine rich motif. The RGS domain possesses the GAP (GTPase activating protein) activity towards G proteins. However, we found that Dhit exerts not only the GAP activity but also the GDI (guanine nucleotide dissociation inhibitor) activity towards Gao. The unexpected GDI activity is preserved in GAIP/RGS19 - a mammalian homologue of Dhit. Further experiments confirmed the GDI activity of Dhit and GAIP/RGS19 in Drosophila and mammalian cell models. Therefore, we propose that Dhit and its mammalian homologues modulate GPCR signaling by a double suppression of Ga subunits - suppression of their nucleotide exchange with GTP and acceleration of their hydrolysis of GTP. Kermit/GEPC was first identified as a binding partner of GAIP/RGS19 in a yeast two- hybrid screen. Instead of interacting with the Drosophila homologue of GAIP/RGS19 (Dhit), Kermit binds to Gao in vivo and in vitro. The functional consequence of Kermit/Gao interaction is the regulation of localization of Vang (one of the planar cell polarity core components) at the apical membrane. Overall, my work elaborated the action of Gao with its two interaction partners in Gao- mediated signaling pathway. Conceivably, the understanding of GPCR signaling including Gao and its regulators or effectors will ultimately shed light on future pharmaceutical research. - Les voies de signalisation médiées par les protéines G transmettent des signaux extracellulaires à l'intérieur des cellules pour réguler des réponses physiologiques distinctes. Cette voie de signalisation consiste en trois composants majeurs : les récepteurs couplés aux protéines G (GPCRs), les protéines G hétérotrimériques (G-proteins) et les effecteurs en aval. Suite à la liaison du ligand, les GPCRs activent les protéines G hétérotrimériques qui initient la cascade de signalisation. Des dysfonctions dans la signalisation médiée par les GPCRs sont corrélées avec de nombreuses maladies comme le diabète, des déficiences immunes et nerveuses, ainsi que le cancer. Puisque la voie de signalisation s'active et se désactive, les protéines G (Gs, Gq/11, G12/13 et Gi/o) ont été un sujet de recherche attrayant pendant des décennies. Une protéine G hétérotrimérique est composée de trois sous-unités, la sous-unité a associée au nucléotide guanine, ainsi que les sous-unités ß et y. En général, la durée du signal est déterminée par le temps de demi-vie des sous-unités Ga activées (Ga liées au GTP). Identifier de nouveaux partenaires de communication des sous-unités Ga se révèle être un moyen attractif de comprendre la machinerie de la signalisation par les GPCRs. Dans notre laboratoire nous nous sommes concentrés principalement sur Gao qui est exprimée de manière abondante dans le système nerveux. Nous présentons ici deux nouveaux partenaires qui interagissent avec Gao chez la drosophile: Dhit et Kermit, qui ont été identifiés respectivement par la méthode du yeast two-hybrid et par criblage génétique. Dhit est caractérisé par une petite taille, avec un domaine RGS conservé et un motif N- terminal riche en cystéines. Le domaine RGS contient une activité GAP (GTPase activating protein) pour les protéines G. Toutefois, nous avons découvert que Dhit exerce non seulement une activité GAP mais aussi une activité GDI (guanine nucleotide dissociation inhibitor) à l'égard de Gao. Cette activité GDI inattendue est préservée dans RGS19 - un homologue de Dhit chez les mammifères. Des expériences supplémentaires ont confirmé l'activité GDI de Dhit et de RGS19 chez Drosophila melanogaster et les modèles cellulaires mammifères. Par conséquent, nous proposons que Dhit et ses homologues mammifères modulent la signalisation GPCR par une double suppression des sous-unités Ga - suppression de leur nucléotide d'échange avec le GTP et une accélération dans leur hydrolyse du GTP. Kermit/GIPC a été premièrement identifié comme un partenaire de liaison de RGS19 dans le criblage par yeast two-hybrid. Au lieu d'interagir avec l'homologue chez la drosophile de RGS19 (Dhit), Kermit se lie à Gao in vivo et in vitro. La conséquence fonctionnelle de l'interaction Kermit/Gao est la régulation de la localisation de Vang, un des composants essentiel de la polarité planaire cellulaire, à la membrane apicale. Globalement, mon travail a démontré l'action de Gao avec ses deux partenaires d'interaction dans la voie de signalisation médiée par Gao. La compréhension de la signalisation par les GPCRs incluant Gao et ses régulateurs ou effecteurs aboutira à mettre en lumière de futurs axes dans la recherche pharmacologique.