Papers by Thierry Fischer
European Journal of Pharmacology, Nov 1, 1996
The nature of the pertussis toxin-insensitive G-protein involved in muscarinic-mediated phosphoin... more The nature of the pertussis toxin-insensitive G-protein involved in muscarinic-mediated phosphoinositides breakdown and contraction of isolated smooth muscle cells from the circular layer of the rabbit caecum was investigated, Immunoblotting of membrane proteins using affinity purified antibodies directed against different G-protein o~-subunits revealed the expression of G.q/t t , G. it and G~i2 in these cells. The carbachol-mediated [3H]inositol phosphates accumulation in saponin-permeabilized cells was abolished by anti-G~,q/l i-antibodies whereas anti-G¢~i~.e-antib°dies were ineffective. Moreover, the carbachol-induced contraction of permeabilized cells, as determined by videomicrocopic measurements, was reversed by anti-G~q/ll-antibodies but not affected by anti-G~ike-antibodies, From these data, we conclude that carbachol stimulates phosphoinositides hydrolysis and cell contraction through activation of specific muscarinic M 3 receptors coupled to the pertussis toxin-insensitive G~q/ll-protein. This is the first demonstration of Goq/~ implication in the contractile signal transduction pathway of muscarinic M 3 receptors in smooth muscle cells.
We have isolated an RGS-GAIP interacting protein that links RGS proteins to protein degradation. ... more We have isolated an RGS-GAIP interacting protein that links RGS proteins to protein degradation. GIPN (GAIP interacting protein N terminus) is a 38-kDa protein with an N-terminal leucine-rich region, a central RING finger-like domain, and a putative C-terminal transmembrane domain. GIPN binds exclusively to RGS proteins of subfamily A, RGS-GAIP, RGSZ1, and RGSZ2. The N-terminal leucinerich region of GIPN interacts with the cysteine-rich motif of RGS-GAIP. GIPN mRNA is ubiquitously expressed, and GIPN is found on the plasma membrane of transfected HEK293 cells. Endogenous GIPN is concentrated along the basolateral plasma membrane of proximal and distal tubules in rat kidney, where many G proteincoupled receptors and some G proteins are also located. Two immunoreactive species are found in rat kidney, a 38-kDa cytosolic form and an Ϸ94-kDa membrane form. GIPN shows Zn 2؉-and E1͞E2-dependent autoubiquitination in vitro, suggesting that it has E3 ubiquitin ligase activity. Overexpression of GIPN stimulates proteasome-dependent reduction of endogenous G␣i3 in HEK293 cells and reduces the half-life of overexpressed G␣i3-YFP. Thus, our findings suggest that GIPN is involved in the degradation of G␣i3 subunits via the proteasome pathway. RGS-GAIP functions as a bifunctional adaptor that binds to G␣ subunits through its RGS domain and to GIPN through its cysteine string motif. C ells respond to a variety of extracellular stimuli through receptors that are linked to heterotrimeric G proteins. The levels of individual G protein signaling molecules are tightly regulated by modifying their levels of expression or by proteasome-dependent degradation. Many components of G protein signaling pathways have been shown to be ubiquitinated and degraded in proteasomes, including G protein-coupled receptors (GPCRs) (1), kinases (2), and RGS proteins (3). Specific degradation of G alpha subunits (G␣) by the ubiquitin͞ proteasome-dependent pathway was previously shown for the yeast Gpa1, a G␣ equivalent (4, 5), and more recently for G␣o (6) and G␥ subunits (7). RGS proteins are known to bind to G␣ (8-10) and to negatively regulate G protein signaling through their GTPaseactivating activity by stabilizing the GTP to GDP hydrolysis transition state (11). RGS-GAIP is a member of subfamily A of the RGS proteins and acts as a GTPase-activating protein for G␣i subunits. The N terminus of RGS-GAIP contains a cysteine-rich motif, which is most likely the site of reversible palmitoylation responsible for anchoring of RGS-GAIP to clathrin-coated pits (12). Here, we describe an RGS-GAIP interacting protein, GIPN, which binds to the N terminus of GAIP, has E3 ubiquitin ligase activity, and promotes proteasomedependent degradation of G␣i3. This interaction provides a functional link between a G␣ subunit and the ubiquitindependent protein degradation system through an RGS protein intermediate. Materials and Methods Antibodies. Monoclonal antibodies against ubiquitin were purchased from Chemicon, anti-GFP (Jl-8) was purchased from Clontech, and anti-mouse IgG was purchased from Bio-Rad. Anti-ezrin antibody was kindly provided by H. Furthmayr (Stanford University, Stanford, CA), and affinity-purified, anti-G␣i3 (EC) IgG was provided by A. Spiegel (National Institute of Diabetes and Digestive and Kidney Diseases). Anti-GIPN antibodies were raised in rabbits against His-6-GIPN (full length); an IgG fraction was prepared by using the caprylic acid method and affinity-purified on either the CNBr immobilized immunogen or a mouse kidney lysate immobilized on poly(vinylidene difluoride) membranes (13). Cell Culture. HEK293 cells were grown in DMEM-high glucose with 10% (vol͞vol) FBS and 100 units͞ml penicillin G, 100 units͞ml streptomycin sulfate, and 0.3 mg͞ml glutamine (Invitrogen). Sf21 cells (obtained from A. Newton, University of California at San Diego) were grown as monolayers in Hink's TNM (JRH Biosciences, Lenexa, KS) with 10% (vol͞vol) FBS and 100 units/ml penicillin G͞100 units/ml streptomycin sulfate and transfected in serum-free EX-CELL 400 (JRH Biosciences).
Journal of Biological Chemistry, Feb 1, 2001
Specific domains of the G-protein ␣ subunit have been shown to control coupling to heptahelical r... more Specific domains of the G-protein ␣ subunit have been shown to control coupling to heptahelical receptors. The extreme N and C termini and a region between ␣4 and ␣5 helices of the G-protein ␣ subunit are known to determine selective interaction with the receptors. The metabotropic glutamate receptor 2 activated both mouse G␣ 15 and its human homologue G␣ 16 , whereas metabotropic glutamate receptor 8 activated G␣ 15 only. The extreme C-terminal 20 amino acid residues are identical between the G␣ 15 and G␣ 16 and are therefore unlikely to be involved in coupling selectivity. Our data reveal two regions on G␣ 16 that inhibit its coupling to metabotropic glutamate receptor 8. On a three-dimensional model, both regions are found in a close proximity to the extreme C terminus of G␣ 16. One module comprises ␣4 helix, ␣4؊6 loop (L9 Loop), 6 sheet, and ␣5 helix. The other, not described previously, is located within the loop that links the N-terminal ␣ helix to the 1 strand of the Ras-like domain of the ␣ subunit. Coupling of G␣ 16 protein to the metabotropic glutamate receptor 8 is partially modulated by each module alone, whereas both modules are needed to eliminate the coupling fully.
Journal of Biological Chemistry, Jun 1, 2005
In this report, we characterize GIV (G␣-interacting vesicle-associated protein), a novel protein ... more In this report, we characterize GIV (G␣-interacting vesicle-associated protein), a novel protein that binds members of the G␣ i and G␣ s subfamilies of heterotrimeric G proteins. The G␣ interaction site was mapped to an 83-amino acid region of GIV that is enriched in highly charged amino acids. BLAST searches revealed two additional mammalian family members, Daple and an uncharacterized protein, FLJ00354. These family members share the highest homology at the G␣ binding domain, are homologous at the N terminus and central coiled coil domain but diverge at the C terminus. Using affinitypurified IgG made against two different regions of the protein, we localized GIV to COPI, endoplasmic reticulum (ER)-Golgi transport vesicles concentrated in the Golgi region in GH3 pituitary cells and COS7 cells. Identification as COPI vesicles was based on colocalization with -COP, a marker for these vesicles. GIV also codistributes in the Golgi region with endogenous calnuc and the KDEL receptor, which are cis Golgi markers and with G␣ i3-yellow fluorescent protein expressed in COS7 cells. By immunoelectron microscopy, GIV colocalizes with -COP and G␣ i3 on vesicles found in close proximity to ER exit sites and to cis Golgi cisternae. In cell fractions prepared from rat liver, GIV is concentrated in a carrier vesicle fraction (CV2) enriched in ER-Golgi transport vesicles. -COP and several G␣ subunits (G␣ i1-3 , G␣ s) are also most enriched in CV2. Our results demonstrate the existence of a novel G␣-interacting protein associated with COPI transport vesicles that may play a role in G␣-mediated effects on vesicle trafficking within the Golgi and/or between the ER and the Golgi.
Molecular Pharmacology, Jul 1, 2003
Regulators of G-protein signaling (RGS) proteins are GTPaseactivating proteins (GAPs) that bind t... more Regulators of G-protein signaling (RGS) proteins are GTPaseactivating proteins (GAPs) that bind to G␣ subunits and attenuate G protein signaling, but where these events occur in the cell is not yet established. Here we investigated, by immunofluorescence labeling and deconvolution analysis, the site at which endogenous G␣-interacting protein (GAIP) (RGS19) binds to G␣i3-YFP and its fate after activation of ␦-opioid receptor (DOR). In the absence of agonist, GAIP is spatially segregated from G␣i3 and DOR in clathrin-coated domains (CCPs) of the cell membrane (PM), whereas G␣i3-YPF and DOR are located in non-clathrin-coated microdomains of the PM. Upon addition of agonist, G␣i3 partially colocalizes with GAIP in CCPs at the PM. When endocytosis is blocked by expression of a dynamin mutant [dyn(K44A)], there is a striking overlap in the distribution of DOR and G␣i3-YFP with GAIP in CCPs. Moreover, G␣i3-YFP and GAIP form a coprecipitable complex. Our results support a model whereby, after agonist addition, DOR and G␣i3 move together into CCPs where G␣i3 and GAIP meet and turn off G protein signaling. Subsequently, G␣i3 returns to non-clathrin-coated microdomains of the PM, GAIP remains stably associated with CCPs, and DOR is internalized via clathrin-coated vesicles. This constitutes a novel mechanism for regulation of G␣ signaling through spatial segregation of a GAP in clathrin-coated pits. Ligand binding to G protein-coupled receptors (GPCRs) causes activation of G␣ and G␥ subunits that in turn regulate multiple downstream effectors. Signaling is shut off by regulators of G protein signaling (RGS) proteins that bind G␣ subunits through a conserved RGS domain and act as GT-Pase-activating proteins (GAPs), accelerating GTP hydrolysis and inactivation (De Vries et al., 2000; Ross and Wilkie, 2000). The orchestration of these events and their localization at the cellular level is a topic of high current interest. Many GPCRs are thought to be associated in part with lipid rafts or caveolae (Shaul and Anderson, 1998) on the plasma membrane (PM). After ligand binding, they cluster in clathrin-coated pits, undergo dynamin-mediated endocytosis via clathrin coated vesicles (CCVs) (Ferguson, 2001), and traffic to early endosomes, where they are sorted for recycling to the PM or delivered to lysosomes and degraded (Tsao and von Zastrow, 2001). G␣i subunits are assumed to interact with GPCR at the PM (Neubig, 1994; Wedegaertner et al., 1996; Huang et al., 1997; Fishburn et al., 2000) and to remain at the PM after receptor internalization (Wedegaertner, 1998; Hughes et al., 2001). Nothing is known, however, about where interaction between RGS proteins and G␣ proteins occurs or the fate of the GAP after this interaction. We previously showed that GAIP (RGS19), which acts as a GAP for the G␣i subfamily of G proteins (De Vries et al., 1995), is localized on clathrin-coated pits or microdomains (CCPs) on both the PM and the trans-Golgi network (De Vries et al., 1998). The localization of an RGS protein on CCPs raises questions about where GAIP interacts with G␣i and its fate after receptor internalization. To investigate these questions, we used a cell line, 293SFDOR, that stably expresses FLAG-tagged, ␦-opioid receptor (DOR), a G␣i-linked GPCR, because the trafficking of this receptor has been well characterized in these cells (Tsao and von Zastrow, 2001; Whistler et al., 2001). DOR is known to be phosphorylated upon agonist binding (Whistler et al., 2001), internalized in clathrin-coated vesicles (Keith et al., 1996), and subsequently targeted to lysosomes where it is degraded (Tsao and von Zastrow, 2000).
Proceedings of the National Academy of Sciences of the United States of America, Jan 18, 2000
Calnuc (nucleobindin) was previously shown to be present both in the cytosol and in the Golgi and... more Calnuc (nucleobindin) was previously shown to be present both in the cytosol and in the Golgi and to be the major Golgi Ca 2؉ binding protein. In this study we verified the existence of the cytosolic pool of calnuc and investigated its interaction with G␣i3. Cytosolic calnuc was released by mild digitonin permeabilization. In pulsechase experiments, the two pools of calnuc had different mobilities, suggesting different posttranslational modifications. That calnuc interacts with G␣i3 in vivo was verified by the finding that G␣i3 could be crosslinked intracellularly to calnuc and co-immunoprecipitated from NIH 3T3 cells stably overexpressing either activated (Q204L) or inactivated (G203A) G␣i3. Binding was Ca 2؉ and Mg 2؉-dependent. Calnuc and G␣i3-GFP codistributed primarily in the Golgi region. By yeast two-hybrid analysis, the binding site on G␣i3 for calnuc was mapped to the C-terminal region because removal of the last 12 amino acids (but not 11) abolished the interaction. Peptide competition indicated that calnuc, with its coiled-coil domain constituted by the two EF-hands, binds to G␣i3's C-terminal ␣5-helix. These results demonstrate that calnuc may play an important role in G protein-and Ca 2؉-regulated signal transduction events.
Journal of Biological Chemistry, May 1, 1999
Norepinephrine inhibits-conotoxin GVIA-sensitive presynaptic Ca 2؉ channels in chick dorsal root ... more Norepinephrine inhibits-conotoxin GVIA-sensitive presynaptic Ca 2؉ channels in chick dorsal root ganglion neurons through two pathways, one mediated by G o and the other by G i. These pathways desensitize at different rates. We have found that recombinant G␣ interacting protein (GAIP) and regulators of G protein signaling (RGS)4 selectively accelerate the rate of desensitization of G o-and G i-mediated pathways, respectively. Blockade of endogenous RGS proteins using antibodies raised against G␣ interacting protein and RGS4 slows the rate of desensitization of these pathways in a selective manner. These results demonstrate that different RGS proteins may interact with G i and G o selectively, giving rise to distinct time courses of transmitter-mediated effects. Voltage-dependent calcium channels are the primary triggers for electrically evoked release of chemical transmitters. Understanding the mechanisms underlying their regulation is central to the development of a molecular picture of key events in neuronal signaling. Calcium channels are well known targets for inhibition by receptor-G protein pathways, and multiple forms of inhibition have been described (1, 2). The termination of the response or desensitization represents a main mechanism for controlling synaptic strength in an activity-dependent manner. Much of the knowledge about agonist-dependent desensitization comes from studies of ionotropic receptors such as GABAergic 1 and glutamatergic receptors. In addition to the ionotropic receptor-mediated fast synaptic transmission, signaling in the nervous system relies heavily on G protein-coupled receptors for more prolonged and sustained synaptic action. Norepinephrine (NE) inhibits N-type (-conotoxin GVIAsensitive) Ca 2ϩ channels in embryonic dorsal root ganglion (DRG) neurons through two pathways (3, 4), one mediated by
Nucleic Acids Research, Apr 1, 2019
Alternative splicing is facilitated by accessory proteins that guide spliceosome subunits to the ... more Alternative splicing is facilitated by accessory proteins that guide spliceosome subunits to the primary transcript. Many of these splicing factors recognize the RNA polymerase II tail, but SFPQ is a notable exception even though essential for mammalian RNA processing. This study reveals a novel role for Dido3, one of three Dido gene products, in alternative splicing. Binding of the Dido3 amino terminus to histones and to the polymerase jaw domain was previously reported, and here we show interaction between its carboxy terminus and SFPQ. We generated a mutant that eliminates Dido3 but preserves other Dido gene products, mimicking reduced Dido3 levels in myeloid neoplasms. Dido mutation suppressed SFPQ binding to RNA and increased skipping for a large group of exons. Exons bearing recognition sequences for alternative splicing factors were nonetheless included more efficiently. Reduced SFPQ recruitment may thus account for increased skipping of SFPQ-dependent exons, but could also generate a splicing factor surplus that becomes available to competing splice sites. Taken together, our data indicate that Dido3 is an adaptor that controls SFPQ utilization in RNA splicing. Distributing splicing factor recruitment over parallel pathways provides mammals with a simple mechanism to regulate exon usage while maintaining RNA splicing efficiency.
Proceedings of the National Academy of Sciences of the United States of America, Jun 8, 1999
G␣-interacting protein (GAIP) is a member of the RGS (regulators of G protein signaling) family, ... more G␣-interacting protein (GAIP) is a member of the RGS (regulators of G protein signaling) family, which serve as GAPs (GTPase-activating proteins) for G␣ subunits. Previously, we demonstrated that GAIP is localized on clathrin-coated vesicles (CCVs). Here, we tested whether GAIPenriched vesicles could accelerate the GTPase activity of G␣ i proteins. A rat liver fraction containing vesicular carriers (CV2) was enriched (4.5؋) for GAIP by quantitative immunoblotting, and GAIP was detected on some of the vesicles in the CV2 fraction by immunoelectron microscopy. When liver fractions were added to recombinant G␣ i3 and tested for GAP activity, only the CV2 fraction contained GAP activity. Increasing amounts of CV2 increased the activity, whereas immunodepletion of the CV2 fraction with an antibody against the C terminus of GAIP decreased GAP activity. CCV fractions were prepared from rat liver by using a protocol that maintains the clathrin coats. GAIP was enriched in these fractions and was detected on CCVs by immunogold labeling. Addition of increasing amounts of CCV to recombinant G␣ i3 protein increased the GTPase activity. We conclude that CCVs possess GAP activity for G␣ i3 and that membrane-associated GAIP is capable of interacting with G␣ i3. The reconstitution of the interaction between a heterotrimeric G protein and GAIP on CCVs provides biochemical evidence for a model whereby the G protein and its GAP are compartmentalized on different membranes and come into contact at the time of vesicle fusion. Alternatively, they may be located on the same membrane and segregate at the time of vesicle budding.
Biochimica et biophysica acta. Molecular cell research, Apr 1, 1993
GTP-binding proteins are known to play an important role in controlling mast-cell exocytosis and ... more GTP-binding proteins are known to play an important role in controlling mast-cell exocytosis and are described as the primary targets of peptidic mast-cell histamine releasers. The mechanism of inhibition of the mast-cell peptidergic pathway by alkylamines, which are selective inhibitors of this pathway, was investigated using intact or permeabilized rat peritoneal mast cells. Histamine release induced by GTPyS and by mastoparan (a venom peptide activating G proteins) was inhibited by pretreating mast cells with 0.1 to 3/~g/ml of a mixture of benzalkonium chloride containing in majority a twelve-carbon-atom aliphatic chain (BAC (C_ 12)). Pure benzalkonium chloride, with a fourteen-carbon-atom aliphatic chain (BAC (C14)), at 5 to 10 ~M also inhibited histamine release induced by GTPTS and mastoparan. The dose-response curve of mastoparan-induced histamine release from intact mast cells was shifted to the right by various concentrations of BAC (C14). Similar results were obtained with another alkylamine differing from BAC (C14) by the absence of the benzene ring, tetradecyltrimethylammonium bromide, TAB (C14). This illustrates that the presence of the phenyl radical is not required for the inhibitory effect of benzalkonium chloride. BAC (C~ 12) and BAC (C14) inhibited the generation of inositol polyphosphates induced by GTPTS. BAC (C ~ 12) and TAB (C~4) inhibited the mastoparan-stimulated GTPase activity from mast-cell Gi-like proteins. These results suggest that alkylamines exert selectively their inhibitory effect via an interaction with mast-cell Gi-like proteins coupled to phospholipase C, i.e., at an early stage in the stimulus-secretion coupling process.
Cellular Signalling, Dec 1, 2009
The magnitude and duration of G protein-coupled receptor (GPCR) signals are regulated through des... more The magnitude and duration of G protein-coupled receptor (GPCR) signals are regulated through desensitization mechanisms. In leukocytes, ligand binding to chemokine receptors leads to Ca 2+ mobilization and ERK activation through pertussis toxin-sensitive G proteins, as well as to phosphorylation of the GPCR. After interaction with the endocytic machinery (clathrin, adaptin), the adaptor β-arrestin recognizes the phosphorylated GPCR tail and quenches signaling to receptors. The molecular mechanisms that lead to receptor endocytosis are not universal amongst the GPCR, however, and the precise spatial and temporal events in the internalization of the CCR2 chemokine receptor remain unknown. Here we show that after ligand binding, CCR2 internalizes rapidly and reaches early endosomes, and later, lysosomes. Knockdown of clathrin by RNA interference impairs CCR2 internalization, as does treatment with the dynamin inhibitor, dynasore. Our results show that CCR2 internalization uses a combination of clathrindependent and-independent pathways, as observed for other chemokine receptors. Moreover, the use of dynasore allowed us to confirm the existence of a dynamin-sensitive element that regulates ERK1/2 activation. Our results indicate additional complexity in the link between receptor internalization and cell signaling.
Proceedings of the National Academy of Sciences of the United States of America, Dec 19, 2000
Activator of G protein signaling 3 (AGS3) is a newly identified protein shown to act at the level... more Activator of G protein signaling 3 (AGS3) is a newly identified protein shown to act at the level of the G protein itself. AGS3 belongs to the GoLoco family of proteins, sharing the 19-aa GoLoco motif that is a G␣i/o binding motif. AGS3 interacts only with members of the G␣i/o subfamily. By surface plasmon resonance, we found that AGS3 binds exclusively to the GDP-bound form of G␣i3. In GTP␥S binding assays, AGS3 behaves as a guanine dissociation inhibitor (GDI), inhibiting the rate of exchange of GDP for GTP by G␣i3. AGS3 interacts with both G␣i3 and G␣o subunits, but has GDI activity only on G␣i3, not on G␣o. The fourth GoLoco motif of AGS3 is a major contributor to this activity. AGS3 stabilizes G␣i3 in its GDP-bound form, as it inhibits the increase in tryptophan fluorescence of the G␣i3-GDP subunit stimulated by AlF 4 ؊. AGS3 is widely expressed as it is detected by immunoblotting in brain, testis, liver, kidney, heart, pancreas, and in PC-12 cells. Several different sizes of the protein are detected. By Northern blotting, AGS3 shows 2.3-kb and 3.5-kb mRNAs in heart and brain, respectively, suggesting tissue-specific alternative splicing. Taken together, our results demonstrate that AGS3 is a GDI. To the best of our knowledge, no other GDI has been described for heterotrimeric G proteins. Inhibition of the G␣ subunit and stimulation of heterotrimeric G protein signaling, presumably by stimulating G␥, extend the possibilities for modulating signal transduction through heterotrimeric G proteins. H eterotrimeric G proteins (G proteins), consisting of an ␣ subunit (G␣) with GTPase activity and a ␥ dimer (G␥), act as guanine nucleotide-dependent molecular switches in signaling pathways that connect transmembrane receptors with downstream effectors (1, 2). In the classical paradigm at the plasma membrane, the liganded transmembrane receptor activates the G protein by stimulation of GDP dissociation from G␣ and acts as a guanine exchange factor (GEF), thereby enhancing GTP binding and releasing free G␣ and G␥ subunits to interact with their respective effectors (3). Inactivation of G protein signaling takes place by inhibiting G protein activation or by GTP hydrolysis, which leads to reformation of the heterotrimer. Precisely timed activation and inactivation of the G protein, dependent on regulatory factors, is crucial in signal transduction. In the case of the small G proteins, two classes of intracellular proteins can act as inhibitors of G protein activation: GTPase activating proteins (GAPs), which enhance GTP hydrolysis, and guanine dissociation inhibitors (GDIs), which inhibit GDP dissociation (4). GAPs for heterotrimeric G protein ␣ subunits have only recently been discovered and for the most part belong to the RGS (regulator of G protein signaling) protein family (5-7). Until now, GDIs acting on heterotrimeric G proteins have remained elusive. However, several additional G␣-interacting proteins, most of them displaying regulatory-or effector-like functions, have recently been identified. PCP2 and activator of G protein signaling (AGS) 1 are novel GEFs (8, 9) and Rap1GAP is a novel effector (10, 11). AGS3, identified in a functional screen based on G protein signaling in yeast but unrelated to AGS1, was recently shown to bind to G␣ i-GDP and act as an activator of heterotrimeric G protein signaling (12), possibly through effectors of G␥. In contrast to G protein coupled receptors (the classical G protein activators), AGS3 did not enhance GTP␥S binding to the G␣ subunit. Thus, it apparently acts through a different, yet to be elucidated, molecular mechanism (12). Here, we have further characterized AGS3 and have demonstrated that it acts as a GDI for G␣ i3. Materials and Methods Isolation of AGS3 cDNA. For two-hybrid interaction screening, 50 g of a rat GC cell (pituitary) cDNA library in pACT2 was transformed into yeast HF7c(pGBT9G␣ i3) as described (13). Twenty-four positive clones, grouped based on insert size and restriction pattern, were sequenced from the 5Ј or 3Ј end by automated sequencing. One of these was a partial clone for AGS3, encoding the C-terminal half of the molecule (amino acids 361-590), truncated by its last 60 aa. Full length AGS3 (650 aa) cDNA was obtained by reverse transcription (RT)-PCR on rat brain cDNA (kind gift of Dr.
Neuropeptides, May 1, 1992
Molecular Neurodegeneration, 2009
In AtT-20 cells ACTH secretion is regulated by both Ca 2+ and G proteins. We previously demonstra... more In AtT-20 cells ACTH secretion is regulated by both Ca 2+ and G proteins. We previously demonstrated that calnuc, an EF-hand Ca 2+ binding protein which regulates Alzheimer's β-amyloid precursor protein (APP) biogenesis, binds both Ca 2+ as well as Gα subunits. Here we investigate calnuc's role in G protein-mediated regulation of ACTH secretion in AtT-20 neuroendocrine secretory cells stably overexpressing calnuc-GFP. Similar to endogenous calnuc, calnuc-GFP is mainly found in the Golgi, on the plasma membrane (PM), and associated with regulated secretion granules (RSG). By deconvolution immunofluorescence, calnuc-GFP partially colocalizes with Gαi1/ 2 and Gαi3 at the PM and on RSG. Cytosolic calnuc(ΔSS)-CFP with the signal sequence deleted also partially colocalizes with RSG and partially cosediments with Gαi1/2 in fractions enriched in RSG. Overexpression of calnuc-GFP specifically increases the distribution of Gαi1/2 on the PM whereas the distribution of Gβ subunits and synaptobrevin 2 (Vamp 2) is unchanged. Overexpression of calnuc-GFP or cytosolic calnuc(ΔSS)-CFP enhances ACTH secretion twofold triggered by mastoparan or GTPγS but does not significantly affect glycosaminoglycan (GAG) chain secretion along the constitutive pathway or basal secretion of ACTH. Calnuc's facilitating effects on ACTH secretion are decreased after introducing anti-Gαi1/2, Gαi3, Gβ or calnuc IgG into permeabilized cells but not when Gα12 or preimmune IgG is introduced. The results suggest that calnuc binds to Gα subunits on the Golgi and on RSG and that overexpression of calnuc causes redistribution of Gαi subunits to the PM and RSG, indicating that calnuc plays a role in dynamic distribution of only Gα but not Gβ subunits. Thus calnuc may connect G protein signaling and calcium signaling during regulated secretion.
Biochemical Journal, Nov 1, 1995
The effects of elevated levels of cyclic AMP induced by cholera toxin (CTx) were investigated on ... more The effects of elevated levels of cyclic AMP induced by cholera toxin (CTx) were investigated on the differentiated promyelomonocytic cell line U937. After CTx treatment, the initial inhibition of the oxidative burst induced by N-formylmethionylleucyl-phenylalanine (FMLP) was followed by a progressive increase over 20 h, resulting in 4-6-fold potentiation of the initial burst. Various cyclic-AMP-elevating agents produced similar potentiation of the FMLP-or C5a-induced oxidative burst, but the phorbol 12-myristate 13-acetate-induced oxidative burst was not affected by CTx pretreatment of cells. Furthermore, the increase in arachidonate release and intracellular Ca2+ triggered by FMLP were amplified after CTx treatment. ADP-ribosylation
Proceedings of the National Academy of Sciences of the United States of America, Apr 11, 2000
GAIP (G ␣ interacting protein) is a member of the RGS (regulators of G protein signaling) family ... more GAIP (G ␣ interacting protein) is a member of the RGS (regulators of G protein signaling) family and accelerates the turnover of GTP bound to G␣i, G␣q, and G␣13. There are two pools of GAIP-a soluble and a membrane-anchored pool. The membrane-anchored pool is found on clathrin-coated vesicles (CCVs) and pits in rat liver and AtT-20 pituitary cells. By treatment of a GAIP-enriched rat liver fraction with alkaline phosphatase, we found that membranebound GAIP is phosphorylated. By immunoprecipitation carried out on [ 32 P]orthophosphate-labeled AtT-20 pituitary cells stably expressing GAIP, 32 P-labeling was associated exclusively with the membrane pool of GAIP. Phosphoamino acid analysis revealed that phosphorylation of GAIP occurred largely on serine residues. Recombinant GAIP could be phosphorylated at its N terminus with purified casein kinase 2 (CK2). It could also be phosphorylated by isolated CCVs in vitro. Phosphorylation was Mn 2؉-dependent, using both purified CK2 and CCVs. Ser-24 was identified as one of the phosphorylation sites. Our results establish that GAIP is phosphorylated and that only the membrane pool is phosphorylated, suggesting that GAIP can be regulated by phosphorylation events taking place at the level of clathrin-coated pits and vesicles. G proteins ͉ regulator of G protein signaling ͉ casein kinase 2 S ignal transduction components and G-protein coupled receptor (GPCR) signaling pathways in particular undergo phosphorylation and dephosphorylation events that control their effects on multiple physiological processes (1). For example, GPCR are phosphorylated by G protein-coupled receptor kinases (GRKs) (2, 3), and several G proteins undergo phosphorylation in vivo on their ␣ (4-6) and ␥ (7) subunits, which may regulate their membrane targeting (8) and͞or interaction with partners (9-12) Recently, a family of more than 20 members, the RGS proteins (for regulators of G protein signaling), have been identified that act as GTPase activating proteins (GAPs) for G␣ subunits (for reviews see refs. 13 and 14). We localized one member of the RGS family, RGS-GAIP (G ␣ interacting protein), by immunocytochemistry and found it to be associated with clathrincoated pits and vesicles (15, 16) and further demonstrated that CCVs isolated from rat liver possess GAP activity (16). Clathrin-coated vesicles (CCVs) are known to play a role in G-protein coupled receptor-(2) and receptor-mediated endocytosis at the plasma membrane and in sorting of lysosomal enzymes in the trans-Golgi network (TGN) (17-19). Formation of CCVs is known to be tightly regulated by phosphorylation and dephosphorylation of adapters and coat proteins (20-24). GAIP contains nine putative phosphorylation sites-two for protein kinase C (PKC) and seven for casein kinase 2 (CK2) (25). These consensus sites are distributed over all three domains of GAIP: (i) its highly conserved RGS domain (amino acids 86-205) required for GAP activity (26), (ii) its N terminus (amino acids 1-85) most likely responsible for membrane anchoring of GAIP by palmitoylation (27), and (iii) the 12-aa C terminus (amino acids 206-217), which is unique and interacts with GIPC, a PDZ domain-containing protein (28). Up to now, no information has been available on whether GAIP or any other mammalian RGS protein undergoes phosphorylation. We report here the identification of a phosphorylated pool of GAIP that is membrane-anchored in rat hepatocytes and AtT-20 pituitary cells. We also demonstrate that GAIP can be phosphorylated by purified CK2 and isolated CCVs in vitro, and we identified Ser-24 as one of the residues that is phosphorylated. These findings suggest that GAIP is phosphorylated at its N terminus by CK2 on CCVs. Experimental Procedures Materials. Easytag (a mixture of [ 35 S]methionine and [ 35 S]cysteine) was obtained from DuPont͞NEN. Phorbol myristate acetate (PMA) was purchased from Calbiochem, cellulose plates from Analtech, and [ 14 C]methylated protein molecular weight markers from Amersham Pharmacia. Mutant GAIP (S24A) was generated by a PCR according to the manufacturer's instructions by using a QuikChange mutagenesis kit (Stratagene), pET28a GAIP 1-217 (16), and the following primers: 5Ј-GCCCCCT-TCAATGGCCAGTCATGATACAGCC and 3Ј-GGCTGTAT-CATACTGGCCATTGAAGGGGGC. Mutation was verified by automatic sequencing. The EcoRI͞SmaI fragment containing the mutation was subcloned into the wild-type template. His6 GAIP proteins were expressed in BL-21 (DE 3), purified, and dialysed as described (16). Protein was determined by using the BCA assay (Pierce). Antibodies. Anti-hemagglutinin (HA) mAb 16B12 was purchased from Babco (Richmond, CA). Anti-GAIP (N), GAIP (23-217), and anti-GAIP (C) were characterized previously (15).
M S-medecine Sciences, 1997
Molecular Biology of the Cell, May 1, 1998
RGS-GAIP (G␣-interacting protein) is a member of the RGS (regulator of G protein signaling) famil... more RGS-GAIP (G␣-interacting protein) is a member of the RGS (regulator of G protein signaling) family of proteins that functions to down-regulate G␣ i /G␣ q-linked signaling. GAIP is a GAP or guanosine triphosphatase-activating protein that was initially discovered by virtue of its ability to bind to the heterotrimeric G protein G␣ i3 , which is found on both the plasma membrane (PM) and Golgi membranes. Previously, we demonstrated that, in contrast to most other GAPs, GAIP is membrane anchored and palmitoylated. In this work we used cell fractionation and immunocytochemistry to determine with what particular membranes GAIP is associated. In pituitary cells we found that GAIP fractionated with intracellular membranes, not the PM; by immunogold labeling GAIP was found on clathrin-coated buds or vesicles (CCVs) in the Golgi region. In rat liver GAIP was concentrated in vesicular carrier fractions; it was not found in either Golgi-or PM-enriched fractions. By immunogold labeling it was detected on clathrin-coated pits or CCVs located near the sinusoidal PM. These results suggest that GAIP may be associated with both TGN-derived and PM-derived CCVs. GAIP represents the first GAP found on CCVs or any other intracellular membranes. The presence of GAIP on CCVs suggests a model whereby a GAP is separated in space from its target G protein with the two coming into contact at the time of vesicle fusion.
Journal of Immunology, 2011
Autoimmune glomerulonephritis is a common manifestation of systemic lupus erythematosus (SLE). In... more Autoimmune glomerulonephritis is a common manifestation of systemic lupus erythematosus (SLE). In this study, we show that mice lacking macrophage expression of the heterodimeric nuclear receptors PPARg or RXRa develop glomerulonephritis and autoantibodies to nuclear Ags, resembling the nephritis seen in SLE. These mice show deficiencies in phagocytosis and clearance of apoptotic cells, and they are unable to acquire an anti-inflammatory phenotype upon feeding of apoptotic cells, which is critical for the maintenance of self-tolerance. These results demonstrate that stimulation of PPARg and RXRa in macrophages facilitates apoptotic cell engulfment, and they provide a potential strategy to avoid autoimmunity against dying cells and to attenuate SLE.
Proceedings of the National Academy of Sciences of the United States of America, May 24, 2010
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