点击显示 收起
Department of Pharmacology, University of Illinois at Chicago, Chicago, Illinois
Abstract
G protein-regulated inducer of neurite outgrowth 1 (GRIN1) was initially identified as a binding protein for guanosine 5'-3-O-(thio)triphosphateeCbound Gz. GRIN1 is specifically expressed in brain and interacts selectively with activated subunits of the Gi subfamily. GRIN1 colocalizes with Go at the growth cone of neuronal cells and promotes neurite extension in Neuro2a cells when coexpressed with constitutively active mutant GoQ205L. These results suggest that GRIN1 functions as a downstream target for Go. However, GRIN1 does not contain domains that are homologous to known signaling motifs. To understand the mechanisms of Go-GRIN1 pathway, we analyzed functional domains of GRIN1 that are involved in binding with Go or with its targeting to the plasma membrane. Using pull-down assays with glutathione S-transferaseeCfused GRIN1 deletion mutants, Go binding regions were localized to amino acid residues 716 to 746 and 797 to 827 of GRIN1. The Go binding region of GRIN1 did not demonstrate GTPase accelerating activity for Go. GRIN1 localized in the cell periphery in Neuro2a cells, and two cysteine residues at C-terminal region of GRIN1 (Cys818 and Cys819) were shown to be critical for its membrane targeting. Coexpression of GRIN1 with GoQ205L or GRIN1(717eC827), which lacks Go binding region, promoted microspike formation in Swiss 3T3 cells or neurite extension in Neuro2a cells. The dominant-negative mutant of Cdc42 blocked these morphological changes. Coexpression of GRIN1 and GoQ205L stimulated the formation of GTP-bound Cdc42 in Swiss 3T3 cells. These results suggest that the binding of activated Go to GRIN1 induces activation of Cdc42, which leads to morphological changes in neuronal cells.
Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) transduce a variety of signals from a large number of seven-transmembraneeCtype receptors to intracellular effectors (Gilman, 1987; Hepler and Gilman, 1992). Agonist-activated receptors induce dissociation of G protein subunits, generating a GTP-bound form of subunits and free subunits. Both GTP- and - can regulate downstream effectors. Hydrolysis of GTP to GDP on the subunit leads to reassociation of and to form an inactive heterotrimer. More than 30 RGS proteins have been identified as novel regulators of G protein signaling. RGS proteins associate with GTP-bound G and, in most cases, accelerate GTPase activity of G to facilitate the inactivation of G proteins (Berman et al., 1996; Ross and Wilkie, 2000).
Subunits of the Gi subfamily are highly expressed in the nervous system. Particularly, Go and Gi1 constitute nearly 1% of membrane protein in brain. However, the only well-established effectors of these subunits are certain isoforms of adenylyl cyclase (Taussig et al., 1994; Kozasa and Gilman, 1995). We have identified recently G protein-regulated inducer of neurite outgrowth 1 and 2 (GRIN1 and GRIN2, respectively) as novel effector candidates for Go (Chen et al., 1999). GRIN1 was isolated through screening of a mouse embryo cDNA expression library with phosphorylated guanosine 5'-3-O-(thio)triphosphateeCGz as a probe. A homolog of GRIN1, GRIN2 (KIAA0514), was identified by database search. GRIN1 and GRIN2 bind selectively to activated forms of Go, Gz, and Gi. Among different tissues, GRIN1 is specifically expressed in the brain. Furthermore, GRIN1 is enriched in the growth cones of neurites similar to Go and GAP43 (neuromodulin) (Chen et al., 1999). Coexpression of GRIN1 with the constitutively active mutant of Go promoted the extension of neurites in Neuro2a cells. Although GRIN1 does not contain domains that are homologous to other known signaling motifs, these results suggest that the Go-GRIN1 pathway may be involved in the regulation of neurite growth.
Similar to heterotrimeric G proteins, Rho family GTPases, Cdc42, Rac, and Rho, function as molecular switches by cycling between a GDP-bound, inactive state and a GTP-bound, active state. These GTPases are involved in various cellular processes, such as gene expression, cell-cycle progression, cell polarity, or vesicle trafficking (Symons and Settleman, 2000; Etienne-Manneville and Hall, 2002; Schmidt and Hall, 2002). Among them, the most well-established role of Rho family GTPases is the reorganization of the actin cytoskeleton, which is crucial for cellular responses such as cell migration, adhesion, phagocytosis, or axonal guidance (Dickson, 2001; Etienne-Manneville and Hall, 2002).
In this study, we characterized functional domains of GRIN1 to understand the molecular mechanisms of Go-GRIN1 signaling. We have identified regions in GRIN1 that are involved in Go binding or membrane targeting. We also identified a possible link of the Go-GRIN1 pathway to Cdc42 activation.
Materials and Methods
Materials. The anti-GST monoclonal antibody and anti-GFP polyclonal antibody were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-Flag (M2) antibody was purchased from Upstate Biotechnology (Lake Placid, NY) and Sigma-Aldrich (St. Louis, MO). The anti-Go monoclonal antibody was purchased from Calbiochem (San Diego, CA). The anti-Rac1 monoclonal antibody was purchased from Transduction Laboratories (Lexington, KY). Rhodamine phalloidin and Alexa Fluor 350-F(ab')2 fragment of goat anti-mouse IgG were from Molecular Probes (Eugene, OR). [-32P]GTP was purchased from MP Biomedicals (Irvine, CA). Antibodies against GRIN1 (T116) and Go (U1901) were described previously (Chen et al., 1999). VECTASHIELD mounting medium was purchased from Vector Laboratories (Burlingame, CA). Fibronectin and laminin were from Sigma-Aldrich. Plasmids for GST-PAK-PBD and Rho family GTPases were kindly provided by Drs. G. Bokoch (Scripps Research Institute, La Jolla, CA) and T. Satoh (Kobe University, Kobe, Japan), respectively.
Plasmid Constructs. Mouse GRIN1 cDNA (Chen et al., 1999) was used for the construction of plasmids for GST or GFP fusion proteins. The corresponding fragments of GRIN1 cDNA were subcloned in frame at the C terminus of GST in pGEX-KG or at the C terminus of enhanced GFP in pEGFP-C1 vector. The GRIN1 fragment (797eC827) was generated by PCR using the following primers: 5'-cggaattcagggcgccgccaag-3' and 5'-ccgctcgagttactccgcagtggggcc-3'. C818A and C819A mutations of GRIN1 were generated using QuikChange site-directed mutagenesis (Stratagene, La Jolla, CA) with primers 5'-gtgcgccgaccgcgggctgcatcgcgagcgggcccc-3' and 5'-ggggcccgctcgcgatgcagcccgcggtcggcgcac-3'. Go (wild-type or Q205L mutant) was fused at the N terminus of DsRed protein in pDsRed2-N vector (BD Biosciences CLONTECH, Palo Alto, CA). QuikChange mutagenesis was used to remove a stop codon and add an SmaI site in pCMV-GoQ205L with primers 5'-ggctgtggcttgtcccgggctcttgtcctg-3' and 5'-caggacaagagcccgggacaagccacagcc-3'. Then the EcoRI-SmaI fragment of pCMV-GoQ205L was subcloned into pDsRed2-N to generate pDsRed2N-GoQ205L. PCR reactions were performed by QuikChange mutagenesis using pDsRed2N-GoQ205L as a template with primers 5'-cgttggaggccagcgatctgaac-3' and 5'-gttcagatcgctggcctccaacg-3' to make pDsRed2N-Go (wild type). To construct bacterial expression vector for Go, a hexahistidine tag, a stop codon, and HindIII site were generated at the C terminus of rat Go cDNA by PCR using pQE6-Go as a template with primers 5'-cggcatgcacgagtctctcatgc-3' and 5'-aagctttcaatggtgatggtgatggtggtacaagccacagcc-3'. The PCR fragment was subcloned into pCR4-TOPO, digested with SphI and HindIII, and subcloned into pQE6-Go.
Expression and Purification of Recombinant Proteins. GST-fused GRIN1 mutants, GST-PAK-PBD, or Go-His6 were expressed in BL21 or JM109 by induction with 100 e isopropyl -D-thiogalactoside at 30 or 37°C for 2 to 3 h, respectively. GST fusion proteins were purified using glutathione Sepharose 4B column (Amersham Biosciences, Piscataway, NJ). Go-His6 was purified using a nickel-nitrilotriacetic acid column (QIAGEN, Valencia, CA).
Go Binding Assay. The lysates of Escherichia coli expressing GST-fused GRIN1 mutants were mixed with purified Go in the binding buffer (50 mM HEPES, pH 8.0, 50 mM NaCl, 50 e GDP, 5 mM MgCl2, 2 mM DTT, 0.1% C12E10, and protease inhibitors) in the presence or absence of AlF eC4 (10 mM NaF and 30 e AlCl3) and incubated on ice for 1 h. The resin was washed twice with wash buffer A (50 mM HEPES, pH 8.0, 100 mM NaCl, 50 e GDP, 5 mM MgCl2, 1 mM DTT, 0.1% C12E10, and protease inhibitors) and once with wash buffer B (50 mM HEPES, pH 8.0, 50 e GDP, 5 mM MgCl2, 2 mM DTT, 0.1% C12E10, and protease inhibitors). The bound proteins were eluted by boiling the resin in SDS sample buffer and analyzed by SDS-PAGE, followed by immunoblotting using an anti-Go antibody, U1901 (Chen et al., 1999).
Cell Culture. Neuro2a murine neuroblastoma cells or Swiss 3T3 fibroblast cells were cultured in DMEM containing 10% fetal bovine serum with 10% CO2.
Fluorescence Microscopy. Neuro2a cells on laminin-coated chambered coverslips were transiently transfected with appropriate expression plasmids using LipofectAMINE Plus (Invitrogen, Carlsbad, CA). Two hours after transfection, medium was changed to DMEM with 7.5% fetal bovine serum for 2 h and then to DMEM without serum for 24 h before capturing fluorescence images.
Swiss 3T3 cells plated on fibronectin-coated coverslips were subjected to serum starvation overnight. Then, expression plasmids with total DNA concentration of 0.05 mg/ml were microinjected into the nucleus of cells using computer-assisted microinjection system (Eppendorf - 5 Prime, Inc., Boulder, CO, and Cellbiology Trading, Hamburg, Germany). After 20 to 22 h, cells were rinsed with PBS, fixed in 4% paraformaldehyde/0.5% glutaraldehyde/PBS, permeabilized in 0.1% Triton X-100/PBS, and then stained with rhodamine phalloidin. For indirect immunofluorescence staining, cells were fixed and permeabilized as described above and incubated with anti-Flag antibody (1:200 dilution in PBS/2% bovine serum albumin). Cells were washed in PBS before incubating with secondary antibody Alexa Fluor 350 (anti-mouse IgG, 1:200 dilution in PBS/2% bovine serum albumin). Coverslips were mounted with VECTASHIELD mounting medium. Fluorescence images were captured using an LSM510 confocal microscope (Carl Zeiss GmbH, Jena, Germany).
Cdc42-GTP Pull-Down Assay. For Cdc42-GTP pull-down assay, Swiss 3T3 cells were transfected with Flag-tagged Cdc42 expression plasmid. After 40 h, they were further transfected with expression plasmids for GoQ205L and/or GFP-GRIN1. After 6 h, the cells were lysed with lysis buffer (25 mM HEPES, pH 7.5, 1% Nonidet P-40, 10 mM MgCl2, 100 mM NaCl, 5% glycerol, 1 mM sodium vanadate, and protease inhibitors). The lysates were incubated for 30 min at 4°C with GST-PAK-PBD (15 e) in binding buffer (25 mM HEPES, pH 7.5, 0.5% Nonidet P-40, 30 mM MgCl2, 50 mM NaCl, and 1 mM DTT) (Benard et al., 1999; Royal et al., 2000). The beads were washed five times with washing buffer (25 mM HEPES, pH 7.5, 0.5% Nonidet P-40, 30 mM MgCl2, 40 mM NaCl, and 1 mM DTT) and boiled in SDS sample buffer. The amount of GTP-bound Cdc42 was analyzed by 12.5% SDS-PAGE, followed by immunoblotting with anti-Flag antibody (for Cdc42) or anti-Rac1 antibody.
GAP Assay. GAP assays of Go were performed as described previously (Nagata et al., 2001; Rochdi et al., 2002).
Results
Go Interaction Region of GRIN1. The initial characterization of GRIN1 demonstrated that its carboxyl-terminal region (amino acid residues 555eC827) contains the Go binding domain (Chen et al., 1999). To further characterize the Go-GRIN1 interaction, several GST-tagged deletion mutants of GRIN1 were constructed. The lysates of E. coli expressing these GST-GRIN1 constructs were mixed with purified Go in the presence or absence of AlF eC4, a reversible activator of G subunits. Their interaction was assessed by GST pull-down experiments. As shown previously, in the presence of AlF eC4, the binding of Go was detected with full-length GRIN1 as well as with GRIN1 (562eC827) (Fig. 1A; data not shown). As shown in Fig. 1B, with further deletion, the binding of Go was detected in the region of GRIN1 (716eC827). In contrast, the mutant without this carboxyl-terminal region, GRIN1 (562eC716), did not interact with Go. Within amino acids 716 to 827 of GRIN1, Go binding was detected in the region of 716 to 746 or 797 to 827 but not in the region of 746 to 771, although the interaction with these short segments was weaker than that with GRIN1 (716eC827). These experiments indicate that the both regions of GRIN1 (716eC746 and 797eC827) are involved in the association with Go. RGS protein recognizes the activated form of G and accelerates its GTPase activity. Given that GRIN1 specifically recognizes activated Go, we tested whether the interaction with GRIN1 stimulates its GTPase activity similar to RGS proteins. However, GRIN1 (716eC771) showed no effect on the GTPase activity of Go (data not shown). Thus, GRIN1 interacts with Go without GAP activity.
Membrane Targeting Region of GRIN1. We next investigated the domain of GRIN1 involved in its subcellular localization. Neuro2a cells were transiently transfected with GRIN1 constructs that were fused with GFP at their amino terminus, and their expression was analyzed by fluorescence microscopy (Fig. 2). Full-length GRIN1 localized mainly at the cell periphery. GRIN1 (716eC827) and GRIN1 (766eC827) also demonstrated a membrane distribution pattern. In contrast, GRIN1 (717eC827) and GRIN1 (716eC771), constructs lacking the carboxyl-terminal region, showed cytosolic distribution. These results indicate that the carboxyl-terminal region of GRIN1 (772eC827) is involved in its membrane targeting.
Because the amino acid sequence of GRIN1 does not contain any apparent transmembrane region, the membrane distribution of GRIN1 suggests the possibility of lipid modification of GRIN1. A variety of signaling proteins undergo palmitoylation at cysteine residues. It is known that palmitoylation affects the activity of the protein in addition to its subcellular distribution (Wedegaertner and Bourne, 1994; Tu et al., 1999; Osterhout et al., 2003). Within the membrane-targeting region of GRIN1 (772eC827), potential palmitoylation sites were found at Cys818 and Cys819. These cysteine residues were conserved in both human and mouse GRIN1, suggesting the functional importance of these residues. We thus generated a GRIN1 mutant, GRIN1CCAA, in which both cysteine residues were mutated to alanine. The GRIN1CCAA mutant demonstrated a cytosolic pattern of distribution (Fig. 2). The results indicate that Cys818 and Cys819 residues are critical to target GRIN1 to plasma membrane.
Effect of the Go-GRIN1 Pathway on Cell Morphology. When coexpressed with GoQ205L, GRIN1 induced neurite formation in Neuro2a cells or fine process formation in MA104 cells (Chen et al., 1999). To further characterize this morphological change induced by Go-GRIN1, a GRIN1 mutant lacking the Go binding region, GFP-GRIN1(717eC827), was expressed in Neuro2a cells with or without GoQ205L. As shown in Fig. 3A, expression of GRIN1(717eC827) in Neuro2a cells promoted neurite extension similar to cells coexpressing wild-type GRIN1. Furthermore, this effect of GRIN1(717eC827) did not require coexpression of GoQ205L. These results suggest that the Go binding domain may have an inhibitory effect on GRIN1 activity, and the deletion of the Go binding domain of GRIN1 may change its conformation to a constitutively active form.
In Fig. 3B, we examined the neurite-promoting activity of GRIN1CCAA mutant. GRIN1CCAA demonstrated membrane distribution when coexpressed with GoQL but not with Go wild-type, suggesting the activation-dependent interaction with Go. It also promoted neurite extension in the presence of activated Go. These results suggest that although Cys818 and Cys819 are required for the membrane distribution of GRIN1, they are not critically involved in the interaction with Go or the neurite-promoting activity for GRIN1.
We also examined the specificity of GRIN1-induced morphological changes using activated mutants of several different G subunits. In this experiment, Swiss 3T3 cells were used to eliminate the morphological changes through endogenous GRIN1. As shown in Fig. 4, Swiss 3T3 cells coexpressing GRIN1 and Gi1Q204L formed microspikes similar to cells expressing GoQ205L and GRIN1. However, no such morphological changes were detected in cells coexpressing GRIN1 and GqR183C or GsQ227L. The results are consistent with the results from in vitro experiments showing that GRIN1 specifically interact with subunits of Gi subfamily. The results also support that these morphological changes are induced by the interaction of GRIN1 with Gi/o.
Involvement of Rho Family GTPases in the Go-GRIN1 Signaling Pathway. It is well established that Rho family GTPases, Rho, Rac, and Cdc42, are involved in morphological changes of cells through regulation of the actin cytoskeleton. We thus investigated whether the morphological changes induced by the Go-GRIN1 pathway involves these Rho family GTPases. Swiss 3T3 cells were microinjected with expression plasmids encoding dominant-negative mutants of Rho GTPases, Cdc42N17, Rac1N17, or RhoAN19, along with GoQ205L-DsRed and GFP-GRIN1. As shown in Fig. 5A, coexpression of dominant-negative Cdc42 blocked microspike formation in Swiss 3T3 cells, whereas dominant-negative Rac1 or RhoA had no pronounced effect on Go-GRIN1eCinduced morphological changes. We also conducted similar experiments using Neuro2a cells (Fig. 5B). Both dominant-negative Cdc42 and dominant-negative Rac1 inhibited neurite extension in Neuro2a cells expressing GoQ205L and GRIN1, whereas dominant-negative RhoA showed no effect. These results suggest that Go-GRIN1 signaling may involve the activation of Cdc42 downstream to induce microspike formation.
Coexpression of GoQ205L and GRIN1 Activates Cdc42. Finally, we examined whether the Go-GRIN1 pathway activates Cdc42 in cells. The amount of GTP-Cdc42 in cells was quantified by pull-down assays using GST-PAK-PBD as described under Materials and Methods. As shown in Fig. 6, coexpression of GoQ205L and GRIN1 in Swiss 3T3 cells increased GTP-Cdc42 approximately 2-fold. The expression of GRIN1(717eC827) induced similar levels of increase in GTP-Cdc42. This suggests again that GRIN1(717eC827) is constitutively active and independent of Go. These results agree well with the morphological data shown in Fig. 5 and further support the model that Go-GRIN1 signaling pathway activates Cdc42 to induce morphological changes in cells.
Discussion
In this study, we have characterized the mechanism of the Go-GRIN1 signaling pathway. We identified the Go binding domain of GRIN1 and also the amino acid residues critical for membrane targeting of GRIN1. We also presented evidence suggesting the possible link of the Go-GRIN1 pathway to the activation of Cdc42, which is probably responsible for the induction of neurite growth by Go-GRIN1.
Using deletion mutants of GRIN1, the Go binding domain of GRIN1 was mapped to its carboxyl-terminal regions (716eC746 and 797eC827). Go interacts with both of these regions, and the presence of either region in GRIN1 could show activation-dependent association with Go. Amino acid sequences of these binding regions are highly conserved in GRIN2, suggesting that GRIN2 will probably interact with Go through the corresponding regions. The Go binding domain did not show apparent homology with any known G-interacting motifs, and GRIN1 did not demonstrate GAP activity for Go. Thus, these regions are considered to be novel G interacting motifs. Further characterization and identification of the critical residues of GRIN1 for interaction with Go will be pursued.
The inhibitory effect of GRIN1 on the GAP activity of RGS4 for Go indicates the competition of the interaction of RGS4 and GRIN1 on Go. However, the results do not exclude the possibility that other RGS proteins might associate Go simultaneously with GRIN1 and regulate the Go-GRIN1 signaling pathway. For example, in the retinal phototransduction system, Gt has been shown to interact simultaneously with its effector, PDE, and its GAP, RGS9eC1 (Slep et al., 2001). The presence of RGS9eC1 was critical for the temporal resolution of Gt-mediated visual signal transduction (Skiba et al., 2001). In contrast to RGS4, which is an effective GAP for both Go and Gi, members of R7 subfamily of RGS protein, such as RGS6 and RGS7, show specific GAP activity for Go but not for Gi (Posner et al., 1999). Furthermore, they are expressed only in the brain, similar to GRIN1 (Witherow and Slepak, 2003). It will be important to further characterize the involvement of RGS proteins, such as R7 subfamily members, in the Go-GRIN1 pathway in brain.
We demonstrated that the carboxyl-terminal region of GRIN1 was involved in its membrane targeting. In particular, cysteines 818 and 819 were identified as critical residues for membrane localization. Mutation of these cysteine residues abolished the membrane distribution pattern of GRIN1. However, they are not required for the interaction with Go or the neurite-promoting activity of GRIN1. Similar to other signaling proteins, it is possible that these cysteine residues are modified by palmitoylation. In addition to the regulation of subcellular distribution, palmitoylation also participates in the regulation of biochemical function of various proteins (Wedegaertner and Bourne, 1994; Tu et al., 1999; Osterhout et al., 2003). It will be important to confirm whether GRIN1 is palmitoylated at these residues and, if it is palmitoylated, whether this modification regulates Go-GRIN1eCmediated signaling.
Coexpression of GoQ205L and full-length GRIN1 stimulates microspike formation in Swiss 3T3 cells and promotes neurite extension in Neuro2a cells. Similar morphological changes were observed under conditions of GRIN1(717eC827) overexpression, which lacks the Go binding region. Coexpression of GoQ205L was not required for the morphological changes induced by GRIN1(717eC827). These results suggest that deletion of the Go binding domain may change the conformation of GRIN1 into a constitutively active form. It is possible that the Go binding domain acts to inhibit GRIN1 function and that the binding of Go to GRIN1 releases the autoinhibitory effect of the Go binding domain.
We also presented evidence suggesting the possible link of the Go-GRIN1 pathway to the activation of Cdc42. Overexpression of dominant-negative Cdc42 blocked microspike formation induced by GoQ205L and GRIN1 in Swiss 3T3 cells. Similarly, overexpression of dominant-negative Cdc42 inhibited the neurite growth in Neuro2a cells. Furthermore, GST pull-down assays to detect GTP-bound Cdc42 in cells showed that GRIN1 activated Cdc42 synergistically when coexpressed with GoQ205L. Consistent with the morphological studies, GRIN1(717eC827) stimulated Cdc42 independent of GoQ205L. Thus, the binding of activated Go to the carboxyl-terminal region of GRIN1 is likely to induce activation of Cdc42, which will then lead to these morphological changes.
Rho family GTPases, Rho, Rac, and Cdc42, are well known for playing critical roles in the regulation of synaptogenesis, growth-cone guidance, or neurite outgrowth (Ziv and Smith, 1996; Threadgill et al., 1997; Jay, 2000). Go is expressed most abundantly in the brain, and expression of a constitutively active mutant of Go promoted neurite extension in PC12 cells (Strittmatter et al., 1994). The molecular mechanisms for these Go-mediated effects has remained poorly characterized. As indicated in this study, one possibility is that Go regulates neurite outgrowth through GRIN1 by controlling the activity of Cdc42. Further analysis of the function of endogenous GRIN1 in Go-mediated Cdc42 activation and neurite growth, and the characterization of the mechanism of regulation of Cdc42, such as the identification of a guanine nucleotide exchange factor for Cdc42 down-stream of GRIN1, will be critically important to further understand the physiological role of Go-GRIN1 signaling in neuronal cells.
Acknowledgements
We thank Drs. X. Du and N. Yokoyama for helpful suggestions and Drs. G. Bokoch and T. Satoh for the gift of plasmids for GST-PAK-PBD and Flag-tagged Rho GTPases, respectively. We also thank Dr. S. Nakamura at National Institute of Neuroscience in Japan for encouragement and helpful discussions.
doi:10.1124/mol.104.003913.
1 Current address: National Institute of Neuroscience, Department of Biochemistry and Cellular Biology, 4eC1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan.
References
Benard V, Boh BP, and Bokoch GM (1999) Characterization of Rac and Cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases. J Biol Chem 274: 13198eC13204.
Berman DM, Wilkie TM, and Gilman AG (1996) GAIP and RGS4 are GTPase-activating proteins for the Gi subfamily of G protein alpha subunits. Cell 86: 445eC452.
Chen LT, Gilman AG, and Kozasa T (1999) A candidate target for G protein action in brain. J Biol Chem 274: 26931eC26938.
Dickson BJ (2001) Rho GTPases in growth cone guidance. Current Opin Neurobiol 11: 103eC110.
Etienne-Manneville S and Hall A (2002) Rho GTPases in cell biology. Nature (Lond) 420: 629eC635.
Gilman AG (1987) G proteins: transducers of receptor-generated signals. Annu Rev Biochem 56: 615eC649.
Hepler JR and Gilman AG (1992) G proteins. Trends Biochem Sci 17: 383eC387.
Jay DG (2000) The clutch hypothesis revisited: ascribing the roles of actin-associated proteins in filopodial protrusion in the nerve growth cone. J Neurobiol 44: 114eC125.
Kozasa T and Gilman AG (1995) Purification of recombinant G proteins from Sf9 cells by hexahistidine tagging of associated subunits: characterization of 12 and inhibition of adenylyl cyclase by z. J Biol Chem 270: 1734eC1741.
Nagata Y, Oda M, Nakata H, Shozaki Y, Kozasa T, and Todokoro K (2001) A novel regulator of G-protein signaling bearing GAP activity for Gai and Gaq in megakaryocytes. Blood 97: 3051eC3060.
Osterhout JL, Waheed AA, Hiol A, Ward RD, Davey PC, Nini L, Wang J, Milligan G, Jones TLZ, and Druey KM (2003) Palmitoylation regulates regulator of G-protein signaling (RGS) 16 function. J Biol Chem 278: 19309eC19316.
Posner B, Gilman AG, and Harris B (1999) Regulators of G protein signaling 6 and 7. Purification of complexes with Gbeta5 and assessment of their effects on G protein-mediated signaling pathways. J Biol Chem 274: 31087eC31093.
Rochdi MD, Watier V, Madeleine CL, Nakata H, Kozasa T, and Parent JL (2002) Regulation of GTP-binding protein q (Gq) signaling by the Ezrin-Radixin-Moesin-binding phosphoprotein-50 (EBP50). J Biol Chem 277: 40751eC40759.
Ross EM and Wilkie TM (2000) GTPase-activating proteins for heterotrimeric G proteins: regulators of G protein signaling (RGS) and RGS-like proteins. Annu Rev Biochem 69: 795eC827.
Royal I, Lamarche-Vane N, Lamorte L, Kaibuchi K, and Park M (2000) Activation of Cdc42, Rac, PAK and Rho-kinase in response to hepatocyte growth factor differentially regulates epithelial cell colony spreading and dissociation. Mol Biol Cell 11: 1709eC1725.
Schmidt A and Hall A (2002) Guanine nucleotide exchange factors for Rho GTPases: turning on the switch. Genes Dev 16: 1587eC1609.
Skiba NP, Martemyanov KA, Elfenbein A, Hopp JA, Bohm A, Simonds WF, and Arshavsky VY (2001) RGS9-G5 substrate selectivity in photoreceptors. Opposing effects of constituent domains yield high affinity of RGS interaction with the G protein-effector complex. J Biol Chem 276: 37365eC37372.
Slep KC, Kercher MA, He W, Cowan CW, Wensel TG, and Sigler PB (2001) Structural determinants for regulation of phosphodiesterase by a G protein at 2.0 A. Nature (Lond) 409: 1071eC1077.
Strittmatter SM, Fishman MC, and Zhu X-P (1994) Activated mutants of the subunit of Go promote an increased number of neurites per cell. J Neurosci 14: 2327eC2338.
Symons M and Settleman J (2000) Rho family GTPases: more than simple switches. Trends Cell Biol 10: 415eC419.
Taussig R, Tang W-J, Hepler JR, and Gilman AG (1994) Distinct patterns of bidirectional regulation of mammalian adenylyl cyclases. J Biol Chem 269: 6093eC6100.
Threadgill R, Bobb K, and Ghosh A (1997) Regulation of dendritic growth and remodeling by Rho, Rac and Cdc42. Neuron 19: 625eC634.
Tu Y, Popov S, Slaughter C, and Ross EM (1999) Palmitoylation of a conserved cysteine in the regulator of G protein signaling (RGS) domain modulates the GTPase-activating activity of RGS4 and RGS10. J Biol Chem 274: 38260eC38267.
Wedegaertner PB and Bourne HR (1994) Activation and depalmitoylation of Gs alpha. Cell 77: 1063eC1070.
Witherow DS and Slepak VZ (2003) A novel kind of G protein heterodimer: the G5-RGS complex. Receptors Channels 9: 205eC212.
Ziv NE and Smith SJ (1996) Evidence for a role of dendritic filopodia in synaptogenesis and spine formation. Neuron 17: 91eC102.