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• W2035447817 abstract "In the developing forebrain, the migration and positioning of neural progenitor cells (NPCs) are regulated coordinately by various molecules. Mutation of these molecules, therefore, causes cortical malformation. GPR56 has been reported as a cortical malformation-related gene that is mutated in patients with bilateral frontoparietal polymicrogyria. GPR56 encodes an orphan G protein-coupled receptor, and its mutations reduce the cell surface expression. It has also been reported that the expression level of GPR56 is involved in cancer cell adhesion and metastasis. However, it remains to be clarified how GPR56 functions in brain development and which signaling pathways are activated by GPR56. In this study, we showed that GPR56 is highly expressed in NPCs and has the ability to inhibit NPC migration. We found that GPR56 coupled with Gα12/13 and induced Rho-dependent activation of the transcription mediated through a serum-responsive element and NF-κB-responsive element and actin fiber reorganization. The transcriptional activation and actin reorganization were inhibited by an RGS domain of the p115 Rho-specific guanine nucleotide exchange factor (p115 RhoGEF RGS) and dominant negative form of Rho. Moreover, we have demonstrated that a functional anti-GPR56 antibody, which has an agonistic activity, inhibited NPC migration. This inhibition was attenuated by p115 RhoGEF RGS, C3 exoenzyme, and GPR56 knockdown. These results indicate that GPR56 participates in the regulation of NPC movement through the Gα12/13 and Rho signaling pathway, suggesting its important role in the development of the central nervous system. In the developing forebrain, the migration and positioning of neural progenitor cells (NPCs) are regulated coordinately by various molecules. Mutation of these molecules, therefore, causes cortical malformation. GPR56 has been reported as a cortical malformation-related gene that is mutated in patients with bilateral frontoparietal polymicrogyria. GPR56 encodes an orphan G protein-coupled receptor, and its mutations reduce the cell surface expression. It has also been reported that the expression level of GPR56 is involved in cancer cell adhesion and metastasis. However, it remains to be clarified how GPR56 functions in brain development and which signaling pathways are activated by GPR56. In this study, we showed that GPR56 is highly expressed in NPCs and has the ability to inhibit NPC migration. We found that GPR56 coupled with Gα12/13 and induced Rho-dependent activation of the transcription mediated through a serum-responsive element and NF-κB-responsive element and actin fiber reorganization. The transcriptional activation and actin reorganization were inhibited by an RGS domain of the p115 Rho-specific guanine nucleotide exchange factor (p115 RhoGEF RGS) and dominant negative form of Rho. Moreover, we have demonstrated that a functional anti-GPR56 antibody, which has an agonistic activity, inhibited NPC migration. This inhibition was attenuated by p115 RhoGEF RGS, C3 exoenzyme, and GPR56 knockdown. These results indicate that GPR56 participates in the regulation of NPC movement through the Gα12/13 and Rho signaling pathway, suggesting its important role in the development of the central nervous system. In the developing cerebral cortex, neural stem cells divide asymmetrically to generate themselves and neural progenitor cells in the lateral ventricle. Neural progenitor cells (NPCs) 2The abbreviations used are: NPC, neural progenitor cell; GPCR, G protein-coupled receptor; GPS, GPCR proteolytic site; BFPP, bilateral frontoparietal polymicrogyria; SRE, serum-responsive element; NF-κB, nuclear factor-κB; GPR56ECD, GPR56 extracellular domain; p115 RhoGEF RGS, RGS domain of p115 Rho-specific guanine nucleotide exchange factor; mDia1RBD, RhoA binding domain of mDia1; LPA, l-α-lysophosphatidic acid; GFP, green fluorescent protein; GST, glutathione S-transferase; TRAP6, thrombin receptor-activating peptide 6; MAP, microtubule-associated protein; MAPK, MAP kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; E, embryonic day; sh, small hairpin; TG2, tissue transglutaminase 2; PEI, polyethyleneimine; PBS, phosphate-buffered saline; Luc, luciferase; TCF, T cell factor. 2The abbreviations used are: NPC, neural progenitor cell; GPCR, G protein-coupled receptor; GPS, GPCR proteolytic site; BFPP, bilateral frontoparietal polymicrogyria; SRE, serum-responsive element; NF-κB, nuclear factor-κB; GPR56ECD, GPR56 extracellular domain; p115 RhoGEF RGS, RGS domain of p115 Rho-specific guanine nucleotide exchange factor; mDia1RBD, RhoA binding domain of mDia1; LPA, l-α-lysophosphatidic acid; GFP, green fluorescent protein; GST, glutathione S-transferase; TRAP6, thrombin receptor-activating peptide 6; MAP, microtubule-associated protein; MAPK, MAP kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; E, embryonic day; sh, small hairpin; TG2, tissue transglutaminase 2; PEI, polyethyleneimine; PBS, phosphate-buffered saline; Luc, luciferase; TCF, T cell factor. then migrate from the ventricular zone to the cortical plate along the radial glial fiber. In this process, called “radial migration,” NPCs terminally differentiate into mature neurons (1Marin O. Rubenstein J.L. Annu. Rev. Neurosci. 2003; 26: 441-483Crossref PubMed Scopus (779) Google Scholar). These series of movements are coordinately regulated by various factors, namely, extracellular, cytoskeletal, and signaling molecules (2Bielas S. Higginbotham H. Koizumi H. Tanaka T. Gleeson J.G. Annu. Rev. Cell Dev. Biol. 2004; 20: 593-618Crossref PubMed Scopus (106) Google Scholar, 3Marin O. Valdeolmillos M. Moya F. Trends Neurosci. 2006; 29: 655-661Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Several molecules involved in neuronal cell migration have been identified in studies of cortical development disorders. Reelin, a large protein secreted extracellularly that is associated with the extracellular matrix, controls neuronal positioning work as a terminal signal of radial migration. Mice deficient in Reelin develop an inverted cortex (4D'Arcangelo G. Miao G.G. Chen S.C. Soares H.D. Morgan J.I. Curran T. Nature. 1995; 374: 719-723Crossref PubMed Scopus (1474) Google Scholar, 5Hirotsune S. Takahara T. Sasaki N. Hirose K. Yoshiki A. Ohashi T. Kusakabe M. Murakami Y. Muramatsu M. Watanabe S. Nat. Genet. 1995; 10: 77-83Crossref PubMed Scopus (295) Google Scholar, 6Ogawa M. Miyata T. Nakajima K. Yagyu K. Seike M. Ikenaka K. Yamamoto H. Mikoshiba K. Neuron. 1995; 14: 899-912Abstract Full Text PDF PubMed Scopus (769) Google Scholar). Cytoskeleton organization, including the formations of an actin filament and a microtubule, induces the cell shape change and plays a critical role in the regulation of neuronal migration. Two microtubule-associated proteins (MAPs), Lis1 and Dcx, have been shown to be essential for radial migration with the ability to influence microtubule dynamics. The mutation of these two genes results in lissencephaly (7Reiner O. Carrozzo R. Shen Y. Wehnert M. Faustinella F. Dobyns W.B. Caskey C.T. Ledbetter D.H. Nature. 1993; 364: 717-721Crossref PubMed Scopus (894) Google Scholar, 8Hattori M. Adachi H. Tsujimoto M. Arai H. Inoue K. Nature. 1994; 370: 216-218Crossref PubMed Scopus (453) Google Scholar, 9des Portes V. Pinard J.M. Billuart P. Vinet M.C. Koulakoff A. Carrie A. Gelot A. Dupuis E. Motte J. Berwald-Netter Y. Catala M. Kahn A. Beldjord C. Chelly J. Cell. 1998; 92: 51-61Abstract Full Text Full Text PDF PubMed Scopus (667) Google Scholar, 10Gleeson J.G. Allen K.M. Fox J.W. Lamperti E.D. Berkovic S. Scheffer I. Cooper E.C. Dobyns W.B. Minnerath S.R. Ross M.E. Walsh C.A. Cell. 1998; 92: 63-72Abstract Full Text Full Text PDF PubMed Scopus (872) Google Scholar). Small GTPases of the Rho family, Rho, Rac, and Cdc42, contribute to cell movement by regulating the actin cytoskeleton (11Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3794) Google Scholar). The involvement of Lis1 in the regulation of the actin cytoskeleton has been also reported. Lis1 haploinsufficiency resulted in the up-regulation of RhoA activity and a reduction in Rac1 and Cdc42 activities (12Kholmanskikh S.S. Dobrin J.S. Wynshaw-Boris A. Letourneau P.C. Ross M.E. J. Neurosci. 2003; 23: 8673-8681Crossref PubMed Google Scholar). Therefore, the microtubule and actin cytoskeleton coordinately have a crucial role in the regulation of neuronal migration. Although many molecules involved in brain development have been identified, it remains to be clarified how these molecules control NPC behavior by distinct or coordinated signal transduction mechanisms.G protein-coupled receptors (GPCRs) constitute the largest family of cell surface receptors and play an important role in controlling key physiological functions, including neurotransmission, hormone release, cell growth, and cell migration (13Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4682) Google Scholar, 14Marinissen M.J. Gutkind J.S. Trends Pharmacol. Sci. 2001; 22: 368-376Abstract Full Text Full Text PDF PubMed Scopus (832) Google Scholar). Heterotrimeric G proteins transduce signals from GPCRs to an effector (15Kaziro Y. Itoh H. Kozasa T. Nakafuku M. Satoh T. Annu. Rev. Biochem. 1991; 60: 349-400Crossref PubMed Scopus (547) Google Scholar, 16Milligan G. Kostenis E. Br. J. Pharmacol. 2006; 147: S46-S55Crossref PubMed Scopus (293) Google Scholar). Some reports have indicated that G protein signaling is involved in the development of the cerebral cortex by modulating cell migration and proliferation. The γ-aminobutyric acid, type B (GABAB) receptor, which is a member of the GPCR, influences migration from the intermediate zone to the cortical plate during development (17Behar T.N. Schaffner A.E. Scott C.A. Greene C.L. Barker J.L. Cereb. Cortex. 2000; 10: 899-909Crossref PubMed Scopus (180) Google Scholar, 18Behar T.N. Smith S.V. Kennedy R.T. McKenzie J.M. Maric I. Barker J.L. Cereb. Cortex. 2001; 11: 744-753Crossref PubMed Scopus (115) Google Scholar). Stromal cell-derived factor-1 (SDF-1) and its receptor, CXC chemokine receptor 4 (CXCR-4), regulate the tangential migration of interneuron precursors in the developing neocortex. In SDF-1- and CXCR-4-deficient mice, late-generated interneurons fail to integrate into the appropriate neocortical layer (19Stumm R.K. Zhou C. Ara T. Lazarini F. Dubois-Dalcq M. Nagasawa T. Hollt V. Schulz S. J. Neurosci. 2003; 23: 5123-5130Crossref PubMed Google Scholar). We showed previously that endothelin-1 (ET-1), a ligand for the endothelin receptor, negatively regulates NPC migration through Gαq and JNK (20Mizuno N. Kokubu H. Sato M. Nishimura A. Yamauchi J. Kurose H. Itoh H. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12365-12370Crossref PubMed Scopus (31) Google Scholar). Another group has indicated that the G protein signaling through Gαi2 maintains mitogenic activity in the NPCs during brain development (21Shinohara H. Udagawa J. Morishita R. Ueda H. Otani H. Semba R. Kato K. Asano T. J. Biol. Chem. 2004; 279: 41141-41148Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). These studies demonstrated the importance of G protein signaling in the development of the central nervous system.GPR56 is a newly identified orphan GPCR that belongs to the adhesion GPCR family (22Liu M. Parker R.M. Darby K. Eyre H.J. Copeland N.G. Crawford J. Gilbert D.J. Sutherland G.R. Jenkins N.A. Herzog H. Genomics. 1999; 55: 296-305Crossref PubMed Scopus (66) Google Scholar). Adhesion GPCRs have a unique feature in that they contain four highly conserved cysteine residues in a GPCR proteolytic site (GPS) and are thought to function in cell-cell or cell-matrix adhesion because their long N-terminal extracellular regions often contain motifs or domains involved in cell adhesion (23Fredriksson R. Lagerstrom M.C. Hoglund P.J. Schioth H.B. FEBS Lett. 2002; 531: 407-414Crossref PubMed Scopus (73) Google Scholar, 24Fredriksson R. Gloriam D.E. Hoglund P.J. Lagerstrom M.C. Schioth H.B. Biochem. Biophys. Res. Commun. 2003; 301: 725-734Crossref PubMed Scopus (99) Google Scholar, 25Bjarnadottir T.K. Fredriksson R. Hoglund P.J. Gloriam D.E. Lagerstrom M.C. Schioth H.B. Genomics. 2004; 84: 23-33Crossref PubMed Scopus (184) Google Scholar, 26Bjarnadottir T.K. Fredriksson R. Schioth H.B. Cell Mol. Life Sci. 2007; 64: 2104-2119Crossref PubMed Scopus (95) Google Scholar). A recent study identified GPR56 as a cortical development-associated gene. Mutations of GPR56 cause bilateral frontoparietal polymicrogyria (BFPP) (27Piao X. Hill R.S. Bodell A. Chang B.S. Basel-Vanagaite L. Straussberg R. Dobyns W.B. Qasrawi B. Winter R.M. Innes A.M. Voit T. Ross M.E. Michaud J.L. Descarie J.C. Barkovich A.J. Walsh C.A. Science. 2004; 303: 2033-2036Crossref PubMed Scopus (407) Google Scholar). In this disorder, the organization of the frontal cortex is disrupted and shows thinner cortical layers and numerous small folds. Several mutations of GPR56 with BFPP cause impairment of cell surface expression of the receptor (28Jin Z. Tietjen I. Bu L. Liu-Yesucevitz L. Gaur S.K. Walsh C.A. Piao X. Hum. Mol. Genet. 2007; 16: 1972-1985Crossref PubMed Scopus (96) Google Scholar). GPR56 mRNA abundantly localizes in the cerebral cortical ventricular and subventricular zones during periods of neurogenesis, suggesting that GPR56 is expressed in neuronal progenitors (27Piao X. Hill R.S. Bodell A. Chang B.S. Basel-Vanagaite L. Straussberg R. Dobyns W.B. Qasrawi B. Winter R.M. Innes A.M. Voit T. Ross M.E. Michaud J.L. Descarie J.C. Barkovich A.J. Walsh C.A. Science. 2004; 303: 2033-2036Crossref PubMed Scopus (407) Google Scholar, 29Terskikh A.V. Easterday M.C. Li L. Hood L. Kornblum H.I. Geschwind D.H. Weissman I.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7934-7939Crossref PubMed Scopus (250) Google Scholar). It has been reported that the expression level of GPR56 is involved in cancer cell adhesion and metastasis (30Zendman A.J. Cornelissen I.M. Weidle U.H. Ruiter D.J. van Muijen G.N. FEBS Lett. 1999; 446: 292-298Crossref PubMed Scopus (72) Google Scholar, 31Shashidhar S. Lorente G. Nagavarapu U. Nelson A. Kuo J. Cummins J. Nikolich K. Urfer R. Foehr E.D. Oncogene. 2005; 24: 1673-1682Crossref PubMed Scopus (127) Google Scholar, 32Xu L. Begum S. Hearn J.D. Hynes R.O. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 9023-9028Crossref PubMed Scopus (217) Google Scholar, 33Sud N. Sharma R. Ray R. Chattopadhyay T.K. Ralhan R. Cancer Lett. 2006; 233: 265-270Crossref PubMed Scopus (28) Google Scholar, 34Ke N. Sundaram R. Liu G. Chionis J. Fan W. Rogers C. Awad T. Grifman M. Yu D. Wong-Staal F. Li Q.X. Mol. Cancer Ther. 2007; 6: 1840-1850Crossref PubMed Scopus (50) Google Scholar). However, not only its ligand but also its functions in the developing forebrain and mechanisms of signal transduction remain largely unknown.To clarify the role of GPR56 in NPC behavior, we first confirmed that GPR56 is highly expressed in NPCs. Next, we analyzed the signaling pathway using the reporter assay and actin reorganization profile. We found that GPR56 activates serum-responsive element (SRE)- and nuclear factor-kappa B (NF-κB)-responsive element-mediated transcription through Gα12/13 and Rho in HEK293T cells. Ectopic expression of GPR56 in NIH3T3 cells induced F-actin accumulation in a Gα12/13- and Rho-dependent manner. We then found that overexpression of GPR56 inhibited NPC migration. Moreover, the role of endogenous GPR56 on NPC migration was examined using the agonistic antibody against the GPR56 extracellular domain. We demonstrated that GPR56 negatively regulates NPC migration through Gα12/13 and Rho.EXPERIMENTAL PROCEDURESCell Culture, Transfection, and Adenovirus Infection— HEK293T cells and NIH3T3 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 50 μg/ml penicillin, and 50 μg/ml streptomycin. Plasmid DNAs were transfected into cells using the calcium phosphate precipitation method or the Lipofectamine™ 2000 transfection reagent (Invitrogen). For the primary culture of neural progenitor cells, telencephalons of mouse embryo at embryonic day 11.5 (E11.5) were dissected surgically and washed with a buffer containing 124 mm NaCl, 5 mm KCl, 3.2 mm MgCl2, 0.1 mm CaCl2, 26 mm NaHCO3, and 10 mm d-glucose. The dissected telencephalons were treated with 0.05% trypsin for 15 min at 37 °C; this reaction was stopped by adding 70 μg/ml ovomucoid (Sigma). After brief centrifugation, cells were suspended in a Dulbecco's modified Eagle's medium/F12 medium (DF medium; Invitrogen), and 2 × 106 cells were plated in a 100-mm plastic dish coated with 20 μg/ml poly-2-hydroxyethyl methacrylate (poly-HEMA; Sigma). Cells were cultured in DF medium supplemented with B27 (Invitrogen), 20 ng/ml basic fibroblast growth factor, 20 ng/ml epidermal growth factor (Peprotech EC Ltd., London, UK), 1 mg/ml bovine serum albumin, and 2 μg/ml heparin (Sigma) with passage every 2–3 days. All cells were grown in 5% CO2 at 37 °C. Adenoviruses expressing green fluorescent protein (GFP), GPR56, shGPR56, shCNTRL, C3 exoenzyme, and the RGS domain of the p115 Rho-specific guanine nucleotide exchange factor (p115 RhoGEF RGS) were prepared as described below and previously (35Arai K. Maruyama Y. Nishida M. Tanabe S. Takagahara S. Kozasa T. Mori Y. Nagao T. Kurose H. Mol. Pharmacol. 2003; 63: 478-488Crossref PubMed Scopus (73) Google Scholar). Neural progenitor cells were infected with adenoviruses at a multiplicity of infection of 0.3–5 for 3 days. Because these adenoviruses express GFP, the infection of adenoviruses can be monitored by GFP fluorescence.Plasmid Constructs and Adenovirus Preparation—Mouse full-length GPR56 cDNA was obtained from the American Type Culture Collection (IMAGE clone ID 3709247). For pCMV5-GPR56, the cDNA of GPR56 was amplified by PCR with primers 5′-GAAGATCTATGGCTGTCCAGGTGCTGC-3′ and 5′-ACGCGTCGACTTAGATGCGGCTGGAGGAGGTG-3′. The PCR product was cloned into the BglII/SalI sites of pCMV5. For pFastBac-GPR56ECD-His, cDNA was amplified by PCR from pCMV5-GPR56 with primers 5′-GAAGATCTATGGCTGTCCAGGTGCTGC-3′ and 5′-GAAGATCTCTAGTGATGGTGATGGTGATGGAGGTAGTGTTTGTGAGTGG-3′ and cloned into the BglII site of pFastBac1. The DNAs of Gα13 (Q226L) and the RGS domain (including residues 1–252) of p115 RhoGEF were amplified by PCR and subcloned into the pCMV5-FLAG vector. pCMV5-Myc-βARK-ct, pEF-Bos-C3, pCMV5-Gα13(Q226L), pCMV5-FLAG-RhoA, pCMV5-FLAG-RhoA(G14V), pCMV5-FLAG-RhoA(T19N), pCMV5-FLAG-Rac1(T17N), and pCMV5-FLAG-Cdc42(T17N) were prepared as described previously (36Sun Y. Yamauchi J. Kaziro Y. Itoh H. J. Biochem. (Tokyo). 1999; 125: 515-521Crossref PubMed Scopus (17) Google Scholar). The Escherichia coli expression plasmid encoding the RhoA binding domain (RBD) of mDia1 was constructed and introduced into E. coli BL21 CodonPlus (DE3)-RIL as described previously (37Yamauchi J. Hirasawa A. Miyamoto Y. Itoh H. Tsujimoto G. Biochem. Biophys. Res. Commun. 2001; 284: 1199-1203Crossref PubMed Scopus (20) Google Scholar). GST-mDia1RBD was expressed and used for a pulldown assay. The adenovirus expressing GPR56 was made using the AdEasy XL adenoviral vector system (Stratagene, La Jolla, CA). Briefly, GPR56 cDNA was subcloned into the BglII/SalI sites of the pShuttle-IRES-hrGFP-1 shuttle vector from pCMV5-GPR56. The adenoviruses expressing GFP, p115 RhoGEF RGS, and the C3 exoenzyme were kindly provided by Dr. H. Kurose (Kyushyu University). The oligonucleotide sequences used in the construction of the small interference RNA vector were as follows: shGPR56, 5′-CGCGTCCGGTAGAAGCCACTCACAAATTCAAGAGATTTGTGAGTGGCTTCTACCTTTTTA-3′ and 5′-AGCTTAAAAAGGTAGAAGCCACTCACAAATCTCTTGAATTTGTGAGTGGCTTCTACCGGA-3′; shCNTRL, 5′-CGCGTCCAAATGTACTGCGTGGAGACTTCAAGAGAGTCTCCACGCAGTACATTTTTTTTA-3′ and 5′-AGCTTAAAAAAAATGTACTGCGTGGAGACTCTCTTGAAGTCTCCACGCAGTACATTTGGA-3′. The oligonucleotides were annealed and then ligated into MluI/HindIII sites of the pRNAT-H1.1/Adeno vector (GenScript, Edison, NJ). These plasmids were introduced into BJ5183-AD-1-competent cells carrying the pAdEasy1 vector. The recombinant adenoviral plasmid was digested with PacI and transfected into HEK293 cells, where virus particles were produced.GPR56ECD and Antibodies—Mouse GPR56ECD was generated using a baculovirus-Sf9 expression system and purified with ion exchange chromatography and affinity chromatography for His-tagged proteins. Briefly, a pFast-Bac-1 vector containing GPR56ECD was introduced into DH10Bac. The recombinant bacmid was prepared to be transfected into Sf9 cells. Recombinant baculovirus-infected Sf9 cells were cultured for 72 h, and the supernatant of the culture medium was applied to SP-Sepharose FF column chromatography. Elution with 50 mm sodium phosphate, pH 8.0, 500 mm NaCl, and 10 mm imidazole was collected and used for nickel-nitrilotriacetic acid-agarose. GPR56ECD was eluted with 400 mm imidazole and dialyzed against 50 mm sodium phosphate, pH 8.0. A Mono-S column was used for the final purification step. The rabbit polyclonal GPR56 antibody was generated against the recombinant GPR56ECD. The antibody was affinity-purified from the anti-serum using a GPR56ECD-conjugated column and dialyzed with PBS. The control antibody used in this study was prepared using mouse Asef2 as the antigen and affinity-purified following the same method. A mouse monoclonal antibody Rat401 against nestin was obtained from BD Biosciences. A mouse monoclonal antibody TUJ1 against βIII-tubulin was obtained from Babco (Richmond, CA). A mouse monoclonal antibody M2 against FLAG-peptide was obtained from Sigma. A mouse monoclonal antibody C4 against actin was obtained from Chemicon International (Temecula, CA). Antibody against p38 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against p44/42 MAPK, phospho-p44/42 MAPK, phospho-p38, and JNK were obtained from Cell Signaling Technology (Danvers, MA). Antibody against phospho-JNK was obtained from Promega (Madison, WI). Anti-mouse and anti-rabbit IgG antibodies conjugated with horseradish peroxidase were obtained from GE Healthcare. Alexa-594 phalloidin, Alexa-488-conjugated anti-rabbit IgG, and Alexa-594- and Alexa-350-conjugated anti-mouse IgG were obtained from Molecular Probes, Inc. (Eugene, OR).Immunoblotting—Samples in a Laemmli buffer were separated using SDS-polyacrylamide gels. The separated proteins were transferred to polyvinylidene difluoride membranes. After blocking for more than 30 min in 5% skim milk-TBST (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.1% Tween 20), membranes were probed for 1 h with primary antibodies in 5% skim milk-TBST. The membranes were then washed three times with TBST and incubated with secondary antibodies coupled to horseradish peroxidase. Immunoblotting was visualized by using an enhanced chemiluminescence Western blotting detection kit (GE Healthcare).Immunocytochemistry and Immunohistochemistry—NIH3T3 cells were plated onto 13-mm poly-d-lysine-coated coverslips. Two days later, cells were serum-starved for 6 h. Neural progenitor cells were cultured on the polyethyleneimine (PEI)/laminin-coated coverslips with basic fibroblast growth factor for 3 days. Cells were fixed in 4% paraformaldehyde in PBS for 30 min, washed three times with PBS for 10 min each, blocked for 30 min with a blocking solution (10% fetal bovine serum and 0.1% Triton X-100 in PBS for NIH3T3 cells or 10% fetal bovine serum in PBS for NPCs), and incubated with primary antibodies for 1 h. Coverslips were incubated with the appropriate secondary antibodies or Alexa-594 phalloidin for 30 min. For immunohistochemistry, E16.5 mouse brain was dissected, fixed overnight in 4% paraformaldehyde in PBS at 4 °C, and impregnated with 30% sucrose in PBS. Cryosections were cut at a thickness of 30 μm and immunostained with primary antibodies in a blocking solution overnight and with secondary antibodies in PBS overnight. The concentrations of antibodies and dye used were: anti-FLAG antibody, 1 μg/ml; anti-GPR56 antibody, 0.4 μg/ml; anti-nestin antibody, 1 μg/ml; anti-βIII-tubulin, 1 μg/ml; Alexa-488-conjugated anti-rabbit IgG and Alexa-350- and Alexa-594-conjugated anti-mouse IgG, 2 μg/ml; and Alexa-594 phalloidin, 2 units/ml. Samples were mounted and photographed with a Zeiss Axiophoto fluorescence microscope. A confocal image of NPCs was captured by a Zeiss LSM510 and analyzed.Reporter Gene Assay—HEK293T cells were seeded in a 48-well plate with about 70% confluence. Cells were transfected with 30 ng/well firefly luciferase reporter gene plasmids (SRE-Luc, SIE-Luc, CRE-Luc, c-fos-Luc, AP1-Luc, NF-κB-Luc, TCF-Luc, and SRF-Luc) and 0.6 ng/well Renilla-luciferase (Renilla-Luc) plasmid in combinations with other plasmids. For some experiments, cells were treated with 1 μg/ml pertussis toxin (Funakoshi Chemical Ltd., Tokyo, Japan) or a 10 μm Gαq/11 inhibitor YM-254890 (38Takasaki J. Saito T. Taniguchi M. Kawasaki T. Moritani Y. Hayashi K. Kobori M. J. Biol. Chem. 2004; 279: 47438-47445Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar) 1 h after transfection. Anti-GPR56 antibody and GPR56ECD were added 6 h before harvesting the cells. Twenty-four hours after transfection, the cells were resuspended in a passive lysis buffer, and the luciferase activity was then measured with a luminometer (ARVO MX, PerkinElmer Life Sciences) using the Dual-Luciferase reporter assay system (Promega, Madison, WI). Firefly luciferase activity was normalized to the internal control activity of Renilla-luciferase.Pulldown Assays of GTP-Rho—HEK293T cells were transfected with pCMV5-FLAG-RhoA (wild type, G14V, T19N) with or without pCMV5-GPR56. Thirty-six hours after serum starvation, the cells were stimulated with or without 15 μg/ml anti-GPR56 antibody for 10 min and lysed in 300 μl of lysis buffer A (20 mm HEPES-NaOH, pH 7.5, 150 mm NaCl, 20 mm MgCl2, 0.5% Nonidet P-40, 1 μg/ml leupeptin) per-35 mm dish. After centrifugation at 15,000 × g at 4 °C for 10 min, 200 μl of supernatant was incubated with 25 μg of GST-mDia1RBD and glutathione-Sepharose at 4 °C for 30 min. The resins were washed twice with lysis buffer A and boiled in Laemmli sample buffer. Eluted samples were subjected to SDS-PAGE. Bound RhoA was detected by immunoblotting using an anti-FLAG antibody.MAP Kinase Assays—HEK293T cells were transfected with or without pCMV5-GPR56. Thirty-six hours after serum starvation, the cells were stimulated with 15 μg/ml anti-GPR56 antibody for 3, 10, and 20 min, 1 μm lysophosphatidic acid (LPA) for 20 min, or 10 μm thrombin receptor-activating peptide 6 (TRAP6) for 20 min. Cell lysates were prepared with the lysis buffer B (20 mm HEPES-NaOH, pH 7.5, 100 mm NaCl, 3 mm MgCl2, 1 mm dithiothreitol, 1 mm phenylmethanesulfonyl fluoride, 1 mm EGTA, 1 mm Na3VO4, 10 mm NaF, 10 mm β-glycerophosphate, 0.5% Nonidet P-40, 1 μg/ml leupeptin). After sonication and centrifugation at 15,000 × g at 4 °C for 10 min, the supernatants were subjected to SDS-PAGE. Activation of MAPKs (ERK, JNK, and p38) was assessed by immunoblotting using each anti-phospho-MAPK antibody.Measurement of Intracellular Ca2+ Mobilization—Intracellular calcium mobilization was measured using the fluorescent Ca2+ indicator Fura-2 acetoxymethyl ester (Fura-2/AM) (Dojin Kagaku). Briefly, HEK293T cells were washed, detached in a suspension buffer (20 mm HEPES-NaOH, pH 7.5, 140 mm NaCl, 5.4 mm KCl, 1.8 mm CaCl2, 0.8 mm MgCl2, and 5.6 mm glucose). The suspended cells were loaded with 2 μm Fura-2/AM for 30 min at 30 °C and then washed with a suspension buffer to remove the extracellular dye. For the fluorimetric measurement, 1 × 106 cells were placed into a cuvette in a thermostatically controlled cell holder at 37 °C with continuous stirring. The cells were stimulated with 1 μm LPA or 7.5 μg/ml anti-GPR56 antibody. Fluorescence was monitored using an F-2000 fluorescence spectrophotometer (Hitachi) with emission at 510 nm after excitation at 340 and 380 nm.Migration Assay—The migration assay was performed as described previously (20Mizuno N. Kokubu H. Sato M. Nishimura A. Yamauchi J. Kurose H. Itoh H. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12365-12370Crossref PubMed Scopus (31) Google Scholar) with slight modifications. The migration of neural progenitor cells was performed in a 96-well dish precoated with PEI. The 96-well dish was coated with 50 μl of 0.1% PEI in a 30 mm boric acid buffer (pH 8.4) for at least 6 h at room temperature and washed three times with distilled water. Eighty μl of a differentiation medium (a DF medium with B27, 1 mg/ml bovine serum albumin) was added to a well, and 20 μl of a 3 day-cultured neurospheres suspension (including 20–40 neurospheres) was then added to the well. Twenty-four hours later, the number of migrating neurospheres was counted. Migration was assessed by measurement of the distance from the edge of the neurosphere to the leading cell of outgrowth. If this distance was more than the diameter of the neurosphere or the neurosphere was completely dispersed, this sphere was evaluated as a migrating neurosphere. The migration activity was indicated by the ratio of migrating neurospheres to total neurospheres.RESULTSGPR56 Is Highly Expressed in Neural Progenitor Cell Membranes as a Cleaved Form—To analyze the tissue-specific distribution and developmental expression of GPR56, we generated a polyclonal antibody against the baculovirus-expressed N-terminal extracellular region of GPR56 and used it for immunoblotting and immunocytochemistry. It has been reported that the N-terminal extracellular region of GPR56 is cleaved at the GPS motif during processing (32Xu L. Begum S. Hearn J.D. Hynes R.O. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 9023-9028Crossref PubMed Scopus (217) Google Scholar). Thus, the anti-GPR56 antibody recognized both the full-length GPR56 (GPR56FL) at 75 kDa and" @default.
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• W2035447817 title "Orphan G Protein-coupled Receptor GPR56 Regulates Neural Progenitor Cell Migration via a Gα12/13 and Rho Pathway" @default.
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