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Growth Factor Receptor-binding Protein 10 (Grb10) as a Partner of Phosphatidylinositol 3-Kinase in Metabolic Insulin Action

2003; Elsevier BV; Volume: 278; Issue: 41 Linguagem: Inglês

10.1074/jbc.m304599200

ISSN

1083-351X

Autores

Youping Deng, Sujoy Bhattacharya, O. Rama Swamy, Ruchi Tandon, Yong Wang, R Janda, Heimo Riedel,

Tópico(s)

RNA Interference and Gene Delivery

Resumo

The regulation of the metabolic insulin response by mouse growth factor receptor-binding protein 10 (Grb10) has been addressed in this report. We find mouse Grb10 to be a critical component of the insulin receptor (IR) signaling complex that provides a functional link between IR and p85 phosphatidylinositol (PI) 3-kinase and regulates PI 3-kinase activity. This regulatory mechanism parallels the established link between IR and p85 via insulin receptor substrate (IRS) proteins. A direct association was demonstrated between Grb10 and p85 but was not observed between Grb10 and IRS proteins. In addition, no effect of mouse Grb10 was observed on the association between IRS-1 and p85, on IRS-1-associated PI 3-kinase activity, or on insulin-mediated activation of IR or IRS proteins. A critical role of mouse Grb10 was observed in the regulation of PI 3-kinase activity and the resulting metabolic insulin response. Dominant-negative Grb10 domains, in particular the SH2 domain, eliminated the metabolic response to insulin in differentiated 3T3-L1 adipocytes. This was consistently observed for glycogen synthesis, glucose and amino acid transport, and lipogenesis. In parallel, the same metabolic responses were substantially elevated by increased levels of Grb10. A similar role of Grb10 was confirmed in mouse L6 cells. In addition to the SH2 domain, the Pro-rich amino-terminal region of Grb10 was implicated in the regulation of PI 3-kinase catalytic activity. These regulatory roles of Grb10 were extended to specific insulin mediators downstream of PI 3-kinase including PKB/Akt, glycogen synthase kinase, and glycogen synthase. In contrast, a regulatory role of Grb10 in parallel insulin response pathways including p70 S6 kinase, ubiquitin ligase Cbl, or mitogen-activated protein kinase p38 was not observed. The dissection of the interaction of mouse Grb10 with p85 and the resulting regulation of PI 3-kinase activity should help elucidate the complexity of the IR signaling mechanism. The regulation of the metabolic insulin response by mouse growth factor receptor-binding protein 10 (Grb10) has been addressed in this report. We find mouse Grb10 to be a critical component of the insulin receptor (IR) signaling complex that provides a functional link between IR and p85 phosphatidylinositol (PI) 3-kinase and regulates PI 3-kinase activity. This regulatory mechanism parallels the established link between IR and p85 via insulin receptor substrate (IRS) proteins. A direct association was demonstrated between Grb10 and p85 but was not observed between Grb10 and IRS proteins. In addition, no effect of mouse Grb10 was observed on the association between IRS-1 and p85, on IRS-1-associated PI 3-kinase activity, or on insulin-mediated activation of IR or IRS proteins. A critical role of mouse Grb10 was observed in the regulation of PI 3-kinase activity and the resulting metabolic insulin response. Dominant-negative Grb10 domains, in particular the SH2 domain, eliminated the metabolic response to insulin in differentiated 3T3-L1 adipocytes. This was consistently observed for glycogen synthesis, glucose and amino acid transport, and lipogenesis. In parallel, the same metabolic responses were substantially elevated by increased levels of Grb10. A similar role of Grb10 was confirmed in mouse L6 cells. In addition to the SH2 domain, the Pro-rich amino-terminal region of Grb10 was implicated in the regulation of PI 3-kinase catalytic activity. These regulatory roles of Grb10 were extended to specific insulin mediators downstream of PI 3-kinase including PKB/Akt, glycogen synthase kinase, and glycogen synthase. In contrast, a regulatory role of Grb10 in parallel insulin response pathways including p70 S6 kinase, ubiquitin ligase Cbl, or mitogen-activated protein kinase p38 was not observed. The dissection of the interaction of mouse Grb10 with p85 and the resulting regulation of PI 3-kinase activity should help elucidate the complexity of the IR signaling mechanism. Grb10 was originally discovered as a partner of the epidermal growth factor (EGF) 1The abbreviations used are: EGF, epidermal growth factor; IR, insulin receptor; IRS-1/2, insulin receptor substrate-1/2; Grb10, growth factor receptor-binding protein 10; SH2, Src homology domain 2; PI 3-kinase, phosphatidylinositol 3-kinase; MAP kinase, mitogen-activated protein kinase; GSK3, glycogen synthase kinase 3; PKB/Akt, protein kinase B; HA, hemagglutinin; IP, immunoprecipitation; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; PY, phosphotyrosine; IgG, immunoglobulin G; PMSF, phenylmethylsulfonyl fluoride; BSA, bovine serum albumin; BPS, between the PH and SH2 domains; MOPS, 4-morpholinepropanesulfonic acid; ERK, extracellular signal-related kinase.1The abbreviations used are: EGF, epidermal growth factor; IR, insulin receptor; IRS-1/2, insulin receptor substrate-1/2; Grb10, growth factor receptor-binding protein 10; SH2, Src homology domain 2; PI 3-kinase, phosphatidylinositol 3-kinase; MAP kinase, mitogen-activated protein kinase; GSK3, glycogen synthase kinase 3; PKB/Akt, protein kinase B; HA, hemagglutinin; IP, immunoprecipitation; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; PY, phosphotyrosine; IgG, immunoglobulin G; PMSF, phenylmethylsulfonyl fluoride; BSA, bovine serum albumin; BPS, between the PH and SH2 domains; MOPS, 4-morpholinepropanesulfonic acid; ERK, extracellular signal-related kinase. receptor (1Ooi J. Yajnik V. Immanuel D. Gordon M. Moskow J.J. Buchberg A.M. Margolis B. Oncogene. 1995; 10: 1621-1630PubMed Google Scholar), however, a role in EGF action remains unsupported. Grb10 has been shown to associate with IR (2Liu F. Roth R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10287-10291Crossref PubMed Scopus (150) Google Scholar, 3Hansen H. Svensson U. Zhu J. Laviola L. Giorgino F. Wolf G. Smith R.J. Riedel H. J. Biol. Chem. 1996; 271: 8882-8886Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 4Frantz J.D. Giorgetti-Peraldi S. Ottinger E.A. Shoelson S.E. J. Biol. Chem. 1997; 272: 2659-2667Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) and the insulin-like growth factor-I (IGF-I) receptor (IGF-IR) (5Morrione A. Valentinis B. Li S. Ooi J.Y.T. Margolis B. Baserga R. Cancer Res. 1996; 56: 3165-3167PubMed Google Scholar, 6Dey B.R. Frick K. Lopaczynski W. Nissley S.P. Furlanetto R.W. Mol. Endocrinol. 1996; 10: 631-641Crossref PubMed Scopus (102) Google Scholar, 7Dong L.Q. Farris S. Christal J. Liu F. Mol. Endocrinol. 1997; 11: 1757-1765Crossref PubMed Scopus (39) Google Scholar, 8Dong L.Q. Du H. Porter S.G. Kolakowski L.F. Lee A.V. Mandarino J. Fan J. Yee D. Liu F. J. Biol. Chem. 1997; 272: 29104-29112Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) that carry out important metabolic and mitogenic functions, respectively. A preference was observed for IR in a direct comparison (9Laviola L. Giorgino F. Chow J.C. Baquero J.A. Hansen H. Ooi J. Zhu J. Riedel H. Smith R.J. J. Clin. Invest. 1997; 99: 830-837Crossref PubMed Scopus (87) Google Scholar). At least seven splice variants have been identified in the human and mouse Grb10 gene, located on chromosomes 7 (human) or 11 (mouse) (10Jerome C.A. Scherer S.W. Tsui L.C. Gietz R.D. Triggs-Raine B. Genomics. 1997; 40: 215-216Crossref PubMed Scopus (31) Google Scholar, 11Angrist M. Bolk S. Bently K. Nallasamy S. Halushka M.K. Chakravarti A. Oncogene. 1998; 17: 3065-3070Crossref PubMed Scopus (20) Google Scholar). This specific chromosomal location implicates a role of Grb10 in Silver-Russel syndrome (12Miyoshi N. Kuroiwa Y. Kohda T. Shitara H. Yonekawa H. Kawabe T. Hasegawa H. Barton S.C. Surani M.A. Kaneko-Ishino T. Ishino F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1102-1107Crossref PubMed Scopus (136) Google Scholar, 13Cattanach B.M. Shibata H. Hayashizaki Y. Townsend K.M.S. Ball S. Beechey C.V. Cytogenet. Cell Genet. 1998; 80: 41-47Crossref PubMed Google Scholar), which remains to be demonstrated (14McCann J.A. Zheng H. Islam A. Goodyer C.G. Polychronakos C. Biochem. Biophys. Res. Commun. 2001; 286: 943-948Crossref PubMed Scopus (38) Google Scholar). Three mouse Grb10 variants have been reported, two of which have not been identified in humans (1Ooi J. Yajnik V. Immanuel D. Gordon M. Moskow J.J. Buchberg A.M. Margolis B. Oncogene. 1995; 10: 1621-1630PubMed Google Scholar, 9Laviola L. Giorgino F. Chow J.C. Baquero J.A. Hansen H. Ooi J. Zhu J. Riedel H. Smith R.J. J. Clin. Invest. 1997; 99: 830-837Crossref PubMed Scopus (87) Google Scholar). Four human sequences have originally been termed Grb-IR or hGrb10α, Grb10/IR-SV1, or hGrb-IRβ/Grb10, hGrb10γ and -δ (2Liu F. Roth R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10287-10291Crossref PubMed Scopus (150) Google Scholar, 4Frantz J.D. Giorgetti-Peraldi S. Ottinger E.A. Shoelson S.E. J. Biol. Chem. 1997; 272: 2659-2667Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 7Dong L.Q. Farris S. Christal J. Liu F. Mol. Endocrinol. 1997; 11: 1757-1765Crossref PubMed Scopus (39) Google Scholar, 8Dong L.Q. Du H. Porter S.G. Kolakowski L.F. Lee A.V. Mandarino J. Fan J. Yee D. Liu F. J. Biol. Chem. 1997; 272: 29104-29112Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 15O'Neill T.J. Rose D.W. Pillay T.S. Hotta K. Olefsky J.M. Gustafson T.A. J. Biol. Chem. 1996; 271: 22506-22513Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) in the absence of a consistent nomenclature. A new consensus nomenclature has recently emerged (16Daly R.J. Cell Signal. 1998; 10: 613-618Crossref PubMed Scopus (102) Google Scholar, 17Nantel A. Mohammad-Ali K. Sherk J. Posner B.I. Thomas D.Y. J. Biol. Chem. 1998; 273: 10475-10484Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) which has been used in this article.Based on structural similarities the Grb10 isoforms are members of the Grb7 superfamily of signaling mediators which include Grb7, Grb14, and the Caenorhabditis elegans MIG-10 and their splice variants (16Daly R.J. Cell Signal. 1998; 10: 613-618Crossref PubMed Scopus (102) Google Scholar, 18Liu F. Roth R.A. Mol. Cell. Biochem. 1998; 182: 73-78Crossref PubMed Scopus (31) Google Scholar, 19Han D.C. Shen T.L. Guan J.L. Oncogene. 2001; 20: 6315-6321Crossref PubMed Scopus (141) Google Scholar). Superfamily members share a Pro-rich putative SH3 domain binding region at the amino terminus, a region termed GM (Grb/Mig) that contains a Ras-associating-like domain (20Wojcik J. Girault J.A. Labesse G. Chomilier J. Mornon J.P. Callebaut I. Biochem. Biophys. Res. Commun. 1999; 259: 113-120Crossref PubMed Scopus (46) Google Scholar) and is followed by a pleckstrin homology region, and a BPS (or IPS) domain located (between the PH and SH2 domains) (21He W. Rose D.W. Olefsky J.M. Gustafson T.A. J. Biol. Chem. 1998; 273: 6860-6867Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 22Dong L.Q. Porter S. Hu D. Liu F. J. Biol. Chem. 1998; 273: 17720-17725Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). All members carry an SH2 domain at the carboxyl terminus except MIG-10, which contains a Pro-rich region instead (23Manser J. Roonprapunt C. Margolis B. Dev. Biol. 1997; 184: 150-164Crossref PubMed Scopus (71) Google Scholar). Both the SH2 and BPS domains have been implicated in the association with receptor tyrosine kinases including IR (21He W. Rose D.W. Olefsky J.M. Gustafson T.A. J. Biol. Chem. 1998; 273: 6860-6867Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 22Dong L.Q. Porter S. Hu D. Liu F. J. Biol. Chem. 1998; 273: 17720-17725Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 24Wang J. Dai H. Yousaf N. Moussaif M. Deng Y. Boufelliga A. Rama Swamy O. Leone M.E. Riedel H. Mol. Cell. Biol. 1999; 19: 6217-6228Crossref PubMed Scopus (100) Google Scholar). The family of Grb10 variants continues to grow (25Strausberg R.L. Feingold E.A. Grouse L.H. Derge J.G. Klausner R.D. Collins F.S. Wagner C.M. Shenmen C.M. Schuler G.D. et al.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16899-16903Crossref PubMed Scopus (1547) Google Scholar).Grb10 and its SH2 domain are dimeric in solution and the crystal structure of the Grb10 SH2 domain reveals a noncovalent dimeric conformation unique to the Grb7 family that will favor binding of dimeric, turn-containing phospho-Tyr sequences typical for IR and IGF-IR (26Stein E.G. Ghirlando R. Hubbard S.R. J. Biol. Chem. 2003; 278: 13257-13264Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The Grb10 SH2 domain has been reported to associate, independently of phosphotyrosine, with Raf1 constitutively, and with MEK1 in response to insulin (17Nantel A. Mohammad-Ali K. Sherk J. Posner B.I. Thomas D.Y. J. Biol. Chem. 1998; 273: 10475-10484Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 27Nantel A. Huber M. Thomas D.Y. J. Biol. Chem. 1999; 274: 35719-35724Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Basal phosphorylation on serine has been reported for Grb10, which was stimulated in response to EGF; similarly platelet-derived growth factor and fibroblast growth factor caused a mobility shift in the migration of Grb10 on SDS gels that was reversible by phosphatase treatment (1Ooi J. Yajnik V. Immanuel D. Gordon M. Moskow J.J. Buchberg A.M. Margolis B. Oncogene. 1995; 10: 1621-1630PubMed Google Scholar). Basal serine phosphorylation of one isoform was also stimulated by insulin, which was reversible by phosphatase, the MEK1 inhibitor PD98059, or the PI 3-kinase inhibitor wortmannin (8Dong L.Q. Du H. Porter S.G. Kolakowski L.F. Lee A.V. Mandarino J. Fan J. Yee D. Liu F. J. Biol. Chem. 1997; 272: 29104-29112Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Tyrosine phosphorylation of Grb10 has been reported in response to insulin or vanadate that involves Src family kinases such as Src and Fyn (28Langlais P. Dong L.Q. Hu D. Liu F. Oncogene. 2000; 19: 2895-2903Crossref PubMed Scopus (36) Google Scholar). The underlying signaling mechanism may involve PI 3-kinase, p60 GAP-associated protein, the mitogen-activated protein kinase signaling cascade, and other pathways/mediators including vascular endothelial growth factor (29Giorgetti-Peraldi S. Murdaca J. Mas J.C. Van Obberghen E. Oncogene. 2001; 20: 3959-3968Crossref PubMed Scopus (30) Google Scholar), tyrosine kinases such as Jak2, Tec, and the oncogene Bcr-Abl (reviewed in Ref. 30Riedel H. Braun B.R. Grunberger G. Zick Y. Insulin Signaling: From Cultured Cells to Animal Models. Taylor and Francis, London2001: 89-105Google Scholar). A role in mitogenesis has also been described for the related Grb7 and Grb14 both of which are also direct partners of IR (16Daly R.J. Cell Signal. 1998; 10: 613-618Crossref PubMed Scopus (102) Google Scholar, 19Han D.C. Shen T.L. Guan J.L. Oncogene. 2001; 20: 6315-6321Crossref PubMed Scopus (141) Google Scholar, 31Kasus-Jacobi A. Bereziat V. Perdereau D. Girard J. Burnol A.F. Oncogene. 2000; 19: 2052-2059Crossref PubMed Scopus (44) Google Scholar). This role also extends to metabolic insulin responses for Grb14 (31Kasus-Jacobi A. Bereziat V. Perdereau D. Girard J. Burnol A.F. Oncogene. 2000; 19: 2052-2059Crossref PubMed Scopus (44) Google Scholar).Consequently, a role in the mitogenic response to insulin and other peptide hormones has been demonstrated for several Grb10 isoforms, however, a physiologic role in other insulin actions remained less defined (32 and reviewed in Ref. 30Riedel H. Braun B.R. Grunberger G. Zick Y. Insulin Signaling: From Cultured Cells to Animal Models. Taylor and Francis, London2001: 89-105Google Scholar). The present study introduces mouse Grb10 as a critical component of the metabolic IR signaling complex that provides an alternative functional link between IR and p85 PI 3-kinase in response to insulin-stimulated metabolism.EXPERIMENTAL PROCEDURESAll presented data are based on repeated experiments with the error shown between multiple measurements in bar graphs or with one representative experiment shown for immunoblots.Cell Culture—Mouse L6 cells, rat PC12 cells, and human IR overexpressing Rat1 cells (HIRcB) (33McClain D.A. Maegawa H. Levy J. Huecksteadt T. Dull T.J. Lee J. Ullrich A. Olefsky J.M. J. Biol. Chem. 1988; 263: 8904-8911Abstract Full Text PDF PubMed Google Scholar) were maintained in high glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 1% (v/v) penicillin/streptomycin, in a 5% CO2 environment. Mouse 3T3-L1 cells were maintained in high glucose DMEM with 10% fetal bovine serum and 1% (v/v) penicillin/streptomycin, and were differentiated 2 days post-confluence by addition of 500 μm isobutylxanthine, 25 μm dexamethasone, and 4 μg/ml insulin (34Kayali A.G. Eichhorn J. Haruta T. Morris A.J. Nelson J.G. Vollenweider P. Olefsky J.M. Webster N.J.G. J. Biol. Chem. 1998; 273: 13808-13818Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), and after 3 days cultured with the addition of only insulin. This medium was replaced every 3 days until cells were used after day 10 as well differentiated adipocytes in experiments. To elicit a metabolic response cells were subsequently typically stimulated with 100 nm insulin as specifically described for each procedure.Transient cDNA Transfection—Plasmid pHook2 (Invitrogen) carries a cytomegalovirus constitutive transcriptional promoter and served as a vector for transient expression of mouse Grb10δ (9Laviola L. Giorgino F. Chow J.C. Baquero J.A. Hansen H. Ooi J. Zhu J. Riedel H. Smith R.J. J. Clin. Invest. 1997; 99: 830-837Crossref PubMed Scopus (87) Google Scholar). The complete protein coding cDNA of Grb10 had been removed from plasmid pRK5 (1Ooi J. Yajnik V. Immanuel D. Gordon M. Moskow J.J. Buchberg A.M. Margolis B. Oncogene. 1995; 10: 1621-1630PubMed Google Scholar) as a NruI-HindIII restriction fragment. Both sites were end-filled and inserted into the pHook2 expression plasmid (Invitrogen) at a unique SmaI restriction site to produce a mouse Grb10δ expression construct in pHook2. Subconfluent mouse L6 cells in 8-cm culture plates were rinsed with antibiotic-free medium before 3 ml of transfection mixture including 5–6 μg of Grb10 expression plasmid or the corresponding control plasmid, 20 μl of Plus reagent, and 30 μl of LipofectAMINE were added according to the instructions of the manufacturer (Invitrogen). Five hours later the transfection medium was replaced with complete culture medium supplemented with 10% fetal bovine serum. The effect of Grb10 expression (after cell starvation) was tested about 2 days posttransfection.Immunoprecipitation and Immunoblotting—Immunoprecipitation and immunoblotting were modified as described in Ref. 24Wang J. Dai H. Yousaf N. Moussaif M. Deng Y. Boufelliga A. Rama Swamy O. Leone M.E. Riedel H. Mol. Cell. Biol. 1999; 19: 6217-6228Crossref PubMed Scopus (100) Google Scholar. Cells were cultured to quiescence for 20 h in serum-free DMEM supplemented with 0.1% BSA and stimulated with or without 100 nm insulin. Cells were rinsed twice with PBS and detergent cell extracts were prepared in ice-cold lysis buffer containing 50 mm HEPES, pH 7.4, 1% Triton X-100, 10% glycerol, 137 mm NaCl, 2 mm EDTA, 10 mm NaF, 100 mm Na3VO4, 10 mm sodium pyrophosphate, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mm PMSF. Samples containing 300–500 μg of total protein were immunoprecipitated in a complex with protein A-Sepharose and antibody directed against Grb10 (Santa Cruz Biotechnology), p85 (Upstate Biotechnology or part of assay kit), IRS-1 (Upstate Biotechnology assay kit), IRS-2 (Upstate Biotechnology assay kit), IR (Upstate Biotechnology assay kit), or c-Cbl (Upstate Biotechnology assay kit). Precipitates were rinsed three times with lysis buffer, separated by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with specific antibodies as listed above or phospho-Tyr-specific antibody PY20 (Transduction Laboratories). For "far western" analysis (35Vidal M. Goudreau N. Cornille F. Cussac D. Gincel E. Garbay C. J. Mol. Biol. 1999; 290: 717-730Crossref PubMed Scopus (41) Google Scholar) the blot was first incubated with TAT-HA-Grb10 fusion protein followed by HA-specific antibody (Santa Cruz Biotechnology) and finally by goat anti-mouse IgG horseradish peroxidase as tertiary antibody (Santa Cruz Biotechnology). Proteins were visualized by the ECL system (Amersham Biosciences).Preparation of Cell Membrane-permeable Fusion Protein—The complete mouse Grb10δ protein-coding cDNA was introduced into Escherichia coli expression plasmid pTAT-HA (kindly provided by Steven F. Dowdy, Washington University School of Medicine) under control of the strong T7 phage transcriptional promoter (36Schwarze S.R. Ho A. Vocero-Akbani A. Dowdy S.F. Science. 1999; 285: 1569-1572Crossref PubMed Scopus (2176) Google Scholar). This plasmid carries amino-terminal coding sequences for a 6-amino acid His tag peptide to facilitate affinity purification of the recombinant protein and an 11-amino acid HIV TAT protein-derived peptide (YGRKKRRQRRR) that renders the resulting fusion proteins cell-membrane permeable. The complete protein coding region of mouse Grb10δ (9Laviola L. Giorgino F. Chow J.C. Baquero J.A. Hansen H. Ooi J. Zhu J. Riedel H. Smith R.J. J. Clin. Invest. 1997; 99: 830-837Crossref PubMed Scopus (87) Google Scholar) was assembled starting just upstream of the ATG initiation codon of translation through an XhoI restriction site in the 3′-untranslated region. A 168-bp cDNA fragment encoding the 5′ end of the mouse Grb10δ protein coding sequence was amplified by PCR using primers (5′-GTCTTGGGGGTACCGGTGGTATGAACAACGATATTAACTCGTCC-3′) and (5′-ACGCCTGTGGCTGTCCCCGGGAGCTAGATG-3′) that introduced a 5′ KpnI site and 3′ XmaI site into the final PCR product. The remaining protein coding sequences of mouse Grb10δ (9Laviola L. Giorgino F. Chow J.C. Baquero J.A. Hansen H. Ooi J. Zhu J. Riedel H. Smith R.J. J. Clin. Invest. 1997; 99: 830-837Crossref PubMed Scopus (87) Google Scholar) were retrieved as an XmaI-XhoI restriction fragment (24Wang J. Dai H. Yousaf N. Moussaif M. Deng Y. Boufelliga A. Rama Swamy O. Leone M.E. Riedel H. Mol. Cell. Biol. 1999; 19: 6217-6228Crossref PubMed Scopus (100) Google Scholar). Both fragments, which reconstituted the complete Grb10δ protein-coding region, were joined with a KpnI-XhoI restriction fragment of plasmid pTAT-HA to produce a cell membranepermeable TAT-HA-Grb10 fusion protein in E. coli.Recombinant plasmids were introduced into E. coli high level expression host BL21(DE3)LysS (Novagen). 500 ml of LB medium was inoculated with 100 ml of freshly saturated overnight culture and was induced with 500 μm isopropyl-β-thiogalactopyranoside at 37 °C to produce the fusion protein. After 7 h cells were sedimented and re-suspended in 8 m urea, 100 mm NaCl, 20 mm HEPES, pH 8.0. Cells were disrupted by sonication (Branson Sonifier-450, CT) on ice in a series of pulses for about 2 min until the solution became turbid and lysates were centrifuged at 15,000 × g for 15 min at 4 °C. Grb10 fusion protein was purified from the soluble fraction via the His tag by affinity chromatography on a nickel-nitrilotriacetic acid column (Qiagen) pre-equilibrated with 5 mm imidazole, 500 mm NaCl, 20 mm Tris, pH 8.0, 8 m urea. Fusion protein was eluted by stepwise addition of 5–10 ml of increasing concentrations of 100, 200, and 500 mm and 1 m imidazole in 8 m urea, 100 mm NaCl, 20 mm HEPES, pH 8.0. Fractions with an A 280 above 0.2 were pooled and initially dialyzed (Slide-A-Lyzer, Pierce) with a 3.5-kDa molecular mass cutoff for 2 h at 4 °C in PBS, pH 7.4, 4 m urea. Proteins were concentrated at 4 °C by centrifugation with Centriplus-3 (Millipore) at 3,000 × g at 3-kDa molecular mass cutoff. Samples were repeatedly reconstituted in PBS, pH 7.2, 10% glycerol to reach a peptide concentration of 0.2–0.3 mg/ml. Aliquots were shock frozen in liquid nitrogen and stored at –80 °C. Cell membrane-permeable fusion peptides representing the Grb10 amino-terminal Pro-rich region or SH2 domain had been prepared as described earlier fused with a sequence of the Drosophila melanogaster antennapedia homeoprotein (24Wang J. Dai H. Yousaf N. Moussaif M. Deng Y. Boufelliga A. Rama Swamy O. Leone M.E. Riedel H. Mol. Cell. Biol. 1999; 19: 6217-6228Crossref PubMed Scopus (100) Google Scholar). The SH2 domain had been expressed as a fusion peptide in E. coli and the Pro-rich region was represented by a synthetic peptide mimetic (synthesized by American Peptide Company). A synthetic peptide lacking Grb10 sequences or a dialyzed column eluate of a control E. coli cell extract served as peptide controls.Glycogen Synthesis—A protocol was followed as described below similar to the procedure outlined in Ref. 37Wada T. Sasaoka T. Funaki M. Hori H. Murakami S. Ishiki M. Haruta T. Asano T. Ogawa W. Ishihara H. Kobayashi M. Mol. Cell. Biol. 2001; 21: 1633-1646Crossref PubMed Scopus (152) Google Scholar. Mouse L6 cells (after transfection) or 3T3-L1 adipocytes were seeded into 12-well plates at densities of 4–5 × 105 cells per well. Cells were cultured for 24 h and subsequently deprived of serum for 18–20 h in DMEM supplemented with 0.1% BSA. Cells were rinsed twice with ice-cold PBS followed by a 3-h incubation in 2.5 mm glucose, 0.1% BSA, 25 mm HEPES, pH 7.4. Cells were incubated for 30 min at 37 °C in the presence or absence of 100 nm insulin, cell membrane-permeable peptide mimetics (10 μg/ml), and/or 20 μm PI 3-kinase inhibitor LY 294002 and/or 20 μm p38 MAP kinase inhibitor SB203580. Subsequently, cells were incubated with 2.5–5.0 μl of d-[U-14C]glucose (200 μCi/ml, 2–4 mCi/mmol, Amersham Biosciences) for 1 h at 37 °C (final concentration 0.5–1 μCi/ml). Glycogen synthesis was terminated by rinsing cells three times with ice-cold PBS followed by cell lysis with 0.5 ml of 30% KOH for 1 h at 37 °C. Lysates were transferred to 1.5-ml microcentrifuge tubes, 50 μl of 20 mg/ml carrier glycogen (final concentration 2 mg/ml) was added, and cell lysates were incubated at 95 °C for 30 min. Samples were cooled to room temperature, 0.6 ml of ice-cold ethanol was added, and glycogen was precipitated for 16 h at –20 °C. Samples were sedimented at 3,000 × g for 10 min and the supernatant was aspirated. The precipitate was solubilized in 1 ml of H2O and incorporated radioactivity was determined by liquid scintillation spectroscopy.Glucose Transport—A protocol was followed as described below similar to the procedure outlined in Ref. 38Wiese R.J. Mastick C.C. Lazar D.F. Saltiel A.R. J. Biol. Chem. 1995; 270: 3442-3446Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar. 5 × 105 L6 cells (after transfection) or 1.5 × 105 3T3-L1 adipocytes per well (for 24-well plates) were starved for 3–5 h in serum-free DMEM supplemented with 0.1% BSA and cells were washed twice with KRPH buffer (1% bovine serum albumin, 1 mm MgSO4, 1 mm CaCl2, 136 mm NaCl, 5 mm Na2HPO4, 20 mm HEPES, pH 7.4). Cells were incubated for 15 min at 37 °C with 100 nm insulin and/or 10 μg/ml cell membrane-permeable peptide mimetics. 0.5 μl of 2-deoxy-[3H]glucose (1 mCi/ml, 10–20 Ci/mmol, Amersham Biosciences) was added for 4 more min (final concentration 0.5 μCi/ml) and nonspecific glucose transport was determined in the presence of 10 μm cytochalasin B, which was subtracted for final data presentation. Cells were subsequently rinsed twice with ice-cold PBS and lysed in 0.5 ml of 0.5 n NaOH, followed by neutralization with 0.5 ml of 2 m Tris, pH 6.8. Associated radioactivity was determined by liquid scintillation spectroscopy.Amino Acid Transport—A protocol was followed as described below similar to the procedure outlined in Ref. 39McDowell H.E. Eyers P.A. Hundal H.S. FEBS Lett. 1998; 441: 15-19Crossref PubMed Scopus (45) Google Scholar. 5 × 105 L6 cells (after transfection) or 1.5 × 105 3T3-L1 adipocytes per well (in 24-well plates) were starved in serum-free DMEM supplemented with 0.1% BSA for 16 h. Cells were incubated for 1 h at 37 °C with 100 nm insulin and/or 10 μg/ml cell membrane-permeable peptide mimetics followed by the addition of 1 μl of 2-amino-[1-14C]isobutyric acid (50 μCi/ml, 50–62 mCi/mmol, Amersham Biosciences) for 10 min (final concentration 0.05 μCi/ml). Cells were rinsed three times with ice-cold PBS, lysed in 0.5 ml of 0.2 n NaOH for 30 min at 40 °C, and samples were neutralized with 0.5 ml of 0.2 n HCl. Associated radioactivity was determined by liquid scintillation spectroscopy.Lipogenesis—A protocol was followed as described below similar to the procedure outlined in Ref. 38Wiese R.J. Mastick C.C. Lazar D.F. Saltiel A.R. J. Biol. Chem. 1995; 270: 3442-3446Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar. 4–5 × 105 L6 cells (after transfection) or 3T3-L1 adipocytes per well (in 12-well plates) were starved for 18–20 h in serum-free medium supplemented with 0.1% BSA. Cells were incubated for 1 h at 37 °C with 100 nm insulin and/or 10 μg/ml cell membrane-permeable peptide mimetics in the presence of 0.5 μl of 2-deoxy-[3H]glucose (1 mCi/ml, 10–20 Ci/mmol, Amersham Biosciences) and 3.5 mm glucose (final concentration 0.5 μCi/ml). Cells were rinsed twice with ice-cold PBS, collected by scraping, and transferred to liquid scintillation vials. 5 ml of Toluene-Bray scintillation liquid was added and the mixture was incubated for 16 h at 25 °C. 4 ml of the organic phase was removed to quantify incorporated radioactivity by liquid scintillation spectroscopy.PI 3-Kinase Activity—A protocol was followed as described below similar to the procedure outlined in Refs. 40Whitman M. Kaplan D.R. Schaffhausen B. Cantley L. Roberts T.M. Nature. 1985; 315: 239-242Crossref PubMed Scopus (547) Google Scholar and 41Vlahos C.J. Matter W.F. Hui K.Y. Brown R.F. J. Biol. Chem. 1994; 269: 5241-5248Abstract Full Tex

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