©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Insulin-stimulated GLUT4 Translocation Is Mediated by a Divergent Intracellular Signaling Pathway (*)

(Received for publication, August 1, 1995; and in revised form, September 25, 1995)

Tetsuro Haruta Aaron J. Morris David W. Rose James G. Nelson Michael Mueckler (1) Jerrold M. Olefsky (2)(§)

From the  (1)Department of Medicine, University of California, San Diego, La Jolla, California 92093, Department of Medicine, Washington University, St. Louis, Missouri 63110, and the (2)Veterans Administration Research Service, San Diego, California 92161

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Insulin stimulates glucose transport largely by mediating translocation of the insulin-sensitive glucose transporter (GLUT4) from an intracellular compartment to the plasma membrane. Using single cell microinjection of 3T3-L1 adipocytes, coupled with immunofluorescence detection of GLUT4 proteins, we have determined that inhibition of endogenous p21 or injection of oncogenic p21 has no effect on insulin-stimulated GLUT4 translocation. On the other hand, microinjection of anti-phosphotyrosine antibodies or inhibition of endogenous phosphatidylinositol 3-kinase by microinjection of a GST-p85 SH2 fusion protein markedly inhibits this biologic effect of insulin. These data suggest that the p21/mitogen-activated protein kinase pathway is not involved in this metabolic effect of insulin, whereas tyrosine phosphorylation and stimulation of phosphatidylinositol 3-kinase activity are critical components of this signaling pathway.


INTRODUCTION

Insulin exerts pleiotropic biologic effects. One of insulin's major physiologic effects is the regulation of plasma glucose levels, which is accomplished by suppression of hepatic glucose production and stimulation of glucose uptake into target tissues(1) . The latter effect is primarily due to translocation of insulin-sensitive glucose transporters (GLUT4) from an intracellular vesicular pool to the plasma membrane, where they can lead to glucose uptake(2, 3) . This response is mediated by a metabolic signaling pathway that is divergent from the mitogenic pathway (4) and may, at least in part, utilize distinct signaling molecules. Elucidation of this signaling pathway will yield information rich with implications for a number of human disease states, such as non-insulin-dependent diabetes mellitus, obesity, and polycystic ovarian syndrome. All of these conditions are associated with insulin resistance and feature decreased insulin-stimulated glucose transport as a major manifestation(5) . Thus, the causes of insulin resistance in these pathophysiologic states likely involve defects in the metabolic signaling pathway by which insulin causes an increase in cellular glucose uptake. We have utilized single cell microinjection of 3T3-L1 adipocytes and immunofluorescence microscopy of GLUT4 proteins to assess the transmembrane signaling pathway leading to insulin-stimulated GLUT4 translocation.


EXPERIMENTAL PROCEDURES

Materials

Porcine insulin was kindly provided by Lilly. Polyclonal anti-GLUT4 antibody (F349) was described previously(6) . Monoclonal anti-GLUT4 antibody (1F8) was from East Acres Biologicals(10) . Polyclonal anti-c-Fos antibody was from Oncogene Science. Monoclonal anti-p21 antibody (Y13-259) was from Santa Cruz Biotechnology. Polyclonal anti-Shc antibody and monoclonal anti-phosphotyrosine antibody (PY20) were from Transduction Laboratories. Recombinant T24 Ras (^1)and N17 Ras proteins were generous gifts from J. R. Feramisco and were previously described(8) . GST-p85 SH2 fusion protein was kindly provided by A. R. Saltiel, and its preparation and use were described previously(9) . Sheep IgG and fluorescein isothiocyanate-, tetramethylrhodamine isothiocyanate-, and AMCA-conjugated anti-rabbit, -mouse, -rat, and -sheep IgG antibodies were from Jackson Immunoresearch Laboratories Inc. All other reagents were purchased from Sigma.

Cell Culture and Microinjection

Rat1 fibroblasts that had been stably transfected and that overexpress wild type human insulin receptor (HIRcB cells) were cultured as described previously(10, 11) . 3T3-L1 cells were grown and maintained in Dulbecco's modified Eagle's medium high glucose containing 50 units/ml penicillin, 50 µg/ml streptomycin, and 10% fetal calf serum (FCS) in a 10% CO(2) environment. The cells were allowed to grow to 2 days postconfluency and then differentiated by addition of the same medium containing isobutylmethylxanthine (500 µM), dexamethasone (25 µM), and insulin (4 µg/ml) for 3 days and the medium containing insulin for 3 more days. The medium was then changed every 3 days until the cells were fully differentiated, typically by 10 days. Prior to experimentation, the adipocytes were trypsinized and reseeded onto acid-washed glass coverslips. The cells were cultured in the same medium without serum for 2 h, and microinjection of various reagents was carried out using a semiautomated Eppendorf microinjection system. All reagents were dissolved in a buffer containing 5 mM sodium phosphate (pH 7.2), 100 mM KCl. N17 Ras, T24 Ras, and GST-p85 SH2 proteins were each co-injected with 10 mg/ml sheep IgG for detection purposes. Prior to staining the cells were allowed to recover for a period of 1 h.

Immunostaining and Fluorescence Microscopy

GLUT4 Protein Staining

The cells were stimulated with insulin for 20 min and then fixed in 3.7% formaldehyde in PBS for 5 min on ice and for 5 min at room temperature. Following washing, the cells were permeabilized and blocked with 0.1% Triton X-100 and 1% FCS in PBS for 10 min at room temperature. The cells were then incubated with rabbit polyclonal anti-GLUT4 antibody (F349) (1 µg/ml) that had been raised against a synthetic peptide corresponding to the C-terminal 16 residues of GLUT4 (6) or a monoclonal anti-GLUT4 antibody (1F8) (7) (2 µg/ml) in PBS with 1% FCS overnight at 4 °C. After washing and GLUT4 staining the identity of microinjected cells was determined by incubation with fluorescein-conjugated donkey anti-rabbit IgG for F349 or anti-mouse IgG for 1F8- and AMCA-conjugated donkey anti-sheep IgG. The results were then analyzed and photographed by immunofluorescence microscopy. Each AMCA-positive microinjected cell was evaluated for the presence of plasma membrane-associated GLUT4 staining. As in all microinjection studies, control cells were microinjected with preimmune sheep IgG and then processed in the same way as experimental injected cells.

c-Fos Protein Staining

After staining for GLUT4 and injected IgG, the cells were stained with rabbit anti-c-Fos antibody (1/100) for 90 min at 37 °C followed by incubation with rhodamine-conjugated anti-rabbit IgG antibody.


RESULTS AND DISCUSSION

Individual, living, differentiated 3T3-L1 adipocytes were microinjected with various reagents, followed by insulin stimulation. Translocation of GLUT4 to the cell surface was then visualized by immunofluorescence microscopy using anti-GLUT4 antibody followed by fluorescein-conjugated second antibody. Fig. 1, A and B, shows a field containing 3T3-L1 adipocytes before (A) and after (B) insulin stimulation. In the basal, unstimulated state, GLUT4 staining is almost exclusively relegated to the intracellular compartment, with typical diffuse punctate and discrete perinuclear staining. After insulin stimulation, a marked difference in the staining pattern occurs, with prominent intense staining uniformly distributed at the cell surface, accompanied by decreased intracellular GLUT4 staining, indicative of translocation of GLUT4 proteins from the intracellular vesicular pool to the plasma membrane. In the basal state, less than 10% of cells display surface GLUT4 staining, whereas after insulin treatment, 60-80% of cells are positive for cell surface GLUT4 localization. Using this technique, translocation occurs with a time course (C) and dose-response curve (D) typical for insulin stimulation of glucose transport, indicating that this visual assay of GLUT4 translocation is representative of the physiologic insulin-stimulated process.



Figure 1: Visualization of insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated without (A) or with (B) 10 ng/ml insulin for 20 min and stained with anti-GLUT4 antibody (1F8) followed by incubation with fluorescein-conjugated anti-mouse IgG antibody. Cells positive for GLUT4 translocation show an increase in plasma membrane-associated fluorescein staining that is visualized as a ring around the cell. C, time course of GLUT4 translocation. 3T3-L1 adipocytes on coverslips were stimulated with 10 ng/ml insulin for various times and stained for GLUT4. D, dose response of GLUT4 translocation. 3T3-L1 adipocytes on coverslips were stimulated with the indicated concentrations of insulin for 20 min and stained for GLUT4. The percentage of cells positive for GLUT4 translocation was calculated by counting at least 100 cells at each point. Mean of two experiments is shown.



We then used this system coupled with microinjection studies in an attempt to elucidate the signaling cascade that mediates insulin's effect on GLUT4 translocation. After insulin binds to its receptor, a series of events is initiated, including receptor autophosphorylation and tyrosine phosphorylation of endogenous substrates such as IRS-1 and Shc(12, 13, 14, 15, 16) . Through its multiple tyrosine phosphorylation sites, IRS-1 can serve as a docking protein forming complexes with SH2 domain-containing proteins such as PI3-kinase(17) . Phospho-Shc forms complexes with Grb2bulletSOS leading to activation of p21, which subsequently stimulates the MAP kinase pathway(18) . While a great deal is known about insulin's mitogenic actions, relatively little is known about the signaling processes that mediate GLUT4 translocation. For example, numerous studies have shown that formation of p21-GTP and stimulation of the MAP kinase pathway are essential steps in insulin's mitogenic signaling pathway(17) . However, whether these molecules are involved in insulin's metabolic actions remains unclear. For example, it has been demonstrated that the metabolic and mitogenic signaling pathways diverge(4) , and it is possible that the point of divergence is proximal to formation of p21-GTP. Several studies, using different experimental approaches, have found results against a role for p21 in the stimulation of glucose transport(19, 20, 21, 22, 23) , whereas other studies have found evidence in favor(24, 25) .

Our current technique allows an opportunity to directly test the importance of intracellular molecules in a given signaling pathway. Thus, we microinjected 3T3-L1 adipocytes with a series of Ras-related reagents. As seen in Fig. 2, microinjection of dominant inhibitory N17 Ras (26) had no effect on basal or insulin-stimulated GLUT4 localization at maximal (10 ng/ml) insulin concentrations. Identical microinjections of N17 Ras protein were carried out in cells that were subsequently stimulated with several submaximal concentrations of insulin. When GLUT4 translocation was quantitated in these injected cells, a dose-response curve identical to that obtained with uninjected cells (Fig. 1D) was observed (data not shown), indicating that N17 Ras protein does not shift this dose response. Thus, within the detection limits of the immunofluorescence assay, it does not appear that inhibition of endogenous Ras activity affects the extent to which GLUT4 is translocated or the insulin responsiveness of the translocation event. A similar lack of effect was observed upon microinjection of anti-p21 antibody. In contrast, both of these Ras inhibitory reagents prevented insulin-stimulated DNA synthesis when they were concurrently injected into Rat 1 fibroblasts (HIRc cells) overexpressing human insulin receptors. The percentages of cells positive for BrdUrd staining were 76, 22, 21, and 28% following injections of control IgG, N17 Ras, anti-p21 antibody, and anti-Shc antibody, respectively. Oncogenic T24 Ras (27, 28) is a constitutively active protein that will stimulate downstream effects of p21-GTP. T24 Ras protein was microinjected into 3T3-L1 adipocytes, and its effects upon c-Fos protein expression and translocation of GLUT4 were simultaneously assessed. As demonstrated in Fig. 3, the T24 Ras protein was biologically active within the cells and caused a marked induction of c-Fos protein as visualized by nuclear rhodamine staining with anti-c-Fos antibody. In contrast, T24 Ras failed to influence GLUT4 localization in the presence or absence of insulin ( Fig. 2and Fig. 3, D and H). Furthermore, concurrent injections of this preparation of T24 Ras stimulated DNA synthesis in quiescent HIRc cells to the same extent as insulin. The percentages of BrdUrd-positive cells following control IgG and T24 Ras injections, without stimulation by insulin, were 19 and 72%, respectively. Thus, the signaling pathway leading to c-Fos expression was activated by the microinjected T24 Ras, while stimulation of this pathway did not influence GLUT4 translocation. In addition, we have found that microinjection of anti-Shc antibody (Fig. 2) as well as a Shc SH2-GST fusion protein (data not shown) did not inhibit insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes, whereas anti-Shc antibody inhibited insulin-stimulated DNA synthesis by greater than 80% in HIRc cells, and the Shc SH2-GST protein inhibited epidermal growth factor-stimulated DNA synthesis. Taken together, these results argue that activation of p21 is neither necessary nor sufficient for GLUT4 translocation and indicate that the metabolic pathway mediating this biologic effect diverges from the mitogenic signaling pathway proximal to activated p21.


Figure 2: Effect of microinjection of N17 Ras, anti-p21 antibody, T24 Ras, and anti-Shc antibody on GLUT4 translocation in 3T3-L1 adipocytes. 3T3-L1 adipocytes on coverslips were microinjected with either control sheep IgG (10 mg/ml), dominant negative mutant p21 protein (N17) (2.0 mg/ml), monoclonal anti-p21 antibody (Y13-259) (5 mg/ml), oncogenic p21 (T24 Ras) (2.0 mg/ml), or anti-Shc antibody (5 mg/ml). One hour after microinjection, cells were stimulated in the absence (open bars) or presence (hatched bars) of 10 ng/ml insulin for 20 min. The cells were fixed, permeabilized, and stained for GLUT4 and injected IgG. The percentage of cells positive for GLUT4 translocation was determined in each experiment by analyzing at least 100 cells positive for injected IgG. Error bars represent S.E. for three experiments.




Figure 3: Microinjection of oncogenic T24 Ras induces c-Fos protein expression but not GLUT4 translocation. 3T3-L1 adipocytes on coverslips were microinjected with either control sheep IgG (10 mg/ml) (A-D) or oncogenic p21 (T24 Ras) (2 mg/ml) combined with sheep IgG (10 mg/ml) (E-H). The cells were stained for GLUT4 and injected IgG followed by staining for c-Fos. A and E, phase contrast. B and F, IgG staining, demonstrating injected cells. C and G, c-Fos staining. D and H, GLUT4 staining. Arrows indicate injected cells. 70% of T24 Ras-injected cells demonstrated c-Fos nuclear staining, while none of the control IgG injected or uninjected cells showed this staining pattern.



There is evidence to suggest that PI3-kinase may be an intermediary molecule facilitating insulin-stimulated GLUT4 translocation(29, 30, 31, 32) . When cells are treated with the PI3-kinase inhibitor Wortmannin, insulin stimulation of glucose transport is inhibited(31, 32) . Although this is suggestive of a role for PI3-kinase in this effect of insulin, the specificity of Wortmannin as an inhibitor has recently been questioned(33) . To directly assess the relevance of PI3-kinase in the metabolic effects of insulin, we utilized a GST fusion protein comprising the N-terminal SH2 domain of the p85 subunit of PI3-kinase. In previous studies, we have shown that microinjection of this SH2-GST fusion protein into HIRc cells completely inhibited insulin, insulin-like growth factor-I, and epidermal growth factor-induced DNA synthesis(9) . As seen in Fig. 4, microinjection of the p85 SH2-GST protein inhibited insulin-stimulated GLUT4 translocation by 75%. The specificity of this effect is verified by the fact that a GST fusion protein containing the Shc SH2 domain was without effect on GLUT4 translocation (data not shown). In support of this, we also found that preincubation of cells with Wortmannin (1 µM) completely inhibited GLUT4 translocation (data not shown).


Figure 4: Inhibition of GLUT4 translocation by microinjection of GST-p85 SH2 fusion protein or anti-phosphotyrosine antibody. Either control sheep IgG (10 mg/ml), a GST fusion protein containing the N-terminal SH2 domain of p85 (12 mg/ml) combined with sheep IgG (10 mg/ml), or a monoclonal anti-phosphotyrosine antibody (pY 20) (5 mg/ml) were microinjected into 3T3-L1 adipocytes. One hour after injection, the cells were stimulated without (open bars) or with (hatched bars) 10 ng/ml insulin for 20 min and then stained for GLUT4.



Insulin leads to autophosphorylation of the insulin receptor with tyrosine phosphorylation of IRS-1, which then binds to the SH2 domains of PI3-kinase leading to activation of this enzyme(17) . To evaluate the role of tyrosine phosphorylation in this pathway, we also microinjected anti-phosphotyrosine antibodies into 3T3-L1 cells. The monoclonal anti-phosphotyrosine antibody markedly inhibited GLUT4 translocation indicating the necessity for tyrosine phosphorylation as well as PI3-kinase activity in GLUT4 translocation (Fig. 4).

Previous studies examining whether molecules in the p21/MAP kinase pathway are involved in insulin stimulation of glucose transport have yielded conflicting results(19, 20, 21, 22, 23, 24, 25) . Some of these studies have not specifically measured GLUT4 translocation or have utilized cell lines that do not express GLUT4. Others have used transfection to generate cell lines overexpressing Ras-related molecules. With this latter approach, the possibility always exists that the selected cell lines will have adapted to the expressed transgene in such a way as to make the results non-representative of the physiologic cell context. Single cell microinjection avoids many of these problems, since our studies can be done in relevant insulin target cells (adipocytes), with direct assessment of the biologic event in question (GLUT4 translocation). Furthermore, the acute introduction of test molecules into the cell interior followed by rapid assay of insulin action does not allow cells enough time to undergo adaptive changes to the perturbation.

Single living cell microinjection has been a powerful technique that has proven useful in identifying components of the signaling pathway for growth factor-mediated mitogenesis(8, 9, 11, 34, 35, 36, 37) . The current system provides the means to identify the intracellular molecular components that transduce insulin's major metabolic effect, i.e. stimulation of GLUT4 translocation. In the current report our data have shown that the Ras pathway, which is a critical component of mitogenic signaling, is unnecessary for insulin stimulation of GLUT4 translocation. As such, this provides clear evidence for the divergence of metabolic and mitogenic signaling events. We also find that PI3-kinase is a necessary molecule coupling the tyrosine phosphorylation signal generated by the insulin receptor to glucose transport stimulation. Although our studies do not elucidate the mechanism whereby PI3-kinase facilitates GLUT4 translocation, our results are consistent with recent studies (38) showing that following insulin stimulation, PI3-kinase can be localized to the intracellular GLUT4-containing vesicles, consistent with a role for this enzyme in trafficking of GLUT4 to the cell surface.

Using the approaches contained in this report, we anticipate that further molecules in this key signaling pathway will be identified in a timely manner. As the details of this signaling pathway unfold, new insights into the mechanisms of insulin-resistant glucose transport stimulation in human disease states are likely to emerge.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant DK33651, by the Veterans Administration Medical Research Service, by a mentor-based fellowship grant from the American Diabetes Association (to T. H.), and by a career development award from the American Diabetes Association (to D. W. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Medicine (0673), University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0673. Tel.: 619-534-6651; Fax: 619-534-6653.

(^1)
The abbreviations used are: T24 Ras, oncogenic (valine 12) p21 protein; N17 Ras, dominant-negative mutant p21 protein; AMCA, 7-amino-4-methylcoumarin-3-acetic acid; IRS-1, insulin receptor substrate-1; BrdUrd, 3-bromo-5`-deoxyuridine; GST, glutathione S-transferase; HIRc, Rat1 fibroblast overexpressing the human insulin receptor; PBS, phosphate-buffered saline; PI3-kinase, phosphatidylinositol 3-kinase; FCS, fetal calf serum; MAP, mitogen-activated protein.


REFERENCES

  1. Olefsky, J. M., and Molina, J. M. (1994) Ellenberg and Rifkin's Diabetes Mellitus , 4th Ed., pp. 121-153, Elsevier Science Publishing Co., Inc., New York
  2. Cushman, S. W., and Wardazala, L. J. (1980) J. Biol. Chem. 255, 4758-4762 [Free Full Text]
  3. Suzuki, K., and Kono, T. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 2542-2545 [Abstract]
  4. Olefsky, J. M. (1990) Diabetes 39, 1009-1016 [Abstract]
  5. Kahn, B. B. (1992) J. Clin. Invest. 89, 1367-1374 [Medline] [Order article via Infotrieve]
  6. Haney, P. M., Slot, J. W., Piper, R. C., James, D. E., and Mueckler, M. (1991) J. Cell Biol. 114, 689-699 [Abstract]
  7. James, D. E., Brown, R., Navarro, J., and Pilch, P. F. (1988) Nature 333, 183-185 [CrossRef][Medline] [Order article via Infotrieve]
  8. Jhun, B. H., Meinkoth, J. L., Leitner, J. W., Draznin, B., and Olefsky, J. M. (1994) J. Biol. Chem. 269, 5699-5704 [Abstract/Free Full Text]
  9. Jhun, B. H., Rose, D. W., Seely, B. L., Rameh, L., Cantley, L., Saltiel, A. R., and Olefsky, J. M. (1994) Mol. Cell. Biol. 14, 7466-7475 [Abstract]
  10. McClain, D. A., Maegawa, H., Lee, J., Dull, T. J., Ullrich, A., and Olefsky, J. M. (1987) J. Biol. Chem. 262, 14663-14671 [Abstract/Free Full Text]
  11. Rose, D. W., Saltiel, A. R., Majumdar, M., Decker, S. J., and Olefsky, J. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 797-801 [Abstract]
  12. White, M. F., Maron, R., and Kahn, C. R. (1985) Nature 318, 183-186 [Medline] [Order article via Infotrieve]
  13. Pronk, G. J., McGlade, J., Pelicci, G., Pawson, T., and Bos, J. L. (1993) J. Biol. Chem. 268, 5748-5753 [Abstract/Free Full Text]
  14. Ullrich, A., and Schlessinger, J. (1990) Cell 61, 203-212 [Medline] [Order article via Infotrieve]
  15. Sasaoka, T., Draznin, B., Leitner, J. W., Langlois, W. J., and Olefsky, J. M. (1994) J. Biol. Chem. 269, 10734-10738 [Abstract/Free Full Text]
  16. Aaronson, S. A. (1991) Science 254, 1146-1153 [Medline] [Order article via Infotrieve]
  17. White, M. F., and Kahn, C. R. (1994) J. Biol. Chem. 269, 1-4 [Free Full Text]
  18. Lowenstein, E. J., Daly, R. J., Batzer, A. G., Li, W., Margolis, B., Lammers, R., Ullrich, A., Skolnik, E. Y., Bar-Sagi, D., and Schlessinger, J. (1992) Cell 70, 431-442 [Medline] [Order article via Infotrieve]
  19. Robinson, L. J., Razzack, Z. F., Lawrence, J. C., Jr., and James, D. E. (1993) J. Biol. Chem. 268, 26422-26427 [Abstract/Free Full Text]
  20. Reusch, J. E., Bhuripanyo, P., Carel, K., Leitner, J. W., Hsieh, P., DePaolo, D., and Draznin, B. (1995) J. Biol. Chem. 270, 2036-2040 [Abstract/Free Full Text]
  21. van den Berghe, N., Ouwens, D. M., Maassen, J. A., van Mackelenbergh, M. G., Sips, H. C., and Krans, H. M. (1994) Mol. Cell. Biol. 14, 2372-2377 [Abstract]
  22. Fingar, D. C., and Birnbaum, M. J. (1994) Endocrinology 134, 728-735 [Abstract]
  23. Hausdorff, S. F., Frangioni, J. V., and Birnbaum, M. J. (1994) J. Biol. Chem. 269, 21391-21394 [Abstract/Free Full Text]
  24. Kozma, L., Baltensperger, K., Klarlund, J., Porras, A., Santos, E., and Czech, M. P. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4460-4464 [Abstract]
  25. Manchester, J., Kong, X., Lowry, O. H., and Lawrence, J. C., Jr. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4644-4648 [Abstract]
  26. Stacey, D. W., Roudebush, M., Day, R., Mosser, S. D., Gibbs, J. B., and Feig, L. A. (1991) Oncogene 6, 2297-2304 [Medline] [Order article via Infotrieve]
  27. Feramisco, J. R., Gross, M., Kamata, T., Rosenberg, M., and Sweet, R. W. (1984) Cell 38, 109-117 [Medline] [Order article via Infotrieve]
  28. Tabin, C. J., Bradley, S. M., Bargmann, C. I., Weinberg, R. A., Papageorge, A. G., Scolnick, E. M., Dhar, R., Lowy, D. R., and Chang, E. H. (1982) Nature 300, 143-149 [Medline] [Order article via Infotrieve]
  29. Hara, K., Yonezawa, K., Sakaue, H., Ando, A., Kotani, K., Kitamura, T., Kitamura, Y., Ueda, H., Stephens, L., Jackson, T. R., Hawkins, P. T., Dhand, R., Clark, A. E., Holman, G. D., Waterfield, M. D., and Kasuga, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7415-7419 [Abstract]
  30. Cheatham, B., Vlahos, C. J., Cheatham, L., Wang, L., Blenis, J., and Kahn, C. R. (1994) Mol. Cell. Biol. 14, 4902-4911 [Abstract]
  31. Clarke, J. F., Young, P. W., Yonezawa, K., Kasuga, M., and Holman, G. D. (1994) Biochem. J. 300, 631-635 [Medline] [Order article via Infotrieve]
  32. Okada, T., Kawano, Y., Sakakibara, T., Hazeki, O., and Ui, M. (1994) J. Biol. Chem. 269, 3568-3573 [Abstract/Free Full Text]
  33. Sakaue, H., Hara, K., Noguchi, T., Matozaki, T., Kotani, K., Ogawa, W., Yonezawa, K., Waterfield, M. D., and Kasuga, M. (1995) J. Biol. Chem. 270, 11304-11309 [Abstract/Free Full Text]
  34. Sasaoka, T., Rose, D. W., Jhun, B. H., Saltiel, A. R., Draznin, B., and Olefsky, J. M. (1994) J. Biol. Chem. 269, 13689-13694 [Abstract/Free Full Text]
  35. Xiao, S., Rose, D. W., Sasaoka, T., Maegawa, H., Burke, T. R., Jr., Roller, P. P., Shoelson, S. E., and Olefsky, J. M. (1994) J. Biol. Chem. 269, 21244-21248 [Abstract/Free Full Text]
  36. Lane, H. A., Fernandez, A., Lamb, N. J., and Thomas, G. (1993) Nature 363, 170-172 [CrossRef][Medline] [Order article via Infotrieve]
  37. Roche, S., Koegl, M., and Courtneidge, S. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9185-9189 [Abstract]
  38. Heller-Harrison, R., Morin, M., and Czech, M. P. (1995) Diabetes 44, Suppl. 1, 31 (abstr.) [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.