©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Pleckstrin Homology Domain in Insulin Receptor Substrate-1 Sensitizes Insulin Signaling (*)

Martin G. MyersJr. (1)(§), Timothy C. Grammer (2), Jennifer Brooks (1)(¶), Erin M. Glasheen (1), Ling-Mei Wang (3), Xiao Jian Sun (1)(**), John Blenis (2), Jacalyn H. Pierce (3), Morris F. White (1)(§§)

From the (1) Research Division, Joslin Diabetes Center and Division of Medical Sciences and the (2) Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02215 and the (3) Laboratory of Cell and Molecular Biology, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The NH terminus of insulin receptor substrate-1 (IRS-1) contains a pleckstrin homology (PH) domain. We deleted the PH domain in IRS-1 (IRS-1) and expressed the mutant in Chinese hamster ovary and 32D cells. During insulin stimulation, IRS-1 is poorly tyrosine-phosphorylated in CHO cells, but undergoes serine/threonine phosphorylation. Similarly, IRS-1 fails to undergo insulin-stimulated tyrosine phosphorylation in 32D cells, which uncouples the activation of phosphatidylinositol 3`-kinase and p70 from the endogenous insulin receptors. Overexpression of the insulin receptor in 32D cells, however, restores tyrosine phosphorylation of IRS-1 and rescues insulin responses including mitogenesis. Thus, while the PH domain is not required for the engagement of downstream signals, it is one of the elements in the NH terminus of IRS-1 that is needed for a sensitive coupling to insulin receptors, especially at ordinary receptor levels found in most cells and tissues.


INTRODUCTION

During insulin stimulation, IRS-1() becomes tyrosine-phosphorylated and binds to the Src homology-2 domains in several signaling proteins (SH2 proteins) (1) . As a consequence of docking SH2 proteins, IRS-1 mediates multiple downstream signals, including the direct activation of PI 3`-kinase and SH-PTP2, and the stimulation of mitogen-activated protein kinase and p70; IRS-1 at least partially regulates mitogenesis, chemotactic signaling, and glucose transport (1, 2, 3, 4, 5) . Since IRS-signaling proteins are not engaged by most growth factor receptors, specific interactions between IRS-signaling proteins and receptors must mediate productive coupling.

Deletion of the first 500 amino acids of IRS-1 prevents phosphorylation of the COOH-terminal portion of the molecule expressed in COS cells (6). A pleckstrin homology (PH) domain exists between residues 13 and 115, which is absolutely identical among the rat, mouse, and human isoforms (7, 8, 9) , and is 62% identical in the recently cloned IRS-2.() PH domains were originally recognized as a repeat in pleckstrin and later found in various signal transduction proteins (7) . Although the amino acid sequence of various PH domains is poorly conserved, the PH domain in spectrin and pleckstrin has a common structure composed of three -sheets and an -helix arranged around a hydrophobic core (10, 11) . Although the exact function of PH domains is obscure, recent studies suggest that they mediate protein-protein interactions or bind phospholipids (12-14).

In this study we have investigated the role of the PH domain in IRS-1 by deleting it and studying this mutant (IRS-1) in CHO cells, which contain endogenous IRS-1, and in 32D cells, which lack endogenous IRS-signaling proteins. Our results suggest that the PH domain is not required for the engagement of downstream signals; rather, it is one of the elements that mediates the coupling between the insulin receptor and IRS-signaling proteins.


MATERIALS AND METHODS

Construction of IRS-1

The cDNA for rat IRS-1 in pBluescript (15) was digested with BspEI and BspMI, and the resulting fragment containing the majority of IRS-1 was religated in the presence of linkers formed by annealing the oligonucleotides 5`-CCG/GAG/GAT/CCC/CTT/AAG-3` and 5`-CCT/CCT/TAA/GGG/GAT/CCT-3`. The resulting mutant cDNA was subcloned into pCMVhis for expression using SacI and HindIII (3) .

Cell Lines

Chinese hamster ovary (CHO) cells expressing the human insulin receptor (CHO) or the human insulin receptor and IRS-1 (CHO/IRS-1) have been described (4, 15) . CHO cell lines were maintained in Ham's F-12 medium containing 10% fetal bovine serum. CHO cells were transfected with IRS-1 cDNA by the calcium phosphate method and selected in 10 mM histidinol (4, 15) . Surviving cells were cloned and maintained in the presence of 10 mM histidinol. 32D cells and cell lines expressing IRS-1 (32D/IRS-1), IR (32D), or IR and IRS-1 (32D/IRS-1) have been described previously (2) . 32D cell lines were maintained in RPMI 1640 media supplemented with 10% fetal bovine serum and 5% WEHI-3-conditioned media (a source of IL-3). 32D and 32D cells were electroporated with the cDNA for IRS-1 and selected with 5 mM histidinol to obtain cell lines expressing the PH deletion mutant (2, 3) . Cell lines were selected for expression by immunoblotting lysates of histidinol-resistant cell lines with IRS-1 antibodies. Expression of the human insulin receptor was confirmed by IR immunoblotting cell lines co-expressing the insulin receptor. For experiments, CHO cells were made quiescent by incubation in Ham' F-12 medium supplemented with 0.5% bovine serum albumin for 18-24 h; 32D cells were made quiescent by incubation in unsupplemented Dulbecco's modified Eagle's medium for 4 h.

Antibodies and Growth Factors

IRS-1 antibodies were rabbit polyclonal antisera against a COOH-terminal peptide of IRS-1; they were used at a 1:300 dilution to immunoblot and 1:100 to immunoprecipitate (16) . Being directed against the COOH terminus of IRS-1, this antibody recognizes IRS-1 and IRS-1 equivalently. IR antibodies were rabbit antisera raised against a glutathione S-transferase fusion protein containing the intracellular -subunit of the human insulin receptor; they were used 1:300 to immunoblot. PY antibodies were mouse monoclonal 4G10 purified from tissue culture supernatant by chromatography on Protein A-Sepharose or affinity-purified rabbit polyclonal antibodies (17) ; both antibodies immunoblotted at 1:300 dilution. Rabbit antisera against p70 and their use have been described (18) . Insulin was from Calbiochem (San Diego, CA).

Immunoblotting

Proteins were denatured by boiling in Laemmli sample buffer containing 100 mM dithiothreitol and resolved by SDS-PAGE. Gels were transferred to nitrocellulose membranes (Schleicher & Schuell) in Towbin buffer containing 0.02% SDS and 20% methanol (19) . Membranes were blocked, probed, and developed as described previously, and visualized using I-Protein A (Amersham Corp.) or horseradish peroxidase-conjugated goat anti-mouse secondary antibodies (Cappel) and the Renaissance system (DuPont NEN) (3, 18) . Blots were exposed to Kodak X-AR film or imaged on a Molecular Dynamics PhosphorImager.

Metabolic Labeling of CHOCells

Quiescent CHO cell lines were washed twice with phosphate-free RPMI 1640 medium and incubated for 3 h in phosphate-free RPMI 1640 medium supplemented with 0.2 mCi/ml [P]orthophosphate (DuPont NEN). Cells were stimulated with 100 nM insulin for 10 min and lysed in ice-cold 100 mM Tris-HCl, pH 7.4, containing 1% Triton X-100, 100 mM NaF, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 10 mg/ml each of leupeptin and aprotinin. Insoluble material was removed by centrifugation at 10,000 g for 10 min, and IRS-1 antibodies were added for 1 h at 4 °C. Immune complexes were collected on Protein A-Sepharose (Pharmacia Biotech Inc.) and washed three times in ice-cold 50 mM HEPES, pH 7.4, containing 1% Triton X-100, 150 mM NaCl, 100 mM NaF, and 2 mM sodium orthovanadate. Immune complexes were resolved by 7.5% SDS-PAGE and visualized on a Molecular Dynamics PhosphorImager.

PI 3`-Kinase, p70, and Mitogenesis Assays

In vitro phosphorylation of phosphatidylinositol was carried out in immune complexes as described previously and quantitated on a Molecular Dynamics PhosphorImager (3, 20) . In vitro kinase assays for p70 were carried out as described previously and quantitated on a PhosphorImager (5, 21). Mitogenesis was measured by thymidine incorporation into DNA as described previously (2, 3, 5) .

RESULTS AND DISCUSSION

Alignment of the NH terminus of IRS-1 with various signaling proteins reveals a PH domain between residues 13 and 115 (7, 12) . The function of this PH domain was investigated by deleting it (in-frame) from the IRS-1 cDNA (IRS-1) (Fig. 1A). IRS-1 and IRS-1 were expressed to equivalent levels in 32D cells and in CHO and 32D cells overexpressing the insulin receptor. The 32D cells were used because they do not contain endogenous IRS-signaling proteins. IRS-1 migrated at the expected molecular mass in all of the cell lines during SDS-PAGE, suggesting that it was stably expressed and Ser/Thr phosphorylated in a manner similar to wild-type IRS-1 (Fig. 1B).


Figure 1: PH domain deletion IRS-1. A, a linear model of IRS-1 with the location of interesting domains and potential tyrosine phosphorylation sites is shown. The PH domain of IRS-1 extends from amino acids 13-115 (1, 7). Amino acids 6-155 are removed from IRS-1. The PH domain (IRS homology region (IH)-1) and another region (IH2) share high identity between IRS-1 and IRS-2 (see footnote 2). The location of potential tyrosine phosphorylation sites is shown; asterisks indicate known sites of tyrosine phosphorylation (25). B, parental CHO, 32D, or 32D cells and CHO, 32D, and 32D cell lines expressing IRS-1 or IRS-1 were lysed, resolved by SDS-PAGE, and immunoblotted with IRS-1 antibodies. Note, although endogenous IRS-1 was detectable in CHO cells on long exposure, it is not seen in the figure as a consequence of the short exposure needed to prevent overexposing the exogenous IRS-1 isoforms. No endogenous IRS-1 was detectable in 32D cell lines, as reported previously (2, 3). Migration of molecular size standards, IRS-1, and IRS-1 is indicated.



Insulin stimulated the expected tyrosyl phosphorylation of the insulin receptor -subunit in CHO, CHO/IRS-1, and CHO/IRS-1 cells (Fig. 2A). During insulin stimulation, tyrosine phosphorylation of endogenous IRS-1 was clearly observed in CHO cells, and overexpression of IRS-1 in these cells resulted in a striking increase, as described previously (4) . However, insulin-stimulated tyrosine phosphorylation of IRS-1 was greatly reduced (>95%) in CHO/IRS-1 cells. Normal tyrosine phosphorylation of endogenous IRS-1 was detected in the CHO/IRS-1 cells during insulin stimulation (Fig. 2A). Thus, deletion of the PH domain from IRS-1 abrogates its tyrosine phosphorylation in CHO cells but does not interfere with the phosphorylation of the endogenous wild-type IRS-1.


Figure 2: Phosphorylation of IRS-1 in CHO cells. A, quiescent CHO cell lines were incubated in the absence or presence of 100 nM insulin for 5 min and lysed. Lysates were resolved by SDS-PAGE, and tyrosyl phosphoproteins were analyzed by immunoblotting with PY. B, quiescent CHO cell lines were labeled for 3 h with [P]orthophosphate and incubated in the absence or presence of 100 nM insulin for 10 min. Cells were lysed and immunoprecipitated with IRS-1. Immunoprecipitates were resolved by SDS-PAGE and imaged on a Molecular Dynamics PhosphorImager. Phosphoamino acid analysis confirmed the presence of Ser(P) and Thr(P) on all IRS-1 isotypes, but significant amounts of Tyr(P) were found only in wild-type IRS-1 (not shown). The migration of IRS-1, IRS-1, IRS-1, and molecular size standards is indicated. These experiments are representative of multiple independent assays performed with multiple independently derived clones.



We investigated the Ser/Thr phosphorylation of IRS-1 and IRS-1 in CHO cells by monitoring levels of [P]phosphate incorporated during insulin stimulation (Fig. 2B). Solely as a control for normal levels of Ser/Thr phosphorylation, we included another IRS-1 mutant, IRS-1, which contains an intact PH domain but is not tyrosyl-phosphorylated due to the substitution of phenylalanine for tyrosine in all of its putative tyrosyl phosphorylation sites.() Insulin stimulated the phosphorylation of IRS-1, IRS-1, and IRS-1 as monitored by decreased mobility and increased [P]phosphate content (Fig. 2B). IRS-1 is highly serine-phosphorylated in the basal state and undergoes tyrosine and Ser/Thr phosphorylation during insulin stimulation (4, 15) . Since IRS-1 and IRS-1 are not tyrosine-phosphorylated in either the basal state or during insulin stimulation in CHO cells, the phosphorylation of these species reflects only Ser/Thr phosphorylation. Thus, IRS-1 is recognized normally by Ser/Thr kinases, suggesting that its structure is not globally disrupted. Therefore, removal of the PH domain interfered specifically with the tyrosine phosphorylation of IRS-1.

32D cells are an ideal system for the examination of IRS-1 function, since they contain no endogenous IRS proteins to interfere with the analysis of signaling by exogenous expressed species (2, 3, 5) . Activation of endogenous insulin receptors stimulated tyrosyl phosphorylation of IRS-1 in 32D/IRS-1 cells, whereas disruption of the PH domain reduced tyrosine phosphorylation by more than 95% (Fig. 3A); no tyrosyl phosphorylation of IRS-1 was observed in untransfected 32D cells, since these cells do not contain IRS-1 (2, 3, 5) . Insulin stimulated the association of PI 3`-kinase with IRS-1 in 32D/IRS-1 cells. Moreover, IRS-1 was essential for the activation of p70 by insulin (Fig. 3C); however, disruption of the PH domain of IRS-1 completely inhibited these events in 32D cells (Fig. 2C).


Figure 3: Signaling by IRS-1 and IRS-1 in 32D cells. A, quiescent 32D cell lines were incubated in the absence or presence of insulin for 5 min and lysed. Lysates were resolved by SDS-PAGE and analyzed by immunoblotting with PY antibodies. Migration of molecular size standards and IRS-1 is indicated, along with the expected position of IRS-1. B, quiescent 32D cell lines were stimulated with insulin for 5 min, lysed, and immunoprecipitated with IRS-1 antibodies. Immunoprecipitates were washed and assayed for associated PI 3`-kinase activity. C, quiescent 32D cell lines were stimulated with insulin for 30 min and lysed. p70 was immunoprecipitated and its activity assayed in an in vitro immune complex assay. Activity in B and C was quantified on a PhosphorImager and is shown as arbitrary units. Each assay in this figure is representative of at least three independent experiments with multiple independently derived cell lines.



Unlike CHO cells, overexpression of the insulin receptor in 32D cells restored normal insulin signaling by IRS-1; both IRS-1 and IRS-1 were tyrosine-phosphorylated to equal levels in 32D cells during insulin stimulation (Fig. 4A). During insulin stimulation, IRS-1 and IRS-1 associated normally with PI 3`-kinase (Fig. 4B) and both mediated the activation of p70 (Fig. 4C). Futhermore, insulin stimulated mitogenesis equivalently in 32D cells expressing either wild-type IRS-1 or IRS-1 (Fig. 4D). Thus, overexpression of the insulin receptor in 32D cells restored IRS-1 phosphorylation and, with it, signaling.


Figure 4: Signaling by IRS-1 and IRS-1 in 32D cells. A, quiescent 32D cell lines were incubated in the absence or presence of insulin for 5 min and lysed. Lysates were resolved by SDS-PAGE and analyzed by immunoblotting with PY antibodies. Migration of molecular size standards, IRS-1, IRS-1, and a degradation product of IRS-1 (IRS-1) is indicated. B, quiescent 32D cell lines were stimulated with insulin for 5 min, lysed, and immunoprecipitated with IRS-1 antibodies. Immunoprecipitates were washed and assayed for associated PI 3`-kinase activity. C, quiescent 32D cell lines were stimulated with insulin for 30 min and lysed. p70 was immunoprecipitated and its activity assayed in an in vitro immune complex assay. Activity in B and C was quantified on a PhosphorImager and is shown as arbitrary units. D, cell lines were incubated in various concentrations of insulin for 48 h, followed by the addition of [H]thymidine for another 3 h. Cells were harvested onto glass filters, and incorporated nucleotide was quantified by scintillation counting. Each assay in this figure is representative of at least three independent experiments with multiple independently derived cell lines.



Our results suggest that the PH domain is essential for sensitive coupling between IRS-1 and the insulin receptor. Despite the inability of IRS-1 molecules with PH domain deletions to become tyrosine-phosphorylated and mediate signaling in the low insulin receptor environment of 32D cells, these mutations are rescued by overexpression of the insulin receptor in 32D cells. Therefore, disruption of the PH domain does not damage the ability of IRS-1 to mediate downstream signals, as IRS-1 is phosphorylated normally and signals effectively in the presence of high levels of insulin receptor in 32D cells. Since the PH domain comprises an independent protein module (10, 11) , it is not surprising that the remaining portions of IRS-1 are properly folded and retain biological function. The observation that a mutant IRS-1 molecule containing a small deletion (30 amino acids) at the beginning of the PH domain also abrogates tyrosyl phosphorylation in 32D cells provides further evidence that these results do not stem from the disruption of sequences downstream of the PH domain.()

Why does overexpression of the insulin receptor in CHOcells not rescue insulin-stimulated tyrosyl phosphorylation of IRS-1? Unlike 32D cells, CHO cells contain endogenous IRS-1, which possesses an intact PH domain. The PH domain must provide a significant advantage during competition for a limited number of insulin receptors, possibly by displacing the low affinity IRS-1 away from the receptor.

It remains unclear how the PH domain mediates its function. It could involve direct protein-protein interactions between the receptor and IRS-1; however, our attempts to measure specific interactions between the purified insulin receptor and glutathione S-transferase fusion proteins containing the PH domain have been unsuccessful. Recent evidence suggests that PH domains from other signaling molecules bind specific phospholipids (13) . Further work should shed light on whether the PH domain acts specifically to target IRS-1 to the insulin receptor or more generally to the membrane compartment where it encounters the tyrosine kinase.

IRS-1 contains a region immediately downstream of the PH domain which has also been implicated in the IR/IRS-1 interaction (IH2 region in Fig. 1A) (6) . The IH2 region is similar in IRS-1 and IRS-2, and like the PH domain, contains no tyrosine phosphorylation sites (15). The IH region appears to be a phosphotyrosine binding domain like the one in Shc (26, 27) ; this region binds the NPEY motif in the insulin receptor (6, 22) , and its deletion partially inhibits IR-dependent phosphorylation (data not shown). This supports previous results showing that the NPXY motif is essential for insulin signaling and IRS-1 phosphorylation (15, 23, 24) . The role of the PH domain and the IH2 regions in IRS engagement by other systems, such as IL-4 and growth hormone, remains to be determined.

The PH domain is 100% identical in mouse, human, and rat IRS-1, and 62% similar in IRS-1 and IRS-2. This is the highest level of PH domain conservation between distinct proteins yet observed; this conservation goes well beyond the requirement for similar folding recently revealed by structural studies (10, 11) and is consistent with a specific function. The IH2 regions are also conserved between IRS-1 and IRS-2. The COOH terminus, however, is poorly conserved between IRS-1 and IRS-2, however, with the critical exception of short elements surrounding tyrosine phosphorylation sites. Thus, the PH domain and the IH2 regions are likely to cooperate in mediating IR/IRS recognition. Impairment of PH domain or IH2 region interactions is expected to alter the sensitivity of the insulin response, and could contribute to insulin resistance.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants DK 38712, DK 43808 (to M. F. W.), and CA 46595 (to J. B.). 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.

§
Supported in part by the Albert J. Ryan Foundation at Harvard Medical School and National Institutes of Health National Research Service Award Training Grant DK 07260.

Established Investigator of the American Heart Association and a recipient of Harvard Medical School Funds for Discovery.

**
Fellow of the Juvenile Diabetes Foundation.

§§
To whom correspondence should be addressed: Research Division, 1 Joslin Pl., Boston, MA 02215. Tel.: 617-732-2578; Fax: 617-732-2593; E-mail: whitemor@joslab.harvard.edu.

The abbreviations used are: IRS-1, insulin receptor substrate-1; SH2, Src homology-2; PI, phosphatidylinositol; CHO, Chinese hamster ovary; IL, interleukin; PAGE, polyacrylamide gel electrophoresis; IH, IRS homology region; PH, pleckstrin homology region.

X. J. Sun, L. M. Wang, Y. Zhang, L. Yenush, M. G. Myers, Jr., E. Glasheen, S. Pons, G. Wolf, S. E. Shoelson, W. S. Lane, J. H. Pierce, and M. F. White, submitted for publication.

M. G. Myers, Jr., Y. Zhang, E. Glasheen, T. Grammer, L. Yenush, L. M. Wang, X. J. Sun, J. Blenis, J. H. Pierce, and M. F. White, manuscript in preparation.

M. G. Myers, Jr., J. H. Pierce, and M. F. White, unpublished data.


ACKNOWLEDGEMENTS

We thank Jia-Huai Wang for helpful discussions and a critical reading of the manuscript. We also thank Gus Gustafson, Mary Elizabeth Patti, Ron Kahn, Jon Backer, and Lynne Yenush for helpful discussions and sharing unpublished data. We thank Yitao Zhang and Charles Knickley for excellent technical assistance.


REFERENCES
  1. Myers, M. G., Jr., Sun, X. J., and White, M. F.(1994) Trends Biochem. Sci. 19, 289-294 [CrossRef][Medline] [Order article via Infotrieve]
  2. Wang, L. M., Myers, M. G., Jr., Sun, X. J., Aaronson, S. A., White, M. F., and Pierce, J. H.(1993) Science 261, 1591-1594 [Medline] [Order article via Infotrieve]
  3. Myers, M. G., Jr., Wang, L. M., Sun, X. J., Zhang, Y., Yenush, L. P., Schlessinger, J., Pierce, J. H., and White, M. F.(1994) Mol. Cell. Biol. 14, 3577-3587 [Abstract]
  4. Sun, X. J., Miralpeix, M., Myers, M. G., Jr., Glasheen, E. M., Backer, J. M., Kahn, C. R., and White, M. F.(1992) J. Biol. Chem. 267, 22662-22672 [Abstract/Free Full Text]
  5. Myers, M. G., Jr., Grammer, T. C., Wang, L. M., Sun, X. J., Pierce, J. H., Blenis, J., and White, M. F.(1994) J. Biol. Chem. 269, 28783-28789 [Abstract/Free Full Text]
  6. O'Neill, T. J., Craparo, A., and Gustafson, T. A.(1994) Mol. Cell. Biol. 14, 6433-6442 [Abstract]
  7. Musacchio, A., Gibson, T., Rice, P., Thompson, J., and Saraste, M. (1993) Trends Biochem. Sci. 18, 343-348 [CrossRef][Medline] [Order article via Infotrieve]
  8. Keller, S. R., Aebersold, R., Garner, C. W., and Lienhard, G. E.(1993) Biochim. Biophys. Acta 1172, 323-326 [Medline] [Order article via Infotrieve]
  9. Araki, E., Sun, X. J., Haag, B. L., Zhang, Y., Chuang, L. M., Yang-Feng, T., White, M. F., and Kahn, C. R.(1993) Diabetes 42, 1041-1054 [Abstract]
  10. Yoon, H. S., Hajduk, P. J., Petros, A. M., Olejniczak, E. T., Meadows, R. P., and Fesik, S. W.(1994) Nature 369, 672-675 [CrossRef][Medline] [Order article via Infotrieve]
  11. Macias, M. J., Musacchio, A., Ponstingl, H., Nilges, M., Saraste, M., and Oschkinat, H.(1994) Nature 369, 675-677 [CrossRef][Medline] [Order article via Infotrieve]
  12. Gibson, T. J., Hyvonen, M., Musacchio, A., and Saraste, M.(1994) Trends Biochem. Sci. 19, 349-353 [CrossRef][Medline] [Order article via Infotrieve]
  13. Harlan, J. E., Hajduk, P. J., Yoon, H. S., and Fesik, S. W.(1994) Nature 371, 168-170 [CrossRef][Medline] [Order article via Infotrieve]
  14. Touhara, K., Inglese, J., Pitcher, J. A., Shaw, G., and Lefkowitz, R. J.(1994) J. Biol. Chem. 14, 10217-10220
  15. Sun, X. J., Rothenberg, P., Kahn, C. R., Backer, J. M., Araki, E., Wilden, P. A., Cahill, D. A., Goldstein, B. J., and White, M. F.(1991) Nature 352, 73-77 [CrossRef][Medline] [Order article via Infotrieve]
  16. Saad, M. J. A., Araki, E., Miralpeix, M., Rothenberg, P. L., White, M. F., and Kahn, C. R.(1992) J. Clin. Invest. 90, 1839-1849 [Medline] [Order article via Infotrieve]
  17. White, M. F. and Backer, J. M.(1991) Methods Enzymol. 201, 65-79 [Medline] [Order article via Infotrieve]
  18. Chung, J., Kuo, C. J., Crabtree, G. R., and Blenis, J.(1992) Cell 69, 1227-1236 [Medline] [Order article via Infotrieve]
  19. Towbin, H., Staehelin, T., and Gordon, G.(1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354 [Abstract]
  20. Ruderman, N., Kapeller, R., White, M. F., and Cantley, L. C.(1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1411-1415 [Abstract]
  21. Varticovski, L., Daley, G. Q., Jackson, P., Baltimore, D., and Cantley, L. C.(1991) Mol. Cell. Biol. 11, 1107-1113 [Medline] [Order article via Infotrieve]
  22. Gustafson, T. A., He, W., Craparo, A., Schaub, C. D., and O'Neill, T. J.(1995) Mol. Cell Biol., in press
  23. White, M. F., Livingston, J. N., Backer, J. M., Lauris, V., Dull, T. J., Ullrich, A., and Kahn, C. R.(1988) Cell 54, 641-649 [Medline] [Order article via Infotrieve]
  24. Yonezawa, K., Ando, A., Kaburagi, Y., Yamamotohonda, R., Kitamura, T., Hara, K., Nakafuku, M., Okabayashi, Y., Kadowaki, T., Kaziro, Y., and Kasuga, M.(1994) J. Biol. Chem. 269, 4634-4640 [Abstract/Free Full Text]
  25. Sun, X. J., Crimmins, D. L., Myers, M. G., Jr., Miralpeix, M., and White, M. F.(1993) Mol. Cell. Biol. 13, 7418-7428 [Abstract]
  26. Bork, P., and Margolis, B.(1995) Cell 80, 693-694 [Medline] [Order article via Infotrieve]
  27. Karanaugh, W. M., and Williams, L. T.(1994) Science 266, 1862-1865 [Medline] [Order article via Infotrieve]

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