©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
G Subunits Mediate Mitogen-activated Protein Kinase Activation by the Tyrosine Kinase Insulin-like Growth Factor 1 Receptor (*)

Louis M. Luttrell , Tim van Biesen (§) , Brian E. Hawes , Walter J. Koch , Kazushige Touhara , Robert J. Lefkowitz (¶)

From the (1)From The Howard Hughes Medical Institute and the Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The receptors for insulin-like growth factor 1 (IGF1) and insulin are related heterotetrameric proteins which, like the epidermal growth factor (EGF) receptor, possess intrinsic ligand-stimulated tyrosine protein kinase activity. In Rat 1 fibroblasts, stimulation of mitogen-activated protein (MAP) kinase via the IGF1 receptor and the G-coupled receptor for lysophosphatidic acid (LPA), but not via the EGF receptor, is sensitive both to pertussis toxin treatment and to cellular expression of a specific G subunit-binding peptide. The IGF1, LPA, and EGF receptor-mediated signals are all sensitive to inhibitors of tyrosine protein kinases, require p21 activation, and are independent of protein kinase C. These data suggest that some tyrosine kinase growth factor receptors (e.g. IGF1 receptor) and classical G protein-coupled receptors (e.g. LPA receptor) employ a similar mechanism for mitogenic signaling that involves both tyrosine phosphorylation and G subunits derived from pertussis toxin-sensitive G proteins.


INTRODUCTION

Activation of the ubiquitous mitogen-activated protein (MAP)()kinase pathway is thought to proceed through a defined sequence of protein phosphorylations and protein-protein interactions. The best understood example is that triggered by the receptor for epidermal growth factor (EGF). Ligand binding promotes EGF receptor dimerization and tyrosine autophosphorylation. Assembly of a multiple protein complex at the cell membrane directed by Src homology (SH) 2 and SH3 domain interactions results in p21 GTP-exchange and activation of the Raf-1 kinase. In the ensuing phosphorylation cascade, activation of MAP kinases follows their phosphorylation by the mixed function threonine/tyrosine kinase, MEK (MAP kinase/extracellular signal-regulated kinase)(1, 2) .

Recent work has shown that some G protein-coupled receptors (e.g. the lysophosphatidic acid (LPA) receptor) which interact with Bordetella pertussis toxin (PT)-sensitive G proteins can also promote p21-dependent activation of MAP kinase(3, 4, 5, 6, 7, 8) . This pathway is sensitive to inhibitors of tyrosine protein kinases (6, 7, 9) and requires G protein G subunits(3, 10, 11) .

Insulin and insulin-like growth factor 1 (IGF1), in contrast to EGF, behave as relatively weak mitogens. Their receptors are closely related proteins which share an heterotetrameric structure and, like the EGF receptor, possess ligand-stimulated tyrosine kinase activity(12) . Moreover, receptor-catalyzed tyrosine phosphorylation of exogenous proteins, such as insulin receptor substrate 1 (IRS-1) (13, 14) or Shc(15) , is one of the earliest steps in mitogenesis triggered by these receptors. Once phosphorylated, these proteins are felt to function as platforms for assembly of the mitogenic signaling protein complex, which then proceeds following the EGF receptor paradigm. Here we present the surprising observation that, in contrast to the classical EGF receptor paradigm, stimulation of the MAP kinase pathway by the tyrosine kinase IGF1 receptor also requires the participation of G subunits derived from PT-sensitive G proteins. Like the G protein-coupled receptor-mediated pathway, the IGF1 signal can be blocked by either PT treatment or by an inhibitor of G subunit-mediated signaling. Thus, mitogenic signals originating from this class of tyrosine kinase growth factor receptors converge with the G protein-coupled receptor signaling pathway at a point upstream of p21 activation.


EXPERIMENTAL PROCEDURES

DNA Constructs

The cDNA encoding hemagglutinin-tagged p44 (p44) in the pcDNA1 eukaryotic expression plasmid was provided by J. Pouysségur. The dominant negative mutant p21 in the pRS eukaryotic expression plasmid was provided by D. Altschuler and M. Ostrowski.

Cell Culture and Transfection

Rat 1 cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 100 µg/ml gentamicin at 37 °C in a humidified, 5% CO atmosphere. Stably transfected ARK1-CT peptide-expressing Rat 1 mutant 277 cells (10) were maintained under identical conditions in the presence of 1000 µg/ml Geneticin (Life Technologies, Inc.).

Transient transfection of Rat 1 cells was performed using LipofectAMINE (Life Technologies, Inc.). Briefly, confluent monolayers in 6-well tissue culture plates were incubated at 37 °C with a transfection mixture composed of 1 ml of serum-free DMEM containing 1.2 µg of DNA/well and 7 µl of LipofectAMINE. After 2 h, the mixture was aspirated and replaced with 2 ml of DMEM containing 10% fetal bovine serum. Assays were performed 48 h after transfection. Empty pRK5 vector was added to transfections as needed to keep the total mass of DNA added per well constant within an experiment. Coexpression of the ARK1-CT peptide was confirmed by protein immunoblotting of whole cell detergent lysates using rabbit anti-ARK1 carboxyl terminus serum as described(16) . Endogenous IGF1 receptor expression was quantified by equilibrium binding of I-IGF1 (DuPont NEN) as described(17) .

Measurement of MAP Kinase Phosphorylation State and Kinase Activity

Agonist-stimulated phosphorylation of endogenous p42 (erk2) was determined by electrophoretic mobility shift as described(10) . Activation of p44 (erk1) was measured using a modification of the procedure of Meloche et al.(18) . Transfected Rat 1 cells were preincubated overnight in serum-free medium and stimulated for 5 min (IGF1, EGF, and PMA) or 10 min (LPA). Monolayers were washed with ice-cold calcium- and magnesium-free phosphate-buffered saline, lysed in 200 µl of ice-cold lysis buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% v/v Nonidet P-40, 0.5% w/v sodium deoxycholate, 0.1% SDS, 10 mM NaF, 10 mM sodium pyrophosphate, 0.1 mM phenylmethylsulfonyl fluoride), and clarified by centrifugation. Coexpressed p44 was immunoprecipitated from the supernatant using 6.5 µg of 12CA5 antibody and 30 µl of a 50% slurry of protein A-agarose. Washed immune complexes were resuspended in 40 µl of kinase buffer (20 mM HEPES, pH 7.4, 10 mM MgCl, 1 mM dithiothreitol) containing 250 µg/ml myelin basic protein (MBP), 20 µM ATP, 2.5 µCi of [-P]ATP and incubated for 20 min at 30 °C. The reaction was terminated by the addition of 40 µl of 2 Laemmli sample buffer, and P-labeled MBP was resolved by SDS-PAGE. Phosphorylation was quantitated using a Molecular Dynamics PhosphorImager.


RESULTS

Effects of Pertussis Toxin Treatment, Tyrosine Kinase Inhibitors, and PKC Depletion on IGF1 Receptor-mediated MAP Kinase Activation

Fig. 1depicts the effects of PT treatment on the time course of ligand-stimulated phosphorylation of MAP kinase mediated by endogenous receptors in wild-type Rat 1 cells. Lysophosphatidic acid (LPA), the simplest naturally occurring phospholipid, provoked a transient increase in MAP kinase phosphorylation, which was abolished by PT pretreatment (Fig. 1A). MAP kinase phosphorylation in response to insulin or IGF1 exposure followed a similar time course and was also PT-sensitive (Fig. 1, B and C). In contrast, MAP kinase phosphorylation stimulated by platelet-derived growth factor, acidic fibroblast growth factor, or EGF (Fig. 1, D-F), each of which is mediated by membrane receptors with intrinsic tyrosine kinase activity, exhibited a more robust and sustained activation which was not significantly affected by PT treatment. A similar pattern of PT sensitivity was observed in C3H10T1/2 murine fibroblasts. Thus, the responses mediated by tyrosine kinase growth factor receptors apparently segregated into two groups: one PT-sensitive, resembling the Gi-coupled LPA receptor, and one PT-insensitive.


Figure 1: Effect of PT treatment on MAP kinase activation via endogenously expressed receptors in Rat 1 cells. Rat 1 cells, cultured as described (15), were incubated overnight in serum-free medium in the presence or absence of PT (100 ng/ml) prior to determination of agonist-stimulated phosphorylation of endogenous p42 as described. Upper panel, representative autoradiograph demonstrating the effects of PT treatment on the p42 phosphorylation state following a 5-min stimulation with LPA (10 µM), IGF1 (100 ng/ml), or EGF (10 ng/ml). Lower panel, the time course of agonist-induced phosphorylation of MAP kinase in control () and PT-treated () cells was determined following stimulation with LPA (A), insulin (100 nM) (B), IGF1 (C), platelet-derived growth factor (10 ng/ml) (D), fibroblast growth factor (10 ng/ml) (E), or EGF (F). Data are presented as the percent of total p42 present in the phosphorylated state. Each point represents the mean ± S.E. for three to six separate experiments.



Insulin, IGF1, and insulin-like growth factor 2 (IGF2) have distinct cell surface receptors, each of which can bind insulin, IGF1, and IGF2 with varying affinity. The receptors can be discriminated based upon the rank order of potency displayed by each agonist. The receptor for IGF2 consists of a single membrane-spanning polypeptide that lacks intrinsic tyrosine kinase activity(19) . While not considered a mediator of mitogenic signals, the IGF2 receptor can reportedly interact with PT-sensitive G proteins(20) . Dose-response curves for MAP kinase phosphorylation in Rat 1 cells revealed a rank order of potency IGF1 > IGF2 insulin (data not shown), indicating that the effects of all three agonists were mediated predominantly by IGF1 receptors, as has been previously reported(21) .

Several authors have reported that the PT-sensitive activation of MAP kinase by G protein-coupled receptors is also sensitive to inhibitors of tyrosine protein kinases (6, 7) and is independent of PKC(3, 4, 5, 6, 7, 8) . As shown in Fig. 2A, preincubation of cells with the tyrosine kinase inhibitor genistein resulted in significant inhibition of both LPA- and IGF1-mediated MAP kinase phosphorylation at concentrations (100 µM) which did not impair EGF- or direct PKC-mediated effects. At higher concentrations (300 µM), EGF-, LPA-, and IGF1-mediated, but not PKC-mediated, MAP kinase phosphorylation was inhibited (data not shown). MAP kinase activation resulting from expression of the constitutively activated mutant p21(22) was unaffected by genistein concentrations up to 300 µM,()indicating that the tyrosine kinase inhibitor-sensitive step lies upstream of p21 activation. In contrast to the effects of genistein, the potent tyrosine kinase and PKC inhibitor staurosporine nonspecifically inhibited MAP kinase activation by each agent.


Figure 2: Effects of protein kinase inhibitors and PKC depletion on MAP kinase phosphorylation in Rat 1 cells. A, serum-starved Rat 1 cells were preincubated for 15 min with genistein (100 µM) or staurosporine (1 µM) prior to stimulation for 5 min with LPA, IGF1, EGF, or the PKC activating phorbol ester, phorbol 12-myristate 13-acetate (PMA) (1 µM). Basal (NS) and agonist-stimulated phosphorylation of p42 was determined as described. B, Rat 1 cells were preincubated overnight in serum-free medium in the presence or absence of PMA (0.1 µM) to down-regulate cellular PKC expression (14), preincubated for 15 min in the absence of PMA, and stimulated for 5 min with LPA, IGF1, EGF, or PMA. Phosphorylation of p42 was then determined as described. Each column represents mean ± S.E. values for percent of total p42 present in the phosphorylated state in three separate experiments performed in duplicate. * signifies value less than control, p < 0.05.



Fig. 2B depicts the effects of cellular PKC depletion on agonist-induced MAP kinase phosphorylation. Rechallenge with phorbol ester following PKC depletion provoked no increase in MAP kinase phosphorylation. In contrast, the effects of LPA, IGF1, and EGF were not significantly inhibited. These data suggest that stimulation of MAP kinase phosphorylation by the G-coupled LPA receptor and the tyrosine kinase IGF1 receptor are qualitatively similar; each is sensitive to inhibitors of tyrosine kinases and is independent of PKC activation.

Effect of the GSubunit-sequestrant ARK1-CT Peptide on IGF1 Receptor-mediated MAP Kinase Activation

Recently, G protein G subunits have been implicated in the direct p21-dependent activation of MAP kinase mediated by PT-sensitive G protein-coupled receptors including the LPA receptor. Coexpression of G subunits in COS-7 cells leads directly to MAP kinase activation(3, 11) . We have previously shown that cellular expression of a specific G subunit binding peptide derived from the carboxyl terminus of the -adrenergic receptor kinase 1 (ARK1-CT) (23, 24) specifically antagonizes G subunit-mediated MAP kinase activation (10, 25) in stably and transiently transfected cell lines. To determine whether the PT-sensitive phosphorylation of MAP kinase stimulated by the IGF1 receptor was mediated by G subunits, we studied the effects of IGF1 in a Rat 1 cell line which stably overexpressed the ARK1-CT peptide (277 cells)(10) . As shown in Fig. 3, compared with the parental Rat 1 cells, LPA-stimulated MAP kinase phosphorylation was attenuated by about 50% (Fig. 3A). IGF1-stimulated phosphorylation was undetectable in the 277 cells (Fig. 3B), while the EGF signal was unaffected (Fig. 3C). Thus, sensitivity to inhibition by PT and a G subunit-binding peptide are properties shared by the LPA- and IGF1-mediated pathways.


Figure 3: Time course of agonist-stimulated MAP kinase phosphorylation Rat 1 cells which stably overexpress the G subunit-binding ARK1-CT peptide. ARK1-CT peptide-expressing Rat 1 mutant 277 cells (15) were incubated overnight in serum-free medium in the presence () or absence () of PT prior to determination of the time course of agonist-induced phosphorylation of endogenous p42 following stimulation with LPA (A), IGF1 (B), or EGF (C). The time course observed in the parental Rat 1 cells () is shown for reference. Data are presented as the percent of total p42 present in the phosphorylated state. Each point represents the mean ± S.E. for three separate experiments. Endogenous IGF1 receptor density was 8.5-10.0 fmol/mg of whole cell protein in both the parental Rat 11 and 277 cell lines.



To confirm that the inhibition of MAP kinase phosphorylation by the G subunit antagonist reflected impaired kinase activation, MAP kinase activity was determined in Rat 1 cells transiently cotransfected with a peptide minigene encoding the ARK1-CT peptide and hemagglutinin-tagged p44 (18). Kinase activity of immunoprecipitated p44 following stimulation was determined to assess MAP kinase activation in the transfected cell pool. As shown in Fig. 4A, results of the p44 kinase assay resembled the findings in 277 cells. LPA and IGF1 receptor-mediated MAP kinase activation was significantly attenuated both by PT treatment and by expression of the ARK1-CT peptide; the EGF receptor signal, as well as that produced by direct activation of PKC, was not affected.


Figure 4: Effects of PT treatment, ARK1-CT peptide, and dominant negative p21 expression on MAP kinase activity in transiently transfected Rat 1 cells. A, Rat 1 cells were transiently transfected with hemagglutinin-tagged p44 (24) with or without a peptide minigene encoding the ARK1-CT peptide (15, 23). The effects of PT treatment and ARK1-CT peptide expression on basal (NS) and agonist-stimulated MAP kinase activity, assessed as phosphorylation of myelin basic protein (MBP) by immunoprecipitated p44, was determined following exposure to LPA, IGF1, EGF, or PMA. B, Rat 1 cells were transiently transfected with hemagglutinin-tagged p44 with or without a plasmid encoding the dominant negative mutant p21 (41). Basal (NS) and agonist-stimulated MAP kinase activity was determined following exposure to LPA, IGF1, EGF, or PMA. MBP phosphorylation is presented in arbitrary units such that 1 unit equals the amount of [P]phosphate incorporated in unstimulated control cells. Data shown represent mean ± S.E. values for three separate experiments performed in duplicate. * signifies value less than control, p < 0.05.



The pathways of both G-coupled receptor- and EGF receptor-mediated mitogenesis can also be discriminated from the PKC-dependent pathway based upon their dependence upon activation of p21 GTP-exchange. As shown in Fig. 4B, transient coexpression of the dominant negative mutant p21 in Rat 1 cells resulted in inhibition of LPA, IGF1, and EGF receptor-stimulated p44 activity, with no significant effect upon phorbol ester-induced activation.


DISCUSSION

We have characterized the activation of MAP kinase by tyrosine kinase growth factor receptors, G-coupled receptors, and phorbol ester in wild-type and mutant Rat 1 fibroblasts. The data suggest that two tyrosine kinase growth factor receptors, those for EGF and IGF1, employ distinct mechanisms of MAP kinase activation. Unlike the well described EGF receptor pathway, activation of MAP kinase by the IGF1 receptor proceeds via a G subunit-dependent activation of p21. This pathway has previously been implicated only in MAP kinase activation mediated by classical G-coupled receptors.

Insulin and IGF1 receptor-mediated p21 activation follows the formation of a protein complex involving the growth factor receptor-binding protein, GRB2, the guanine nucleotide exchange factor, Sos1, and tyrosine-phosphorylated Shc or IRS-1(13, 14, 15) . The molecular mechanism of G subunit-dependent p21 activation is unknown. Recently, however, the G-coupled receptors for thyrotropin-releasing factor (9) and endothelin 1 (26) have been shown to mediate tyrosine phosphorylation of Shc and Shc-GRB2 complex formation and the -thrombin receptor to activate the p60 tyrosine kinase(27) . These observations, along with the data presented here, suggest that G protein-coupled receptors and some tyrosine kinase growth factor receptors may share a common pathway of mitogenic signal transduction.

Interaction between G subunits and the carboxyl terminus of ARK is responsible for agonist-induced translocation of the kinase from the cytosol to the plasma membrane, where it initiates the process of homologous desensitization(23, 24) . It is tempting to speculate that a similar G subunit-mediated protein translocation or localization event, involving some component of the mitogenic signaling complex, facilitates transmission of receptor signals to p21 and MAP kinase. The G subunit-binding carboxyl terminus of ARK contains a region of protein sequence homology, termed a pleckstrin homology (PH) domain (28, 29), which is also shared by several proteins which participate in the regulation of p21. In vitro binding of G subunits to PH domain-containing peptides derived from several of these proteins, including Ras-GRF, Ras-GAP, IRS-1(30) , and the Bruton tyrosine kinase(31) , has been reported. Thus, noncovalent interactions between G subunits and the PH domains of one or more proteins involved in the regulation of p21 may provide the structural basis for G subunit-mediated activation of the MAP kinase pathway. In this regard, it is noteworthy that in transfected Rat 1 cells which stably overexpress human insulin receptors at high density, we have found that insulin treatment provokes a more robust and sustained phosphorylation of MAP kinase which, like the EGF receptor signal, is insensitive to PT treatment or ARK1-CT peptide expression (data not shown). This may reflect an ability of insulin receptors at high density to utilize another, G subunit-independent pathway. Alternatively, it may indicate that G subunits are required for efficient signal transduction at low endogenous levels of receptor expression, but that high receptor density surmounts the requirement for a G subunit-directed protein localization event.

Previous work has suggested that in certain cell types, PT-sensitive G proteins may play a role in several aspects of insulin receptor signaling(32, 33, 34, 35) . The mechanism whereby the insulin or IGF1 receptor might promote the generation of free G subunits is unclear, although indirect evidence suggests that a direct protein-protein interaction may occur between receptor and G protein (36-39). Recently, peptides derived from the insulin receptor subunit have been shown to directly activate G in phospholipid vesicles(40) .

The data in this report suggest that at endogenous levels of receptor expression, the tyrosine kinase IGF1 receptor and G-coupled LPA receptor each require both tyrosine phosphorylation and the release of G subunits for MAP kinase activation. Understanding how mitogenic signals generated by these divergent classes of membrane receptor interact may be of central importance to the development of strategies to modulate cellular proliferative responses to injury such as those mediated by platelet-derived mitogens that bind to G protein-coupled receptors or the accelerated microvascular and macrovascular disease associated with hyperinsulinemia in patients with diabetes mellitus.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant HL 16037. 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.

§
Recipient of a postdoctoral award from the Alberta Heritage Foundation for Medical Research.

To whom correspondence and reprint requests should be addressed. Tel.: 919-684-2974; Fax: 919-684-8875.

The abbreviations used are: MAP, mitogen-activated protein; IGF1, insulin-like growth factor 1; EGF, epidermal growth factor; LPA, lysophosphatidic acid; PT, pertussis toxin; IRS-1, insulin receptor substrate 1; DMEM, Dulbecco's modified Eagle's medium; PMA, phorbol 12-myristate 13-acetate; MBP, myelin basic protein; PKC, protein kinase C; PI, phosphatidylinositol.

T. van Biesen and R. J. Lefkowitz, unpublished observation.


ACKNOWLEDGEMENTS

We thank S. T. Exum for excellent technical assistance and M. Holben and D. Addison for secretarial assistance.


REFERENCES
  1. Schlessinger, J., and Ullrich, A. (1992) Neuron9, 383-391 [Medline] [Order article via Infotrieve]
  2. Medema, R. H., and Bos, J. L. (1993) Crit. Rev. Oncogenesis4, 615-661 [Medline] [Order article via Infotrieve]
  3. Faure, M., Voyno-Yasenetskaya, T. A., and Bourne, H. R. (1994) J. Biol. Chem.269, 7851-7854 [Abstract/Free Full Text]
  4. Howe, L. R., and Marshall, C. J. (1993) J. Biol. Chem.268, 20717-20720 [Abstract/Free Full Text]
  5. Winitz, S., Russell, M., Qian, N.-X., Gardner, A., Dwyer, L., and Johnson, G. L. (1993) J. Biol. Chem.268, 19196-19199 [Abstract/Free Full Text]
  6. Alblas, J., van Corven, E. J., Hordijk, P. L., Milligan, G., and Moolenaar, W. H. (1993) J. Biol. Chem.268, 22235-22238 [Abstract/Free Full Text]
  7. Hordijk, P. L., Verlaan, I., van Corven, E. J., and Moolenaar, W. H. (1994) J. Biol. Chem.269, 645-651 [Abstract/Free Full Text]
  8. van Corven, E. J., Hordijk, P. L., Medema, R. H., Bos, J. L., and Moolenaar, W. H. (1993) Proc. Natl. Acad. Sci. U. S. A.90, 1257-1261 [Abstract]
  9. Omichi, M., Sawada, T., Kanda, Y., Koike, K., Hirota, K., Miyake, A., and Saltiel, A. R. (1994) J. Biol. Chem.269, 3783-3788 [Abstract/Free Full Text]
  10. Koch, W. J., Hawes, B. E., Allen, L. F., and Lefkowitz, R. J. (1994) Proc. Natl. Acad. Sci. U. S. A.91, 12706-12710 [Abstract/Free Full Text]
  11. Crespo, P., Xu, N., Simonds, W. P., and Gutkind, J. S. (1994) Nature369, 418-420 [CrossRef][Medline] [Order article via Infotrieve]
  12. Rosen, O. M. (1987) Science237, 1452-1458 [Medline] [Order article via Infotrieve]
  13. Wang, L. M., Myers, M. G., Jr., Sun, X.-J., Aaronson, S. A., White, M. F., and Pierce, J. H. (1993) Science261, 1591-1594 [Medline] [Order article via Infotrieve]
  14. White, M. F., and Kahn, C. R. (1994) J. Biol. Chem.269, 1-4 [Free Full Text]
  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. Koch, W. J., Hawes, B. E., Inglese, J., Luttrell, L. M., and Lefkowitz, R. J. (1994) J. Biol. Chem.269, 6193-6197 [Abstract/Free Full Text]
  17. Dubler, R. E., Whipple, J. H., and Larner, J. (1985) Arch. Biochem. Biophys.236, 119-129 [Medline] [Order article via Infotrieve]
  18. Meloche, S., Pages, G., and Pouyssegur, J. (1992) Mol. Biol. Cell3, 63-71 [Abstract]
  19. Morgan, D. O., Edman, J. C., Standring, D. N., Fried, V. A., Smith, M. C., Roth, R. A., and Rutter, W. J. (1987) Nature329, 301-307 [CrossRef][Medline] [Order article via Infotrieve]
  20. Okamoto, T., Katada, T., Murayama, Y., Ui, M., Ogata, E., and Nishimoto, I. (1990) Cell62, 709-717 [Medline] [Order article via Infotrieve]
  21. van Obberghen-Schilling, E., and Pouyssegur, J. (1983) Exp. Cell. Res.147, 369-378 [Medline] [Order article via Infotrieve]
  22. Spandidos, D. A., and Wilkie, N. M. (1984) Nature310, 469-475 [Medline] [Order article via Infotrieve]
  23. Pitcher, J. A., Inglese, J., Higgins, J. B., Arriza, J. L., Casey, P. J., Kim, C., Benovic, J. L., Kwatra, M. M., Caron, M. G., and Lefkowitz, R. J. (1992) Science257, 1264-1267 [Medline] [Order article via Infotrieve]
  24. Inglese, J., Koch, W. J., Caron, M. G., and Lefkowitz, R. J. (1992) Nature359, 147-150 [CrossRef][Medline] [Order article via Infotrieve]
  25. Luttrell, L. M., Hawes, B. E., Touhara, K., van Biesen, T., Koch, W. J., and Lefkowitz, R. J. (1995) J. Biol. Chem.270, 12984-12989 [Abstract/Free Full Text]
  26. Cazaubon, S. M., Ramos-Morales, F., Fischer, S., Schweighoffer, F., Strosberg, A. D., and Couraud, P.-O. (1994) J. Biol. Chem.269, 24805-24809 [Abstract/Free Full Text]
  27. Chen, Y.-H., Pouyssegur, J., Courtneidge, S. A., and Van Obberghen-Schilling, E. (1994) J. Biol Chem.269, 27372-27377 [Abstract/Free Full Text]
  28. Mayer, B. J., Ren, R., Clark, K. L., and Baltimore, D. (1993) Cell73, 629-630 [Medline] [Order article via Infotrieve]
  29. Shaw, G. (1993) Biochem. Biophys. Res. Commun.195, 1145-1151 [CrossRef][Medline] [Order article via Infotrieve]
  30. Touhara, K., Inglese, J., Pitcher, J. A., Shaw, G., and Lefkowitz, R. J. (1994) J. Biol. Chem.269, 10217-10220 [Abstract/Free Full Text]
  31. Tsukada, S., Simon, M. I., Witte, O. N., and Katz, A. (1994) Proc. Natl. Acad. Sci. U. S. A.91, 11256-11260 [Abstract/Free Full Text]
  32. Goren, H. J., Northrup, J. K., and Hollenberg, M. D. (1985) Can. J. Physiol. Pharmacol.63, 1017-1022 [Medline] [Order article via Infotrieve]
  33. Heyworth, C. M., Grey, A. M., Wilson, S. R., Hanski, E., and Houslay, M. D. (1986) Biochem. J.235, 145-149 [Medline] [Order article via Infotrieve]
  34. Vila, M. C., Milligan, G., Standaert, M. L., and Farese, R. V. (1990) Biochemistry29, 8735-8740 [Medline] [Order article via Infotrieve]
  35. Luttrell, L. M., Hewlett, E. L., Romero, G., and Rogol, A. D. (1988) J. Biol. Chem.263, 6134-6141 [Abstract/Free Full Text]
  36. Krupinski, J., Rajaram, R., Lakonishok, M., Benovic, J. L., and Cerione, R. A. (1988) J. Biol. Chem.263, 12333-12341 [Abstract/Free Full Text]
  37. Rothenberg, P. L., and Kahn, C. R. (1988) J. Biol. Chem.263, 15546-15552 [Abstract/Free Full Text]
  38. Luttrell, L. M., Kilgour, E., Larner, J., and Romero, G. (1990) J. Biol. Chem.265, 16873-16879 [Abstract/Free Full Text]
  39. Kellerer, M., Obermaier-Kusser, B., Profrock, A., Schleicher, E., Seffer, E., Mushack, J., Ermal, B., and Haring, H.-U. (1991) Biochem. J.276, 103-108 [Medline] [Order article via Infotrieve]
  40. Okamoto, T., Okamoto, T., Murayama, Y., Hayashi, Y., Ogata, E., and Nishimoto, I. (1993) FEBS Lett.334, 143-148 [CrossRef][Medline] [Order article via Infotrieve]
  41. Feig, L. A., and Cooper, G. M. (1991) Mol. Cell. Biol.8, 4822-4829

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