• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of embojLink to Publisher's site
EMBO J. Sep 15, 1994; 13(18): 4269–4277.
PMCID: PMC395354

Compartmentalization of SHC, GRB2 and mSOS, and hyperphosphorylation of Raf-1 by EGF but not insulin in liver parenchyma.


Rat liver parenchyma harbors equal numbers of epidermal growth factor (EGF) and insulin receptors. Following administration of a saturating dose of EGF (10 micrograms/100 g body weight), there was a rapid (t1/2 approximately 1.1 min) internalization of receptor coincident with its tyrosine phosphorylation at residue 1173 and receptor recruitment of the adaptor protein SHC, its tyrosine phosphorylation and its association with GRB2 and the Ras guanine nucleotide exchange factor, mSOS, largely in endosomes. This led to a cytosolic pool of a complex of tyrosine-phosphorylated SHC, GRB2 and mSOS. It was demonstrated that these constituents were linked to Ras activation by the characteristic decrease in Raf-1 mobility on SDS-PAGE, which was maintained for 60 min after a single bolus of administered EGF. While insulin administration (15 micrograms/100 g body weight) led to insulin receptor beta-subunit tyrosine phosphorylation and internalization, there was little detectable tyrosine phosphorylation of SHC, recruitment of GRB2, association of a complex with mSOS or any detectable change in the mobility of Raf-1. Therefore, in normal physiological target cells in vivo, distinct signaling pathways are realized after EGF or insulin receptor activation, with regulation of this specificity most probably occurring at the locus of the endosome.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (2.4M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Authier F, Rachubinski RA, Posner BI, Bergeron JJ. Endosomal proteolysis of insulin by an acidic thiol metalloprotease unrelated to insulin degrading enzyme. J Biol Chem. 1994 Jan 28;269(4):3010–3016. [PubMed]
  • Backer JM, Kahn CR, White MF. The dissociation and degradation of internalized insulin occur in the endosomes of rat hepatoma cells. J Biol Chem. 1990 Sep 5;265(25):14828–14835. [PubMed]
  • Baltensperger K, Kozma LM, Cherniack AD, Klarlund JK, Chawla A, Banerjee U, Czech MP. Binding of the Ras activator son of sevenless to insulin receptor substrate-1 signaling complexes. Science. 1993 Jun 25;260(5116):1950–1952. [PubMed]
  • Bangalore L, Tanner AJ, Laudano AP, Stern DF. Antiserum raised against a synthetic phosphotyrosine-containing peptide selectively recognizes p185neu/erbB-2 and the epidermal growth factor receptor. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11637–11641. [PMC free article] [PubMed]
  • Batzer AG, Rotin D, Ureña JM, Skolnik EY, Schlessinger J. Hierarchy of binding sites for Grb2 and Shc on the epidermal growth factor receptor. Mol Cell Biol. 1994 Aug;14(8):5192–5201. [PMC free article] [PubMed]
  • Baudhuin P, Evrard P, Berthet J. Electron microscopic examination of subcellular fractions. I. The preparation of representative samples from suspensions of particles. J Cell Biol. 1967 Jan;32(1):181–191. [PMC free article] [PubMed]
  • Bergeron JJ, Rachubinski R, Searle N, Borts D, Sikstrom R, Posner BI. Polypeptide hormone receptors in vivo: demonstration of insulin binding to adrenal gland and gastrointestinal epithelium by quantitative radioautography. J Histochem Cytochem. 1980 Aug;28(8):824–835. [PubMed]
  • Bergeron JJ, Searle N, Khan MN, Posner BI. Differential and analytical subfractionation of rat liver components internalizing insulin and prolactin. Biochemistry. 1986 Apr 8;25(7):1756–1764. [PubMed]
  • Buday L, Downward J. Epidermal growth factor regulates p21ras through the formation of a complex of receptor, Grb2 adapter protein, and Sos nucleotide exchange factor. Cell. 1993 May 7;73(3):611–620. [PubMed]
  • Burgess JW, Wada I, Ling N, Khan MN, Bergeron JJ, Posner BI. Decrease in beta-subunit phosphotyrosine correlates with internalization and activation of the endosomal insulin receptor kinase. J Biol Chem. 1992 May 15;267(14):10077–10086. [PubMed]
  • Chang CP, Lazar CS, Walsh BJ, Komuro M, Collawn JF, Kuhn LA, Tainer JA, Trowbridge IS, Farquhar MG, Rosenfeld MG, et al. Ligand-induced internalization of the epidermal growth factor receptor is mediated by multiple endocytic codes analogous to the tyrosine motif found in constitutively internalized receptors. J Biol Chem. 1993 Sep 15;268(26):19312–19320. [PubMed]
  • Chardin P, Camonis JH, Gale NW, van Aelst L, Schlessinger J, Wigler MH, Bar-Sagi D. Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2. Science. 1993 May 28;260(5112):1338–1343. [PubMed]
  • Cohen S, Fava RA, Sawyer ST. Purification and characterization of epidermal growth factor receptor/protein kinase from normal mouse liver. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6237–6241. [PMC free article] [PubMed]
  • Doherty JJ, 2nd, Kay DG, Lai WH, Posner BI, Bergeron JJ. Selective degradation of insulin within rat liver endosomes. J Cell Biol. 1990 Jan;110(1):35–42. [PMC free article] [PubMed]
  • Donaldson RW, Cohen S. Epidermal growth factor stimulates tyrosine phosphorylation in the neonatal mouse: association of a M(r) 55,000 substrate with the receptor. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8477–8481. [PMC free article] [PubMed]
  • Downward J, Parker P, Waterfield MD. Autophosphorylation sites on the epidermal growth factor receptor. Nature. 1984 Oct 4;311(5985):483–485. [PubMed]
  • Egan SE, Giddings BW, Brooks MW, Buday L, Sizeland AM, Weinberg RA. Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation. Nature. 1993 May 6;363(6424):45–51. [PubMed]
  • Faure R, Baquiran G, Bergeron JJ, Posner BI. The dephosphorylation of insulin and epidermal growth factor receptors. Role of endosome-associated phosphotyrosine phosphatase(s). J Biol Chem. 1992 Jun 5;267(16):11215–11221. [PubMed]
  • Felder S, Miller K, Moehren G, Ullrich A, Schlessinger J, Hopkins CR. Kinase activity controls the sorting of the epidermal growth factor receptor within the multivesicular body. Cell. 1990 May 18;61(4):623–634. [PubMed]
  • Gale NW, Kaplan S, Lowenstein EJ, Schlessinger J, Bar-Sagi D. Grb2 mediates the EGF-dependent activation of guanine nucleotide exchange on Ras. Nature. 1993 May 6;363(6424):88–92. [PubMed]
  • Geuze HJ, Slot JW, Strous GJ, Peppard J, von Figura K, Hasilik A, Schwartz AL. Intracellular receptor sorting during endocytosis: comparative immunoelectron microscopy of multiple receptors in rat liver. Cell. 1984 May;37(1):195–204. [PubMed]
  • Hsuan JJ, Totty N, Waterfield MD. Identification of a novel autophosphorylation site (P4) on the epidermal growth factor receptor. Biochem J. 1989 Sep 1;262(2):659–663. [PMC free article] [PubMed]
  • Jhappan C, Stahle C, Harkins RN, Fausto N, Smith GH, Merlino GT. TGF alpha overexpression in transgenic mice induces liver neoplasia and abnormal development of the mammary gland and pancreas. Cell. 1990 Jun 15;61(6):1137–1146. [PubMed]
  • Kahn CR, Freychet P, Roth J, Neville DM., Jr Quantitative aspects of the insulin-receptor interaction in liver plasma membranes. J Biol Chem. 1974 Apr 10;249(7):2249–2257. [PubMed]
  • Karnovsky MJ. The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J Cell Biol. 1967 Oct;35(1):213–236. [PMC free article] [PubMed]
  • Kay DG, Lai WH, Uchihashi M, Khan MN, Posner BI, Bergeron JJ. Epidermal growth factor receptor kinase translocation and activation in vivo. J Biol Chem. 1986 Jun 25;261(18):8473–8480. [PubMed]
  • Kelly KL, Ruderman NB. Insulin-stimulated phosphatidylinositol 3-kinase. Association with a 185-kDa tyrosine-phosphorylated protein (IRS-1) and localization in a low density membrane vesicle. J Biol Chem. 1993 Feb 25;268(6):4391–4398. [PubMed]
  • Khan MN, Savoie S, Bergeron JJ, Posner BI. Characterization of rat liver endosomal fractions. In vivo activation of insulin-stimulable receptor kinase in these structures. J Biol Chem. 1986 Jun 25;261(18):8462–8472. [PubMed]
  • Khan MN, Baquiran G, Brule C, Burgess J, Foster B, Bergeron JJ, Posner BI. Internalization and activation of the rat liver insulin receptor kinase in vivo. J Biol Chem. 1989 Aug 5;264(22):12931–12940. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Lai WH, Cameron PH, Wada I, Doherty JJ, 2nd, Kay DG, Posner BI, Bergeron JJ. Ligand-mediated internalization, recycling, and downregulation of the epidermal growth factor receptor in vivo. J Cell Biol. 1989 Dec;109(6 Pt 1):2741–2749. [PMC free article] [PubMed]
  • Lai WH, Cameron PH, Doherty JJ, 2nd, Posner BI, Bergeron JJ. Ligand-mediated autophosphorylation activity of the epidermal growth factor receptor during internalization. J Cell Biol. 1989 Dec;109(6 Pt 1):2751–2760. [PMC free article] [PubMed]
  • Li N, Batzer A, Daly R, Yajnik V, Skolnik E, Chardin P, Bar-Sagi D, Margolis B, Schlessinger J. Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling. Nature. 1993 May 6;363(6424):85–88. [PubMed]
  • Lowenstein EJ, Daly RJ, Batzer AG, Li W, Margolis B, Lammers R, Ullrich A, Skolnik EY, Bar-Sagi D, Schlessinger J. The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling. Cell. 1992 Aug 7;70(3):431–442. [PubMed]
  • Margolis BL, Lax I, Kris R, Dombalagian M, Honegger AM, Howk R, Givol D, Ullrich A, Schlessinger J. All autophosphorylation sites of epidermal growth factor (EGF) receptor and HER2/neu are located in their carboxyl-terminal tails. Identification of a novel site in EGF receptor. J Biol Chem. 1989 Jun 25;264(18):10667–10671. [PubMed]
  • Marti U, Burwen SJ, Jones AL. Biological effects of epidermal growth factor, with emphasis on the gastrointestinal tract and liver: an update. Hepatology. 1989 Jan;9(1):126–138. [PubMed]
  • McCormick F. Signal transduction. How receptors turn Ras on. Nature. 1993 May 6;363(6424):15–16. [PubMed]
  • Mead JE, Fausto N. Transforming growth factor alpha may be a physiological regulator of liver regeneration by means of an autocrine mechanism. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1558–1562. [PMC free article] [PubMed]
  • Morrison DK, Kaplan DR, Escobedo JA, Rapp UR, Roberts TM, Williams LT. Direct activation of the serine/threonine kinase activity of Raf-1 through tyrosine phosphorylation by the PDGF beta-receptor. Cell. 1989 Aug 25;58(4):649–657. [PubMed]
  • Mortimore GE, King E, Jr, Mondon CE, Glinsmann WH. Effects of insulin on net carbohydrate alterations in perfused rat liver. Am J Physiol. 1967 Jan;212(1):179–183. [PubMed]
  • O'Keefe E, Hollenberg MD, Cuatrecasas P. Epidermal growth factor. Characteristics of specific binding in membranes from liver, placenta, and other target tissues. Arch Biochem Biophys. 1974 Oct;164(2):518–526. [PubMed]
  • Olivier JP, Raabe T, Henkemeyer M, Dickson B, Mbamalu G, Margolis B, Schlessinger J, Hafen E, Pawson T. A Drosophila SH2-SH3 adaptor protein implicated in coupling the sevenless tyrosine kinase to an activator of Ras guanine nucleotide exchange, Sos. Cell. 1993 Apr 9;73(1):179–191. [PubMed]
  • Pelicci G, Lanfrancone L, Grignani F, McGlade J, Cavallo F, Forni G, Nicoletti I, Grignani F, Pawson T, Pelicci PG. A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction. Cell. 1992 Jul 10;70(1):93–104. [PubMed]
  • Pronk GJ, McGlade J, Pelicci G, Pawson T, Bos JL. Insulin-induced phosphorylation of the 46- and 52-kDa Shc proteins. J Biol Chem. 1993 Mar 15;268(8):5748–5753. [PubMed]
  • Rothenberg PL, Lane WS, Karasik A, Backer J, White M, Kahn CR. Purification and partial sequence analysis of pp185, the major cellular substrate of the insulin receptor tyrosine kinase. J Biol Chem. 1991 May 5;266(13):8302–8311. [PubMed]
  • Rozakis-Adcock M, McGlade J, Mbamalu G, Pelicci G, Daly R, Li W, Batzer A, Thomas S, Brugge J, Pelicci PG, et al. Association of the Shc and Grb2/Sem5 SH2-containing proteins is implicated in activation of the Ras pathway by tyrosine kinases. Nature. 1992 Dec 17;360(6405):689–692. [PubMed]
  • Rozakis-Adcock M, Fernley R, Wade J, Pawson T, Bowtell D. The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1. Nature. 1993 May 6;363(6424):83–85. [PubMed]
  • Ruff-Jamison S, McGlade J, Pawson T, Chen K, Cohen S. Epidermal growth factor stimulates the tyrosine phosphorylation of SHC in the mouse. J Biol Chem. 1993 Apr 15;268(11):7610–7612. [PubMed]
  • Simionescu N, Simionescu M. Galloylglucoses of low molecular weight as mordant in electron microscopy. I. Procedure, and evidence for mordanting effect. J Cell Biol. 1976 Sep;70(3):608–621. [PMC free article] [PubMed]
  • Simon MA, Dodson GS, Rubin GM. An SH3-SH2-SH3 protein is required for p21Ras1 activation and binds to sevenless and Sos proteins in vitro. Cell. 1993 Apr 9;73(1):169–177. [PubMed]
  • Skolnik EY, Batzer A, Li N, Lee CH, Lowenstein E, Mohammadi M, Margolis B, Schlessinger J. The function of GRB2 in linking the insulin receptor to Ras signaling pathways. Science. 1993 Jun 25;260(5116):1953–1955. [PubMed]
  • Skolnik EY, Lee CH, Batzer A, Vicentini LM, Zhou M, Daly R, Myers MJ, Jr, Backer JM, Ullrich A, White MF, et al. The SH2/SH3 domain-containing protein GRB2 interacts with tyrosine-phosphorylated IRS1 and Shc: implications for insulin control of ras signalling. EMBO J. 1993 May;12(5):1929–1936. [PMC free article] [PubMed]
  • Smith DE, Fisher PA. Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in Drosophila embryos: application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots. J Cell Biol. 1984 Jul;99(1 Pt 1):20–28. [PMC free article] [PubMed]
  • Sorkin A, Carpenter G. Interaction of activated EGF receptors with coated pit adaptins. Science. 1993 Jul 30;261(5121):612–615. [PubMed]
  • Ueki K, Matsuda S, Tobe K, Gotoh Y, Tamemoto H, Yachi M, Akanuma Y, Yazaki Y, Nishida E, Kadowaki T. Feedback regulation of mitogen-activated protein kinase kinase kinase activity of c-Raf-1 by insulin and phorbol ester stimulation. J Biol Chem. 1994 Jun 3;269(22):15756–15761. [PubMed]
  • Wada I, Lai WH, Posner BI, Bergeron JJ. Association of the tyrosine phosphorylated epidermal growth factor receptor with a 55-kD tyrosine phosphorylated protein at the cell surface and in endosomes. J Cell Biol. 1992 Jan;116(2):321–330. [PMC free article] [PubMed]
  • Walton GM, Chen WS, Rosenfeld MG, Gill GN. Analysis of deletions of the carboxyl terminus of the epidermal growth factor receptor reveals self-phosphorylation at tyrosine 992 and enhanced in vivo tyrosine phosphorylation of cell substrates. J Biol Chem. 1990 Jan 25;265(3):1750–1754. [PubMed]
  • Wood KW, Sarnecki C, Roberts TM, Blenis J. ras mediates nerve growth factor receptor modulation of three signal-transducing protein kinases: MAP kinase, Raf-1, and RSK. Cell. 1992 Mar 20;68(6):1041–1050. [PubMed]

Articles from The EMBO Journal are provided here courtesy of The European Molecular Biology Organization


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • Compound
    PubChem Compound links
  • MedGen
    Related information in MedGen
  • Pathways + GO
    Pathways + GO
    Pathways, annotations and biological systems (BioSystems) that cite the current article.
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...