Insulin-like Growth Factor-1 (IGF-1) Receptor-Insulin Receptor Substrate Complexes in the Uterus

• DOI: 10.1074/jbc.273.19.11962
• Publication Date: 2002-07-26T15:04:43Z
• Authors: R. Gregg Richards; Michael P. Walker; Joseph Sebastian; Richard P. DiAugu...
• Keywords: Insulin receptor substrate; IRS2; IRS1

Some of the actions estradiol occur through stimulation growth factor pathways in target organs. Tyrosine-phosphorylated (Tyr(P)) insulin-like factor-1 receptor (IGF-1R) and insulin substrate (IRS)-1 are found uterus mice treated with estradiol. Immunoprecipitates uterine Tyr(P) IRS-1 contained both p85, regulatory subunit phosphatidylinositol (PI) 3-kinase, PI 3-kinase catalytic activity. Estradiol also stimulated binding to IGF-1R. Depletion from extracts reduced associated receptor, which suggests that enzyme IGF-1R occurs primarily a complex contains IRS-1. Following treatment estradiol, formation IGF-1R, IRS-1, p85·IRS-1 was very weak IGF-1m/m mice, severely deficient IGF-1. This indicated most, if not all, estradiol-stimulated Tyr phosphorylation originates ligand activation kinase. IRS-2 Tyr-phosphorylated normal bound more p85 response estradiol; however, marked decrease levels occurred 12–24 h after Since present precipitates recombinant form IGF-1 (long R3IGF-1) IRS-2, hormonal this docking protein probably In summary, our findings show estrogen kinase results enhanced (PI 3-kinase) IRS-2. The one or these complexes may be important for potent mitogenic action steroid. That but proteins have different roles hormone-induced signaling uterus. exerts effects on gene expression cell proliferation Several earlier studies local (reviewed Refs. 1Murphy L.J. Ghahary A. Endocrine Rev. 1990; 11: 443-453Crossref PubMed Scopus (281) Google Scholar, 2Brigstock D.R. Robertson D.M. Herington A.C. Bailliere's Clinical Endocrinology Metabolism: Growth Factors Endocrinology. 5. Bailliere Tindall, London1991: 791-808Google 3Giudice L.C. Fertil. Steril. 1994; 61: 1-17Abstract Full Text PDF (327) Scholar). 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Wong A.J. 379: 560-564Crossref (594) modules functional properties similar those Tyrosine sites IRS provide motifs distinct proteins, Grb-2 27Sun 28Lavan 30Lavan 31Holgado-Madruga 32Sun Crimmins D.L. Miralpeix Cell. 13: 7418-7428Crossref Shc another (33Sasaoka Draznin 269: 13689-13694Abstract When tyrosine-phosphorylated, (34Skolnik E.Y. Batzer Li C.H. Lowenstein Mohammadi Margolis Schlessinger 260: 1953-1955Crossref (498) which, turn, GTP exchange Sos activate Ras downstream mitogen-activated cascade (35Lowenstein E.J. Daly R.J. A.G. Lammers Ullrich 70: 431-442Abstract (1326) 36Rozakis-Adcock Fernley Wade Bowtell 363: 83-85Crossref (834) To identify immediately ligand-activated following exposure uterus, tissues were examined We utilized profoundly growth-retarded due low (37Lembo G. Rockman H.A. Hunter Steinmetz Koch W.J. Ma Prinz Ross Chien K.R. Powell-Braxton Invest. 98: 2648-2655Crossref (114) evaluate relative importance mediating hormone (p85), Grb-2, Syp determined association Finally, changes phosphorylation, interaction, SH2-containing 17β-Estradiol, l-α-PI,l-α-PI-4-monophosphate, ATP purchased Sigma. Long R3 obtained Diagnostic Systems Laboratories, Inc. (Webster, TX). Protein A-Sepharose Amersham Pharmacia Biotech. precast minigels electrophoresis NOVEX (San Diego, CA) Bio-Rad. Prestained molecular weight standards Bio-Rad NOVEX, polyvinylidene fluoride membrane (Immobilon-P) Millipore Corp. (Bedford, MA). Reagents chemiluminescence, Hyperfilm-ECL [32P]ATP Silica (LHPKDF 60-Å) TLC plates Whatman (Maidstone, United Kingdom). Polyclonal antibodies against extreme C terminus (amino acids 1222–1235) (number 06–248), glutathione S-transferase fusion containing residues 6–125 pleckstrin 06–524), 976–1094 human Upstate Biotechnology, (Lake Placid, NY). Antiserum raised amino 618–747 gift Morris (Joslin Diabetes Center, Boston, (β-subunit), SHP-2 Grb-10 Santa Cruz (Santa Cruz, CA). Horseradish peroxidase-conjugated anti-phosphotyrosine monoclonal antibody (PY20) ICN (Costa Mesa, Agarose-conjugated anti-Tyr(P) Calbiochem. donkey anti-rabbit IgG Biotech; rabbit Vector Laboratories (Burlingame, Procedures experimental animals followed guidelines NIEHS, National Institutes Health, Animal Care Use Committee. CD-1 (Charles River Laboratories) ovariectomized at 76–82 days age 13–48 castration. Mice homozygous (IGF-1m/m) site-specific insertional mutation igf1 allele Lyn (Genentech, Inc.; San Francisco, These 36% smaller than their counterparts contrast null mutants (38Liu J.-P. Baker Perkins A.S. 75: 59-72Abstract (2536) 39Baker 73-82Abstract (2019) 40Powell-Braxton Hollingshead Warburton Dowd Pitts-Meek Dalton Gillett Stewart Genes Dev. 7: 2609-2617Crossref (663) viable fertile, they produce small amount Serum 30% values 100–135 14–47 Each subcutaneously either single injection 1 μg 0.1 ml PBS, 1% ethanol three successive injections, 5-min intervals, 200 long 0.2% bovine albumin. injected (20 μg/kg) ethanol. Uterine all collected times treatment; controls received vehicle only. Tissue homogenized 4 °C previously After centrifugation homogenates 21,000 × g min, aliquots supernatants (2–4 protein/μl) boiled Laemmli sample buffer 5 equivalent volume (10 μl) each evaluated Western blot analysis detailed below. concentrations BCA assay. additional (400–800 protein), reported using specific (5 μg), antiserum (1:80), nonspecific μg). immunoprecipitates resolved SDS-PAGE transferred membrane. blocked Tris-buffered saline, 0.1% Tween 20, 5% albumin 3% nonfat dry milk probed appropriate antibodies. Immunoreactive detected chemiluminescence. Supernatants estradiol- vehicle-treated incubated anti-IRS-1 (C terminus) IgG. adsorption immune A-Sepharose, immunoprecipitation precipitated again second round immunoprecipitation, anti-IGF-1R antibody. All subjected analysis, activity assay (19Myers 6 washed successively Triton X-100, 100 μm Na3VO4 PBS (three times); 500 mm LiCl, μmNa3VO4 Tris-HCl, pH 7.5 NaCl, EDTA, 10 mmTris-HCl, (two times). pellets resuspended 50 μl EDTA 7.5, combined mmMgCl2 (4 μg/μl) sonicated EGTA 7.5. reaction started adding 440 300 μCi [γ-32P]ATP. min 22 constant shaking Eppendorf thermomixer, stopped 20 8 n HCl 160 CHCl3:methanol (1:1). centrifugation, lower organic phase recovered and, along PI-4-monophosphate standard, applied silica plate. developed CHCl3/CH3OH/H2O/NH4OH (99:78:18:5.3), dried, visualized autoradiography.32P incorporation into PI-3-monophosphate quantified PhosphorImager (Molecular Dynamics, Inc.). juxtamembrane motif (NPEY) result ligand-induced (41Tartare-Deckert 23456-23460Abstract (103) induces coordinated β-subunit we whether steroid stimulates Immunoblot (β-subunit) showed increased 110 170 kDa; 185-kDa evident 12 (Fig. A). 110-kDa treatment, whereas 170- still 72 revealed 110- 170-kDa correspond respectively identity coprecipitated has been determined. At time period examined, there no apparent change As (Fig.1 B). control (0 h), content displayed mobility kDa shifted 48 h, migration observed h) samples comparable throughout investigated IGF-1R·IRS-1 since potential tyrosine-phosphorylate wanted determine catalyzed predominantly Therefore, mice. exposure, (Fig.2 A) B) much less Although negligible mutants, immunoreactive band exhibited same hormone-dependent wild types uteri vehicle- estradiol-treated mutant weights about 2-fold hormone. sections groups light microscopy, gross difference histology observed. action, possible interactions between proteins. IGF-1R- IRS-1-associated samples, (Fig.3 exposure. Additionally, immunoreactivity 3 C). There interaction (6 (Fig.4 A); did (data shown). estrogen-enhanced lack confirmed derived regions molecule, region (Figs. 3, andB, N shown).Figure 4Grb-2 do prepared castrates (E) (C).Panel A, (IP) (Ext) separated SDS-15% PAGE immunoblotted Syp. (panel C) SDS-7.5% phosphotyrosine (P-Tyr), panel doublet precipitates, (∼25 kDa) migrated slightly faster (∼27 ut