Title: Insulin Stimulates Sequestration of β-Adrenergic Receptors and Enhanced Association of β-Adrenergic Receptors with Grb2 via Tyrosine 350
Abstract: G-protein-linked receptors, such as the β2-adrenergic receptor, are substrates for growth factor receptors with intrinsic tyrosine kinase activity (Karoor, V., Baltensperger, K., Paul, H., Czech, M. P., and Malbon C. C. (1995) J. Biol. Chem. 270, 25305–25308). In the present work, the counter-regulatory action of insulin on catecholamine action is shown to stimulate enhanced sequestration of β2-adrenergic receptors in either DDT1MF-2 smooth muscle cells or Chinese hamster ovary cells stably expressing β2-adrenergic receptors. Both insulin and insulin-like growth factor-1 stimulate internalization of β-adrenergic receptors, contributing to the counter-regulatory effects of these growth factors on catecholamine action. In combination with β-adrenergic agonists, insulin stimulates internalization of 50–60% of the complement of β-adrenergic receptors. Insulin administration in vitroand in vivo stimulates phosphorylation of Tyr-350 of the β-adrenergic receptor, creating an Src homology 2 domain available for binding of the adaptor molecule Grb2. The association of Grb2 with β-adrenergic receptors was established using antibodies to Grb2 as well as a Grb2-glutathione S-transferase fusion protein. Insulin treatment of cells provokes binding of Grb2 to β2-adrenergic receptors. Insulin also stimulates association of phosphatidylinositol 3-kinase and dynamin, via the Src homology 3 domain of Grb2. Both these interactions as well as internalization of the β-adrenergic receptor are shown to be enhanced by insulin, β-agonist, or both. The Tyr-350 → Phe mutant form of the β2-adrenergic receptor, lacking the site for tyrosine phosphorylation, fails to bind Grb2 in response to insulin, fails to display internalization of β2-adrenergic receptor in response to insulin, and is no longer subject to the counter-regulatory effects of insulin on cyclic AMP accumulation. These data are the first to demonstrate the ability of a growth factor insulin to counter-regulate G-protein-linked receptor, the β-adrenergic receptor, via a new mechanism, i.e. internalization. G-protein-linked receptors, such as the β2-adrenergic receptor, are substrates for growth factor receptors with intrinsic tyrosine kinase activity (Karoor, V., Baltensperger, K., Paul, H., Czech, M. P., and Malbon C. C. (1995) J. Biol. Chem. 270, 25305–25308). In the present work, the counter-regulatory action of insulin on catecholamine action is shown to stimulate enhanced sequestration of β2-adrenergic receptors in either DDT1MF-2 smooth muscle cells or Chinese hamster ovary cells stably expressing β2-adrenergic receptors. Both insulin and insulin-like growth factor-1 stimulate internalization of β-adrenergic receptors, contributing to the counter-regulatory effects of these growth factors on catecholamine action. In combination with β-adrenergic agonists, insulin stimulates internalization of 50–60% of the complement of β-adrenergic receptors. Insulin administration in vitroand in vivo stimulates phosphorylation of Tyr-350 of the β-adrenergic receptor, creating an Src homology 2 domain available for binding of the adaptor molecule Grb2. The association of Grb2 with β-adrenergic receptors was established using antibodies to Grb2 as well as a Grb2-glutathione S-transferase fusion protein. Insulin treatment of cells provokes binding of Grb2 to β2-adrenergic receptors. Insulin also stimulates association of phosphatidylinositol 3-kinase and dynamin, via the Src homology 3 domain of Grb2. Both these interactions as well as internalization of the β-adrenergic receptor are shown to be enhanced by insulin, β-agonist, or both. The Tyr-350 → Phe mutant form of the β2-adrenergic receptor, lacking the site for tyrosine phosphorylation, fails to bind Grb2 in response to insulin, fails to display internalization of β2-adrenergic receptor in response to insulin, and is no longer subject to the counter-regulatory effects of insulin on cyclic AMP accumulation. These data are the first to demonstrate the ability of a growth factor insulin to counter-regulate G-protein-linked receptor, the β-adrenergic receptor, via a new mechanism, i.e. internalization. G-protein-linked receptor growth factor receptor with intrinsic tyrosine kinase activity β2-adrenergic receptor phosphatidylinositol 3-kinase Src homology Dulbecco's modified Eagle's medium insulin receptor substrate polyacrylamide gel electrophoresis insulin-like growth factor Chinese hamster ovary glutathione S-transferase. G-protein-linked receptors (GPLRs)1 and growth factor receptors with intrinsic tyrosine kinase activity (TKR) represent two prominent pathways for cellular signaling (1Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4611) Google Scholar, 2Hausdorff W.P. Caron M.G. Lefkowitz R.J. FASEB J. 1990; 4: 2881-2889Crossref PubMed Scopus (1087) Google Scholar). Study of the integration of signaling between GPLR and TKR pathways has revealed recently the existence of cross-talk at the most proximal point, receptor-to-receptor interaction with GPLRs acting as substrates for TKRs (3Hadcock J.R. Port J.D. Gelman M.S. Malbon C.C. J. Biol. Chem. 1992; 267: 26017-26022Abstract Full Text PDF PubMed Google Scholar, 4Karoor V. Baltensperger K. Paul H. Czech M.P. Malbon C.C. J. Biol. Chem. 1995; 270: 25305-25308Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 5Baltensperger K. Karoor V. Paul H. Ruoho A. Czech M.P. Malbon C.C. J. Biol. Chem. 1996; 271: 1061-1065Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Insulin stimulates the phosphorylation of the β2-adrenergic receptor (β2AR) on tyrosyl residues 350/354 and 364, both in vivo (3Hadcock J.R. Port J.D. Gelman M.S. Malbon C.C. J. Biol. Chem. 1992; 267: 26017-26022Abstract Full Text PDF PubMed Google Scholar, 4Karoor V. Baltensperger K. Paul H. Czech M.P. Malbon C.C. J. Biol. Chem. 1995; 270: 25305-25308Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and in vitro (5Baltensperger K. Karoor V. Paul H. Ruoho A. Czech M.P. Malbon C.C. J. Biol. Chem. 1996; 271: 1061-1065Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) using recombinant, purified β2AR and insulin receptors. Tyrosyl residue 350, a prominent residue for insulin receptor-catalyzed phosphorylation, is embedded in a sequence motif (Tyr-Gly-Asn-Gly) which is similar to the motifs known to interact withCaenorhabditis elegans sem5 Src homology 2 (SH2) domains when phosphorylated (6Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.J. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2384) Google Scholar). Phosphorylation of sites on the β2AR by the insulin receptor and the IGF-1 receptor include a motif for TKR at Tyr-364 (7Geahlen R.L. Harrison M.L. Peptides and Protein Phosphorylation. CRC Press, Boca Raton, FL1989: 239-254Google Scholar), the Grb2 binding site at Tyr-350 (4Karoor V. Baltensperger K. Paul H. Czech M.P. Malbon C.C. J. Biol. Chem. 1995; 270: 25305-25308Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 6Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.J. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2384) Google Scholar), and a potential SHC binding site at Tyr-132 (4Karoor V. Baltensperger K. Paul H. Czech M.P. Malbon C.C. J. Biol. Chem. 1995; 270: 25305-25308Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 5Baltensperger K. Karoor V. Paul H. Ruoho A. Czech M.P. Malbon C.C. J. Biol. Chem. 1996; 271: 1061-1065Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 8Songyang Z. Shoelson S.E. McGlade J. Oliver P. Pawson T. Bustelo X.R. Barbacid M. Sabe H. Hanafusa H. Yi T. Ren R. Baltimore D. Ratnofsky S. Feldman R.A. Cantley L.C. Mol. Cell. Biol. 1994; 14: 2777-2785Crossref PubMed Scopus (833) Google Scholar). For insulin action, activation of phosphatidylinositol 3-kinase (PI 3-kinase) is an early event, following temporally the phosphorylation of the insulin receptor and IRS-1 (for reviews, see Refs. 9Myers M. Sun X.J. White M. Trends Biochem. Sci. 1994; 19: 289-292Abstract Full Text PDF PubMed Scopus (290) Google Scholar, 10Quon M. Butte A. Taylor S. Trends Endocrinol. Metab. 1994; 5: 369-376Abstract Full Text PDF PubMed Scopus (48) Google Scholar, 11White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar). For the insulin pathway, the p85 regulatory subunit of PI 3-kinase binds the IRS-1 via SH2 domain(s), activating the catalytic p110 subunit, which phosphorylates various phosphoinositides at the 3′-position of the inositol ring (12Backer J. Myers M. Shoelson S. Chin D. Sun X.J. Miralpeix M. Hu P. Margolis B. Skolnik E. Schlessinger J. White M. EMBO J. 1992; 11: 3469-3479Crossref PubMed Scopus (820) Google Scholar, 13Rordorf-Nikolic T. Van Horn D.J. Chen D. White M.F. Backer J.M. J. Biol. Chem. 1995; 270: 3662-3666Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Ample reports support the premise that PI 3-kinase and its 3′-phosphoinositide products are critical to intracellular trafficking of membrane-bound elements in general (14Carlberg K. Tapley P. Haystead C. Rohrschneider L. EMBO J. 1991; 10: 877-883Crossref PubMed Scopus (54) Google Scholar,15Schu P. Takegawa K. Fry M. Stack J. Waterfield M. Emr S. Science. 1993; 260: 88-91Crossref PubMed Scopus (806) Google Scholar) and of downstream elements of TKR signaling, particularly insulin (16Okada T. Kawano Y. Sakakibara T. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3568-3573Abstract Full Text PDF PubMed Google Scholar, 17Cheatham B. Vlahos C.J. Cheatham L. Wang L. Blenis J. Kahn C.R. Mol. Cell. Biol. 1994; 14: 4902-4911Crossref PubMed Scopus (1001) Google Scholar, 18Heller-Harrison R.A. Morin M. Guilherme A. Czech M.P. J. Biol. Chem. 1996; 271: 10200-10204Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Dynamin, a GTPase regulating formation and trafficking of clathrin-coated vesicles (19Shpetner H.S. Vallee R.B. Cell. 1989; 59: 421-432Abstract Full Text PDF PubMed Scopus (341) Google Scholar, 20Shpetner H.S. Vallee R.B. Nature. 1992; 355: 733-735Crossref PubMed Scopus (171) Google Scholar, 21Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1039) Google Scholar, 22Shpetner H.S. Herskovitz J.S. Valee R.B. J. Biol. Chem. 1996; 271: 13-16Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), plays a key role in the sequestration of β-adrenergic receptors in response to chronic stimulation with β-adrenergic agonist (23Zhang J. Feguson S.S.G. Barak L.S. Menard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). Based upon our findings that insulin receptor catalyzes the phosphorylation of Tyr-350 of the β2AR in an insulin-dependent manner and thereby creates an SH2 domain capable binding Grb2 (3Hadcock J.R. Port J.D. Gelman M.S. Malbon C.C. J. Biol. Chem. 1992; 267: 26017-26022Abstract Full Text PDF PubMed Google Scholar, 4Karoor V. Baltensperger K. Paul H. Czech M.P. Malbon C.C. J. Biol. Chem. 1995; 270: 25305-25308Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 5Baltensperger K. Karoor V. Paul H. Ruoho A. Czech M.P. Malbon C.C. J. Biol. Chem. 1996; 271: 1061-1065Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), we hypothesized that PI 3-kinase and dynamin may play a role in intracellular trafficking of the β2AR and perhaps an internalization of this G-protein-linked receptor as a component of the counter-regulatory effects of insulin with respect to catecholamine action. Our data are the first to demonstrate insulin-stimulated internalization of β-adrenergic receptors and associations between β2AR and both dynamin and PI 3-kinase via Grb2 binding that are dependent upon prior insulin receptor-catalyzed phosphorylation of tyrosyl residue at position 350 of the β2AR. Furthermore, insulin is shown to enhance β-adrenergic agonist-induced internalization of β2ARs in a manner that is sensitive to inhibition of PI 3-kinase or to substitution of phenylalanine for tyrosine at position 350 of the receptor (Y350F). DDT1 MF-2 hamster vas deferens smooth muscle cells and Chinese hamster ovary (CHO) cells stably expressing wild-type and mutant β2ARs (4Karoor V. Baltensperger K. Paul H. Czech M.P. Malbon C.C. J. Biol. Chem. 1995; 270: 25305-25308Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 5Baltensperger K. Karoor V. Paul H. Ruoho A. Czech M.P. Malbon C.C. J. Biol. Chem. 1996; 271: 1061-1065Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine serum. Recombinant insulin receptors were purified from CHO-T cells overexpressing the insulin receptor by affinity chromatography on wheat germ agglutinin coupled to agarose, as described (24Baltensperger K. Lewis R.E. Woon C.W. Vissavajjhala P. Ross A.H. Czech M.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7885-7888Crossref PubMed Scopus (58) Google Scholar). CH-Sepharose was obtained from Amersham Pharmacia Biotech. Antibodies to PI 3-kinase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and those to Grb2 and dynamin from Transduction Labs (Lexington, KY). The GST-Grb2 construct was a gift from Dr. James Bliska (SUNY, Stony Brook, NY) and the fusion protein was expressed in E. coli and purified using standard protocols. The matrix-immobilized GST-Grb2 Sepharose was purchased from Amersham Pharmacia Biotech. Phosphatidylinositol bisphosphate was a kind gift from Dr. Andrew Morris (Department of Pharmacology, SUNY, Stony Brook, NY). Antipeptide antibodies to the hamster β2-adrenergic receptor were either developed in the laboratory (CM-04; Refs. 3Hadcock J.R. Port J.D. Gelman M.S. Malbon C.C. J. Biol. Chem. 1992; 267: 26017-26022Abstract Full Text PDF PubMed Google Scholar, 4Karoor V. Baltensperger K. Paul H. Czech M.P. Malbon C.C. J. Biol. Chem. 1995; 270: 25305-25308Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 5Baltensperger K. Karoor V. Paul H. Ruoho A. Czech M.P. Malbon C.C. J. Biol. Chem. 1996; 271: 1061-1065Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) or obtained from a commercial supplier (Santa Cruz Biotechnology) and employed throughout these studies. Confluent cultures of DDT1MF-2 cells were serum-starved for 18 h prior to each experiment. Cells were treated with hormones in serum-free DMEM for the indicated times and then lysed in a buffer containing 20 mm Tris-HCl (pH 7.5), 150 mmNaCl, 100 μm sodium vanadate, 40 mm NaF, 1% Nonidet P-40, 1 mm CaCl2, 1 mmMgCl2, 10% glycerol, 20 μm phenylarsine oxide, and a mixture of protease inhibitors (50 μg/ml leupeptin, 50 μg/ml aprotinin, 100 μg/ml phenylmethylsulfonyl fluoride, 100 μg/ml bacitracin, and 100 μg/ml benzamidine). β2AR was immunoprecipitated with antipeptide antibody CM-04 for 5 h at 4 °C (3Hadcock J.R. Port J.D. Gelman M.S. Malbon C.C. J. Biol. Chem. 1992; 267: 26017-26022Abstract Full Text PDF PubMed Google Scholar, 4Karoor V. Baltensperger K. Paul H. Czech M.P. Malbon C.C. J. Biol. Chem. 1995; 270: 25305-25308Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 5Baltensperger K. Karoor V. Paul H. Ruoho A. Czech M.P. Malbon C.C. J. Biol. Chem. 1996; 271: 1061-1065Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The CM-04 antibody was generated to a synthetic peptide corresponding to Trp-99–Thr-110 located in the exofacial domain of the β2-adrenergic receptor (3Hadcock J.R. Port J.D. Gelman M.S. Malbon C.C. J. Biol. Chem. 1992; 267: 26017-26022Abstract Full Text PDF PubMed Google Scholar, 4Karoor V. Baltensperger K. Paul H. Czech M.P. Malbon C.C. J. Biol. Chem. 1995; 270: 25305-25308Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 5Baltensperger K. Karoor V. Paul H. Ruoho A. Czech M.P. Malbon C.C. J. Biol. Chem. 1996; 271: 1061-1065Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), recognizing the Y350F mutant β2-adrenergic receptor as it does the wild-type counterpart (data not shown). In the case of the GST-Grb2 Sepharose beads, aliquots of whole-cell lysates were incubated with 20 μl of the beads for 2 h at 4 °C. The beads were washed exhaustively and the adsorbed proteins solubilized and subjected to SDS-PAGE as described by the commercial supplier. The immunoprecipitates were collected by centrifugation and subjected to SDS-PAGE (4Karoor V. Baltensperger K. Paul H. Czech M.P. Malbon C.C. J. Biol. Chem. 1995; 270: 25305-25308Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 5Baltensperger K. Karoor V. Paul H. Ruoho A. Czech M.P. Malbon C.C. J. Biol. Chem. 1996; 271: 1061-1065Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The resolved proteins were transferred onto nitrocellulose blots, probed with the antibodies indicated, and stained with a secondary antibody to which horseradish peroxidase was coupled. The Western blot chemiluminescence reagent (NEN Life Science Products) and autoradiography were used to detect the immunocomplexes, following the instruction procedure of the supplier. Immunoprecipitates and GST-Grb2 treatments were performed with equivalence in the amount of cell extract protein and amount of β2-adrenergic receptor. PI 3-kinase activity of immunoprecipitates of the β2AR was measured directly, essentially as described (25Okamoto M. Hayashi T. Kono S. Inoue G. Kubota M. Okamoto M. Kuzuya H. Imura H. Biochem. J. 1993; 290: 327-333Crossref PubMed Scopus (30) Google Scholar). The immunoprecipitates were washed two times with phosphate-buffered saline containing 1% Nonidet P-40, and 100 μm sodium vanadate, twice with 100 mmTris (pH 7.5) containing 0.5 m LiCl, and twice with the assay buffer (10 mm Tris-HCl (pH 7.5) containing 100 mm NaCl, 1 mm EDTA, and 100 μmsodium vanadate). The kinase assay was performed directly on beads in a total reaction volume of 50 μl in assay buffer containing 10 mm MgCl2 50 μm ATP (containing 10 μCi of [γ-32P]ATP) at 30 °C for 5.0 min. The reaction was stopped by the addition of 50 μl of 0.1 mHCl, and the tubes were transferred to an ice slurry. Lipids then were extracted by the addition of 375 μl of chloroform:methanol:HCl (200:400:5) first, 125 μl of HCl, and 125 μl of chloroform. The tubes were vortexed, and the lower phase was re-extracted with synthetic upper phase chloroform:methanol:0.1 m HCl (1:1:1). The lipids were dried under nitrogen and separated by TLC on oxalate-coated silica gel plates in a solvent system containing chloroform:methanol:ammonia:water (140:200:30:50). For assay of cyclic AMP accumulation, cells were seeded in 96-well plates 48 h prior to determination, at a density of 1 × 104cells/well. On the day of experiment, medium was aspirated, the cells washed and replenished with Krebs-Ringer phosphate medium containing 10 mm RO-201724 (cyclic AMP phosphodiesterase inhibitor), and then treated with the indicated hormones in a total assay volume of 50 μl. The reaction was terminated by the addition of 100 μl of 100% ethanol and the cyclic AMP content measured by the competitive binding assay, as described (26Czech M.P. Malbon C.C. Kerman K. Gitomer W. Pilch P.F. J. Clin. Invest. 1980; 66: 574-583Crossref PubMed Scopus (51) Google Scholar). Radioligand binding was performed on whole cells, as described (27Von Zastrow M. Kobilka B.K. J. Biol. Chem. 1992; 267: 3530-3538Abstract Full Text PDF PubMed Google Scholar). Cultures of DDT1MF-2 cells were treated with indicated hormones in either the presence or the absence of the PI 3-kinase inhibitor wortmannin (100 nm) at 25 °C for 5 min. The cells then were washed with ice-cold phosphate-buffered saline and resuspended in DMEM containing 20 mm HEPES (pH 7.4) and the hydrophilic, membrane-impermeant β-adrenergic antagonist [3H]CGP-12177 (70 nm). Binding was performed at 4 °C for 6 h. The cells were diluted, collected on GF/C membranes at reduced pressure, and washed rapidly. The radioligand bound to the washed cell mass on the filter was counted by liquid scintillation spectrometry. Nonspecific binding was defined as the radioligand binding insensitive to competition by the unlabeled, β-adrenergic antagonist propanolol (10 μm), as reported earlier (27Von Zastrow M. Kobilka B.K. J. Biol. Chem. 1992; 267: 3530-3538Abstract Full Text PDF PubMed Google Scholar, 28Shih M. Malbon C.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12193-12197Crossref PubMed Scopus (91) Google Scholar). Assay of β-adrenergic receptors by binding of iodocyanopindolol to intact cells was performed as described (28Shih M. Malbon C.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12193-12197Crossref PubMed Scopus (91) Google Scholar). Insulin stimulates phosphorylation of β2ARs on specific tyrosyl residues and attenuates the ability of the receptor to activate Gs in response to β-adrenergic agonist (3Hadcock J.R. Port J.D. Gelman M.S. Malbon C.C. J. Biol. Chem. 1992; 267: 26017-26022Abstract Full Text PDF PubMed Google Scholar, 4Karoor V. Baltensperger K. Paul H. Czech M.P. Malbon C.C. J. Biol. Chem. 1995; 270: 25305-25308Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 5Baltensperger K. Karoor V. Paul H. Ruoho A. Czech M.P. Malbon C.C. J. Biol. Chem. 1996; 271: 1061-1065Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). PI 3-kinase activation is critical to intracellular trafficking of membrane-bound receptors (14Carlberg K. Tapley P. Haystead C. Rohrschneider L. EMBO J. 1991; 10: 877-883Crossref PubMed Scopus (54) Google Scholar, 15Schu P. Takegawa K. Fry M. Stack J. Waterfield M. Emr S. Science. 1993; 260: 88-91Crossref PubMed Scopus (806) Google Scholar) and is an early event in insulin action (16Okada T. Kawano Y. Sakakibara T. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3568-3573Abstract Full Text PDF PubMed Google Scholar, 17Cheatham B. Vlahos C.J. Cheatham L. Wang L. Blenis J. Kahn C.R. Mol. Cell. Biol. 1994; 14: 4902-4911Crossref PubMed Scopus (1001) Google Scholar, 18Heller-Harrison R.A. Morin M. Guilherme A. Czech M.P. J. Biol. Chem. 1996; 271: 10200-10204Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). We explored to what extent, if any, does internalization of β2ARs represent a counter-regulatory effect of insulin. Since both insulin and chronic stimulation by β-adrenergic agonists attenuate β-adrenergic action (29Shih M. Malbon C.C. Cell Signalling. 1998; 10: 575-582Crossref PubMed Scopus (21) Google Scholar), we investigated if insulin treatment alters the level of β2AR sequestration from the cell membrane as does β-adrenergic agonist (2Hausdorff W.P. Caron M.G. Lefkowitz R.J. FASEB J. 1990; 4: 2881-2889Crossref PubMed Scopus (1087) Google Scholar, 27Von Zastrow M. Kobilka B.K. J. Biol. Chem. 1992; 267: 3530-3538Abstract Full Text PDF PubMed Google Scholar, 28Shih M. Malbon C.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12193-12197Crossref PubMed Scopus (91) Google Scholar), measured using the hydrophilic, membrane-impermeant radioligand [3H]CGP-12177 (27Von Zastrow M. Kobilka B.K. J. Biol. Chem. 1992; 267: 3530-3538Abstract Full Text PDF PubMed Google Scholar). As shown previously (27Von Zastrow M. Kobilka B.K. J. Biol. Chem. 1992; 267: 3530-3538Abstract Full Text PDF PubMed Google Scholar), treatment of DDT1MF-2 hamster smooth muscle cells in culture with isoproterenol promotes an ∼40% decline in the amount of receptor accessible by the hydrophilic [3H]CGP-12177 radioligand. Notably, treatment with insulin promoted a significant, but lesser internalization of the β2ARs (Fig. 1). IGF-1, which also counter-regulates β2AR-mediated response (3Hadcock J.R. Port J.D. Gelman M.S. Malbon C.C. J. Biol. Chem. 1992; 267: 26017-26022Abstract Full Text PDF PubMed Google Scholar, 4Karoor V. Baltensperger K. Paul H. Czech M.P. Malbon C.C. J. Biol. Chem. 1995; 270: 25305-25308Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 5Baltensperger K. Karoor V. Paul H. Ruoho A. Czech M.P. Malbon C.C. J. Biol. Chem. 1996; 271: 1061-1065Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), promotes internalization of the β2ARs (mean ± S.E., 35 ± 2%,n = 3). In the presence of either insulin or IGF-1, the internalization of β2AR in response to isoproterenol was increased. Stimulation by insulin and isoproterenol in combination promoted internalization of more than 55% of the β2ARs. These observations derived from studies with DDT1MF-2 smooth muscle cells were explored in CHO cells stably transfected to express wild-type β2ARs (Fig. 1). The data from multiple experiments from both the DDT1MF-2 cells and CHO β2AR-expressing cells were in good agreement, demonstrating that this growth factor-stimulated internalization of β2ARs is not unique to the smooth muscle cells. The functional read-out of insulin action on cyclic AMP accumulation in response to stimulation by β-adrenergic agonist was measured in the DDT1MF-2 cells challenged with isoproterenol in the absence and presence of insulin (Fig. 2). The time course for isoproterenol-stimulated cyclic AMP accumulation was rapid, peaking at ∼3 min. As noted earlier (3Hadcock J.R. Port J.D. Gelman M.S. Malbon C.C. J. Biol. Chem. 1992; 267: 26017-26022Abstract Full Text PDF PubMed Google Scholar, 4Karoor V. Baltensperger K. Paul H. Czech M.P. Malbon C.C. J. Biol. Chem. 1995; 270: 25305-25308Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), insulin (100 nm) counter-regulates the ability of isoproterenol to stimulate intracellular accumulation of cyclic AMP. Thus, insulin both counter-regulated β-adrenergic stimulation of cyclic AMP accumulation (Fig. 2) and stimulated internalization of the β2ARs (Fig. 1). Upon tyrosine kinase receptor-catalyzed phosphorylation (3Hadcock J.R. Port J.D. Gelman M.S. Malbon C.C. J. Biol. Chem. 1992; 267: 26017-26022Abstract Full Text PDF PubMed Google Scholar), the Tyr-350 residue located in the cytoplasmic, C-terminal tail of the β2AR creates an SH2 recognition domain capable of binding the adaptor protein Grb2 (4Karoor V. Baltensperger K. Paul H. Czech M.P. Malbon C.C. J. Biol. Chem. 1995; 270: 25305-25308Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 5Baltensperger K. Karoor V. Paul H. Ruoho A. Czech M.P. Malbon C.C. J. Biol. Chem. 1996; 271: 1061-1065Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The Grb2 molecule displays two SH2 domains and one SH3 domain. The similarity of theM r of Grb2 to that of the light chains of the IgG precluded detection of Grb2 by immunoblotting of immunoprecipitates of whole-cell extracts performed with antibodies to β2ARs. As an alternative strategy to probing the possible direct association of Grb2 to β2ARs in the counter-regulatory actions of insulin, whole-cell extracts were subjected to immuneprecipitations with anti-Grb2 antibodies first or treated with insoluble beads to which a GST fusion protein (GST-Grb2) was immobilized. Immunoprecipitations performed with anti-Grb2 antibodies were subjected to SDS-PAGE and then immunoblotting with antibodies specific for β2AR (Fig. 3). A time course of insulin action on Grb2 binding to β2ARs was established. Shown in Fig. 3are representative examples of blots from two time courses in which immunoprecipitates with anti-Grb2 antibodies were performed with extracts of cells challenged with insulin for 1–40 min, subjected to SDS-PAGE, and stained subsequently with antibodies to β2ARs. Both trials reveal Grb2 binding to β2ARs in the whole-cell extracts from untreated, control cells. Insulin treatment of the cells leads to an increase in the amount of β2ARs associated with Grb2, peaking at ∼5 min after challenge with insulin and declining thereafter. Association of β2ARs with Grb2 was probed further using a complementary approach, i.e. challenging cells with either insulin or agonist (or both) and treating extracts prepared from these cells with a GST-Grb2 fusion protein immobilized to insoluble beads. Binding of β2ARs to the Grb2 was detected in immunoblots of the proteins bound to the immobilized Grb2 stained with anti-β2AR antibodies (Fig. 4); these data are in good agreement with the data obtained with immunoprecipitates performed with anti-Grb2 antibodies (Fig. 3). The amount of β2AR associated with the GST-Grb2 fusion protein was quantified from several separate experiments and, as displayed in Fig. 4, was found to increase by more than 2-fold in extracts from those cells challenged with insulin. Isoproterenol, in contrast, stimulates only a small and variable increase in the amount of β2AR-Grb2 binding. Challenge with isoproterenol and insulin, in combination, provokes an increase in the amount of β2ARs associating with GST-Grb2 similar to that obtained with insulin alone. These data extend earlier studies on the role of tyrosine phosphorylation of β2ARs in insulin action (3Hadcock J.R. Port J.D. Gelman M.S. Malbon C.C. J. Biol. Chem. 1992; 267: 26017-26022Abstract Full Text PDF PubMed Google Scholar, 4Karoor V. Baltensperger K. Paul H. Czech M.P. Malbon C.C. J. Biol. Chem. 1995; 270: 25305-25308Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 5Baltensperger K. Karoor V. Paul H. Ruoho A. Czech M.P. Malbon C.C. J. Biol. Chem. 1996; 271: 1061-1065Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) and the ability of insulin to create a Grb2-dependent shift in agonist affinity of β2ARs (28Shih M. Malbon C.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12193-12197Crossref PubMed Scopus (91) Google Scholar). The current studies provide a direct demonstration that the insulin-catalyzed phosphorylation of the β2AR creates a bona fide SH2 domain that can be shown to bind Grb2 in an insulin-dependent manner, using two distinct but complementar