Differences in Signaling Properties of the Cytoplasmic Domains of the Insulin Receptor and Insulin-like Growth Factor Receptor in 3T3-L1 Adipocytes
1999; Elsevier BV; Volume: 274; Issue: 43 Linguagem: Inglês
10.1074/jbc.274.43.30864
ISSN1083-351X
AutoresBirgitte Ursø, Diane L. Cope, Heidi Elisa Kalloo-Hosein, Amanda C. Hayward, Jonathan P. Whitehead, Stephen O’Rahilly, Kenneth Siddle,
Tópico(s)Pancreatic function and diabetes
ResumoInsulin and insulin-like growth factors (IGFs) elicit distinct but overlapping biological effects in vivo. To investigate whether differences in intrinsic signaling capacity of receptors contribute to biological specificity, we constructed chimeric receptors containing the extracellular portion of the neurotrophin receptor TrkC fused to the intracellular portion of the insulin or IGF-I receptors. Chimeras were stably expressed in 3T3-L1 adipocytes at levels comparable to endogenous insulin receptors and were efficiently activated by neurotrophin-3. The wild-type insulin receptor chimera mediated approximately 2-fold greater phosphorylation of insulin receptor substrate 1 (IRS-1), association of IRS-1 with phosphoinositide 3-kinase, stimulation of glucose uptake, and GLUT4 translocation, compared with the IGF-I receptor chimera. In contrast, the IGF-I receptor chimera mediated more effective Shc phosphorylation, association of Shc with Grb2, and activation of mitogen-activated protein kinase compared with the insulin receptor chimera. The two receptors elicited similar activation of protein kinase B, p70S6 kinase, and glycogen synthesis. We conclude that the insulin receptor mediates some aspects of metabolic signaling in adipocytes more effectively than the IGF-I receptor, as a consequence of more efficient phosphorylation of IRS-1 and greater recruitment/activation of phosphoinositide 3-kinase. Insulin and insulin-like growth factors (IGFs) elicit distinct but overlapping biological effects in vivo. To investigate whether differences in intrinsic signaling capacity of receptors contribute to biological specificity, we constructed chimeric receptors containing the extracellular portion of the neurotrophin receptor TrkC fused to the intracellular portion of the insulin or IGF-I receptors. Chimeras were stably expressed in 3T3-L1 adipocytes at levels comparable to endogenous insulin receptors and were efficiently activated by neurotrophin-3. The wild-type insulin receptor chimera mediated approximately 2-fold greater phosphorylation of insulin receptor substrate 1 (IRS-1), association of IRS-1 with phosphoinositide 3-kinase, stimulation of glucose uptake, and GLUT4 translocation, compared with the IGF-I receptor chimera. In contrast, the IGF-I receptor chimera mediated more effective Shc phosphorylation, association of Shc with Grb2, and activation of mitogen-activated protein kinase compared with the insulin receptor chimera. The two receptors elicited similar activation of protein kinase B, p70S6 kinase, and glycogen synthesis. We conclude that the insulin receptor mediates some aspects of metabolic signaling in adipocytes more effectively than the IGF-I receptor, as a consequence of more efficient phosphorylation of IRS-1 and greater recruitment/activation of phosphoinositide 3-kinase. insulin-like growth factor insulin receptor type I insulin-like growth factor receptor TrkC-insulin receptor chimera TrkC-IGF-I receptor chimera phosphoinositide 3-kinase protein kinase B/Akt mitogen-activated protein MAP kinase insulin receptor substrate neurotrophin-3 Dulbecco's modified Eagle's medium polyacrylamide gel electrophoresis Insulin and insulin-like growth factors (IGFs)1 are structurally related polypeptides that exert diverse effects on mammalian tissues. The most prominent actions of insulin in vivo are concerned with the acute regulation of carbohydrate and lipid metabolism in liver, muscle, and fat, whereas IGFs act on skeletal and other tissues to promote growth, differentiation, and survival. The receptors for insulin and IGFs, which mediate these effects (IR and IGFR), are also structurally related and highly homologous, consisting of extracellular α-subunits responsible for ligand binding and transmembrane β-subunits possessing protein-tyrosine kinase activity, in a disulphide linked β-α-α-β configuration (1Ebina Y. Ellis L. Jarnagin K. Edery M. Graf L. Clauser E. Ou J. Masiarz F. Kan Y.W. Goldfine I.D. Cell. 1985; 40: 747-758Abstract Full Text PDF PubMed Scopus (965) Google Scholar, 2Ullrich A. Bell J.R. Chen E.Y. Herrera R. Petruzzeli L.M. Dull T.J. Gray A. Coussens L. Liao Y.-C. Tsubokawa M. Mason A. Seeburg P.H. Grunfeld C. Rosen O.M. Ramachandran J. Nature. 1985; 313: 756-761Crossref PubMed Scopus (1506) Google Scholar, 3Ullrich A. Gray A. Tam A.W. Yang-Feng T. Tsubokawa M. Collins C. Henzel W. LeBon T. Kathuria S. Chen E. Jacobs S. Francke U. Ramachandran J. Fujita-Yamaguchi Y. EMBO J. 1986; 5: 2503-2512Crossref PubMed Scopus (1495) Google Scholar). Within the intracellular portion, the level of sequence identity between the receptors is greatest in the tyrosine kinase domain (84%) and somewhat less in the flanking juxtamembrane and carboxyl-terminal regions (61 and 44%, respectively). Not surprisingly, the signaling mechanisms of the insulin receptor (IR) and IGF-I receptor (IGFR) are broadly similar. Ligand binding activates tyrosine kinase activity, leading to phosphorylation of intracellular substrates, such as IRS and Shc proteins, and the recruitment and/or stimulation of signal transducing molecules, including phosphoinositide 3-kinase (PI 3-kinase) and Grb2·Sos (4White M.F. Diabetologia. 1997; 40: S2-S17Crossref PubMed Scopus (460) Google Scholar, 5White M.F. Yenush L. Curr. Top. Microbiol. Immunol. 1998; 228: 179-208Crossref PubMed Google Scholar). These signal transducers in turn promote activation of protein-serine kinase cascades involving phosphoinositide-dependent kinase/PKB and MAPK/Erk kinase/MAPK, respectively, which modulate the activity of glucose transporters, enzymes, and transcription factors (6Alessi D.R. Cohen P. Curr. Opin. Genet. Dev. 1998; 8: 55-62Crossref PubMed Scopus (674) Google Scholar, 7Denton R.M. Tavaré J.M. Eur. J. Biochem. 1995; 227: 597-611Crossref PubMed Scopus (129) Google Scholar).Given the similarity in structure and signaling mechanism of the respective receptors, obvious questions arise concerning the basis of specificity in the actions of insulin and IGFs in vivo. In part, this specificity must reflect the different patterns of expression of receptors and responsive pathways in different cell types. Indeed, when examined in the same cell background, insulin and IGFs induce the same end point responses (8Steele-Perkins G. Turner J. Edman J.C. Hari J. Pierce S.B. Stove C. Rutter W.J. Roth R.A. J. Biol. Chem. 1988; 263: 11486-11492Abstract Full Text PDF PubMed Google Scholar, 9Hofmann C. Goldfine I.D. Whittaker J. J. Biol. Chem. 1989; 264: 8606-8611Abstract Full Text PDF PubMed Google Scholar, 10Weiland M. Bahr F. Höhne M. Schürmann A. Ziehm D. Joost H.G. J. Cell. Physiol. 1991; 149: 428-435Crossref PubMed Scopus (56) Google Scholar). However, such studies do not rule out the possibility of subtle differences in the coupling of the receptors to different responses, and there are various problems of interpretation in making detailed comparisons of IR and IGFR activity in a given cell type. First, neither receptor is completely specific for its ligand, and at high ligand concentrations, some cross-reaction with the heterologous receptor is inevitable. Second, it is difficult to make comparisons at equivalent occupancy of IR and IGFR because of differences in ligand affinity and levels of expression. Third, in cells that express both IR and IGFR a substantial fraction of receptors assemble as hybrid structures capable of binding both ligands (11Bailyes E.M. Navé B.T. Soos M.A. Orr S.R. Hayward A.C. Siddle K. Biochem. J. 1997; 327: 209-215Crossref PubMed Scopus (236) Google Scholar). Finally, attempts to overcome these problems by overexpression of receptors in transfected cells might distort the signaling specificity, which could be seen at lower and more physiological levels of receptor expression.In order to circumvent such problems, several laboratories have made use of chimeric receptors, in which the intracellular domains of the IR, IGFR, or insulin receptor-related receptor are coupled to a common extracellular ligand binding domain (12Lammers R. Gray A. Schlessinger J. Ullrich A. EMBO J. 1989; 8: 1369-1375Crossref PubMed Scopus (176) Google Scholar, 13Dandekar A.A. Wallach B.J. Barthel A. Roth R.A. Endocrinology. 1998; 139: 3578-3584Crossref PubMed Scopus (14) Google Scholar, 14Kalloo-Hosein H.E. Whitehead J.P. Soos M. Tavaré J.M. Siddle K. O'Rahilly S. J. Biol. Chem. 1997; 272: 24325-24332Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 15Chaika O.V. Chaika N. Volle D.J. Wielden P.A. Pirrucello S.J. Lewis R.E. J. Biol. Chem. 1997; 272: 11968-11974Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). It was reported that the IGFR was more efficient than the IR in mediating stimulation of DNA synthesis in NIH3T3 fibroblasts, with no difference in stimulation of glucose uptake (12Lammers R. Gray A. Schlessinger J. Ullrich A. EMBO J. 1989; 8: 1369-1375Crossref PubMed Scopus (176) Google Scholar). In contrast, in 3T3-L1 fibroblasts we found that the IR mediated a greater stimulation of glycogen synthesis compared with the IGFR, with no difference in the extent or efficiency of stimulation of DNA synthesis (14Kalloo-Hosein H.E. Whitehead J.P. Soos M. Tavaré J.M. Siddle K. O'Rahilly S. J. Biol. Chem. 1997; 272: 24325-24332Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Other work with truncated receptors, site-directed mutants, and chimeras has suggested that the efficiency of "metabolic" and "mitogenic" stimulations may depend on the carboxyl-terminal domains of the respective receptors (16Maegawa H. McClain D.A. Freidenberg G. Olefsky J.M. Napier M. Lipari T. Dull T.J. Lee J. Ullrich A. J. Biol. Chem. 1988; 263: 8912-8917Abstract Full Text PDF PubMed Google Scholar, 17Takata Y. Webster N.J. Olefsky J.M. J. Biol. Chem. 1991; 266: 9135-9139Abstract Full Text PDF PubMed Google Scholar, 18Faria T.N. Blakesley V.A. Kato H. Stannard B. LeRoith D. Roberts Jr., C.T. J. Biol. Chem. 1994; 269: 13922-13928Abstract Full Text PDF PubMed Google Scholar, 19Tartare S. Mothe I. Kowalski-Chauvel A. Breittmayer J.P. Balloti R. VanObberghen E. J. Biol. Chem. 1994; 269: 11449-11455Abstract Full Text PDF PubMed Google Scholar, 20Esposito D.L. Blakesley V.A. Koval A.P. Scrimgeour A.G. Roith D.L. Endocrinology. 1997; 138: 2979-2988Crossref PubMed Google Scholar), although the results have not been entirely consistent (21Tavaré J.M. Siddle K. Biochim. Biophys. Acta. 1993; 1178: 21-39Crossref PubMed Scopus (80) Google Scholar). A serious limitation of all these studies is that they have been carried out in cultured fibroblasts that display only a limited range of rather small responses to insulin and IGF-I compared with physiologically important target tissues. We have therefore examined the question of intrinsic signaling specificity by expressing IR and IGFR chimeras in differentiated 3T3-L1 adipocytes, which are highly insulin-responsive. We show here that glucose uptake is stimulated to a greater extent by IR than IGFR and that differences in the relative phosphorylation of distinct intracellular substrates by the IR and IGFR tyrosine kinases contribute to signaling specificity of the two receptors.DISCUSSIONAlthough insulin and IGFs exert distinct physiological effectsin vivo, the contribution of differences in receptor function, as opposed to receptor distribution, to this specificity remains unclear. Functional differences might arise from the kinetics of ligand association and dissociation, affecting the lifetime and/or intracellular itinerary of the activated receptors (35DeMeyts P. Christofferson C.T. Ursø B. Wallach B. Grønskov K. Yakushiji F. Shymko R.M. Metabolism. 1995; 10: 2-11Abstract Full Text PDF Scopus (56) Google Scholar, 36Zapf A. Hsu D. Olefsky J.M. Endocrinology. 1994; 134: 2445-2452Crossref PubMed Scopus (58) Google Scholar). Alternatively, the receptor intracellular domains might possess different signaling capacities, reflecting their differences in primary sequence (nonidentity, 16% in the tyrosine kinase domain, 39% in the juxtamembrane domain, and 56% in the carboxyl-terminal domain). To focus on the activity of intracellular domains without interference from endogenous receptors and to allow examination of a range of physiologically relevant metabolic responses, we expressed TrkC-IR (TIR) or TrkC-IGFR (TIGR) chimeras in 3T3-L1 adipocytes. Signaling capacities of the IR and IGFR have previously been compared only in transfected fibroblasts, in which reaction of ligands with endogenous as well as transfected receptors complicates interpretation of data, and the spectrum and magnitude of metabolic responses are rather small; the results of such studies have been inconsistent. For instance, it was reported that the IGFR was more effective than the IR in stimulating DNA synthesis and MAP kinase activity (12Lammers R. Gray A. Schlessinger J. Ullrich A. EMBO J. 1989; 8: 1369-1375Crossref PubMed Scopus (176) Google Scholar, 37Sasaoka T. Ishiki M. Sawa T. Ishihara H. Takata Y. Imamura T. Usui I. Olefsky J.M. Kobayashi M. Endocrinology. 1996; 137: 4427-4434Crossref PubMed Scopus (87) Google Scholar), although in other work, no such difference was observed (14Kalloo-Hosein H.E. Whitehead J.P. Soos M. Tavaré J.M. Siddle K. O'Rahilly S. J. Biol. Chem. 1997; 272: 24325-24332Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 38Mastick C.C. Kato H. Roberts C.T. LeRoith D. Saltiel A.R. Endocrinology. 1994; 135: 214-222Crossref PubMed Scopus (43) Google Scholar). The IR appeared to be more effective than IGFR in phosphorylating IRS-1 and activating PI 3-kinase in one study (38Mastick C.C. Kato H. Roberts C.T. LeRoith D. Saltiel A.R. Endocrinology. 1994; 135: 214-222Crossref PubMed Scopus (43) Google Scholar) but not in another (39Myers M.G. Sun X.-J. Cheatham B. Jachna B.R. Glasheen E.M. Backer J.M. White M.F. Endocrinology. 1993; 132: 1421-1430Crossref PubMed Scopus (227) Google Scholar).We found that stimulation of glucose uptake and GLUT4 translocation in 3T3-L1 adipocytes was more effectively mediated by TIR than TIGR (Figs.2 and 3). TIR also mediated greater stimulation of IRS-1 phosphorylation and IRS-1-associated PI 3-kinase activity at both early and late time points (Figs. Figure 4, Figure 5, Figure 6). These differences were consistently observed when paired clones of 3T3-L1 fibroblasts, selected for similar levels of expression of TIR and TIGR, were differentiated on multiple occasions. The differences were not a consequence of unequal or excessive expression of the respective chimeras, as if anything, the TIGR was expressed at a slightly higher level than TIR in the differentiated adipocytes, and the level of expression of both chimeras was of the same order as that of endogenous insulin receptors. We also rule out the possibility that the different responses of TIR and TIGR cells to NT-3 reflect chance clonal variation, as insulin acting via its own receptor elicited very similar responses in both sets of cells. Moreover, phosphorylation of Shc showed the opposite relationship to phosphorylation of IRS-1 in the same cells, being more effectively induced via the TIGR than via TIR (Fig. 4).The more effective stimulation of glucose uptake via the IR than the IGFR chimera was manifest as a difference in maximum response rather than half-maximally effective concentration of NT-3 (EC50approximately 0.2 nm), although the dose-response relationships were not defined with sufficient precision to rule out a difference in sensitivity. It is generally believed that glucose uptake reflects the number of GLUT4 transporters at the plasma membrane (29Holman G.D. Kasuga M. Diabetologia. 1997; 40: 991-1003Crossref PubMed Scopus (187) Google Scholar), and we showed that the translocation of GLUT4 was indeed greater in response to stimulation of TIR than TIGR (Fig. 3). This result implies that the IR signals to a larger intracellular pool of transporters than the IGFR, which might reflect differential efficiency of mobilization from a single pool of transporters, or differential recruitment from more than one pool. In relation to the latter possibility, it has been shown that GLUT4 vesicles cycle through several distinct intracellular compartments in adipocytes (40Millar C.A. Campbell L.C. Cope D.L. Melvin D.R. Powell K.A. Gould G.W. Biochem. Soc. Transact. 1997; 25: 974-977Crossref PubMed Scopus (8) Google Scholar), and in skeletal muscle, discrete pools of GLUT4 vesicles are responsive to stimulation by insulin and contraction (41Coderre L. Kandror K.V. Vallega G. Pilch P.F. J. Biol. Chem. 1995; 270: 27584-27588Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar).There is ample evidence that PI 3-kinase activity is both necessary and sufficient for stimulation of glucose transport (29Holman G.D. Kasuga M. Diabetologia. 1997; 40: 991-1003Crossref PubMed Scopus (187) Google Scholar, 30Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (835) Google Scholar). Differences in glucose uptake and GLUT4 translocation mediated by TIR and TIGR were paralleled by greater IRS-1-associated PI 3-kinase activity, and the stimulations of glucose uptake and IRS-1 phosphorylation showed similar NT-3 concentration dependence (EC50 approximately 0.2 nm) (Figs. Figure 2, Figure 3, Figure 4). In contrast, TIR and TIGR induced similar PI 3-kinase activity measured in anti-phosphotyrosine precipitates, and this activity was increased at very low concentrations of NT-3 (<0.04 nm). Our finding that relative stimulations of glucose transport correlate better with IRS-associated than with anti-phosphotyrosine-precipitable PI 3-kinase activity are at variance with studies suggesting that IRS-1-associated PI 3-kinase was not essential for glucose transport stimulation (42Morris A.J. Martin S.S. Haruta T. Nelson J.G. Vollenweider P. Gustafson T.A. Mueckler M. Rose D.W. Olefsky J.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8401-8406Crossref PubMed Scopus (60) Google Scholar, 43Staubs P.A. Nelson J.G. Reichart D.R. Olefsky J.M. J. Biol. Chem. 1998; 273: 25139-25147Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar).The serine-specific protein kinase PKB/Akt has been identified as a downstream effector of PI 3-kinase, which is activated by phosphoinositide-dependent kinases secondarily to recruitment of both PKB and phosphoinositide-dependent kinase to membranes via association with the PI 3-kinase product phosphatidylinositol 3,4,5 trisphosphate (6Alessi D.R. Cohen P. Curr. Opin. Genet. Dev. 1998; 8: 55-62Crossref PubMed Scopus (674) Google Scholar). Studies of the role of PKB in stimulation of glucose transport have produced conflicting data. On the one hand, glucose uptake was increased in 3T3-L1 cells expressing constitutively active or inducible PKB (44Kohn A.D. Summers S.A. Birnbaum M.J. Roth R.A. J. Biol. Chem. 1996; 271: 31372-31378Abstract Full Text Full Text PDF PubMed Scopus (1086) Google Scholar, 45Hajduch E. Alessi D.R. Hemmings B.A. Hundal H.S. Diabetes. 1998; 17: 1006-1013Crossref Scopus (294) Google Scholar, 46Ueki K. Yamamoto-Honda R. Kaburagi Y. Yamauchi T. Tobe K. Burgering B.M. Th. Coffer P.J. Komuro I. Akanuma Y. Yazaki Y. Kadowaki T. J. Biol. Chem. 1998; 273: 5315-5322Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 47Kohn A.D. Barthel A. Kovacina K.S. Boge A. Wallach B. Summers S.A. Birnbaum M.J. Scott P.H. Lawrence Jr., J.C. Roth R.A. J. Biol. Chem. 1998; 273: 11937-11943Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). However, expression of a dominant negative PKB failed to block insulin-stimulated glucose uptake (48Kitamura T. Ogawa W. Sakaue H. Hino Y. Kuroda S. Takata M. Matsumoto M. Maeda T. Konishi H. Kikkawa U. Kasuga M. Mol. Cell. Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (295) Google Scholar, 49Imanaka T. Hayashi H. Kishi K. Wang L. Ishii K. Hazeki O. Katada T. Ebina Y. J. Biol. Chem. 1998; 273: 25347-25355Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). We found no difference in levels of PKB activity in isoform-specific immunoprecipitates or in phosphorylation of serine 473 at early and late time points, in TIR and TIGR cells stimulated with NT-3 (Figs. 7 and 8), despite the substantial difference in IRS-1-associated p85 and PI 3-kinase activity at the same NT-3 concentration. It may be that a small activation of PI 3-kinase is sufficient for maximum activation of PKB or that the IRS-1-associated pool of PI 3-kinase does not have as much influence on PKB activity as the anti-phosphotyrosine-precipitable pool. Regardless of the relationship between PKB and PI 3-kinase activities, our data suggest that PKB activity alone is not the sole determinant of stimulated glucose uptake, as identical PKB activities were observed in TIR and TIGR cells exhibiting significantly different stimulations of glucose uptake.In contrast to the stimulation of glucose transport, incorporation of glucose into glycogen was stimulated to the same maximum by NT-3 in TIR and TIGR cells, although TIR cells were somewhat more responsive at low NT-3 concentrations (EC50 values in the range of 0.01–0.03 nm) (Fig. 2). The disparity in NT-3 concentration dependence and fold stimulation of glycogen synthesis compared with glucose uptake suggests that the stimulation of synthesis reflected activation of glycogen synthase rather than increased glucose uptake. The similar effectiveness of IR and IGFR in stimulating glycogen synthesis in 3T3-L1 adipocytes contrasts with the substantial difference observed previously in 3T3-L1 fibroblasts (14Kalloo-Hosein H.E. Whitehead J.P. Soos M. Tavaré J.M. Siddle K. O'Rahilly S. J. Biol. Chem. 1997; 272: 24325-24332Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). However, it has been reported that the activation of glycogen synthase involves predominantly inhibition of glycogen synthase kinase-3 in 3T3-L1 fibroblasts but stimulation of PP1 in adipocytes (50Brady M.J. Bourbonais F.J. Saltiel A.R. J. Biol. Chem. 1998; 273: 14063-14066Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), and it may be that this difference in mechanism underlies the difference in responsiveness to IR and IGFR in the two cell types. Like the stimulation of glucose uptake, activation of glycogen synthase requires PI 3-kinase (30Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (835) Google Scholar). In terms of NT-3 concentration dependence and relative effectiveness of IR and IGFR, stimulation of glycogen synthesis correlates better with anti-phosphotyrosine-precipitable than IRS-1-associated PI 3-kinase activity, raising the possibility that separate pools of PI 3-kinase are involved in stimulation of glucose uptake and glycogen synthase. Certainly, the signaling pathways to GLUT4 translocation and glycogen synthase activation appear to diverge downstream of PI 3-kinase. PKB has been proposed as the principal route to inactivation of glycogen synthase kinase-3, which in turn is thought to be the major mechanism of activation of glycogen synthase in many tissues (51Cross D.A.E. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4326) Google Scholar). However, in 3T3-L1 adipocytes, levels of glycogen synthase kinase-3 are low, and glycogen synthesis is not activated by constitutively active PKB (44Kohn A.D. Summers S.A. Birnbaum M.J. Roth R.A. J. Biol. Chem. 1996; 271: 31372-31378Abstract Full Text Full Text PDF PubMed Scopus (1086) Google Scholar, 46Ueki K. Yamamoto-Honda R. Kaburagi Y. Yamauchi T. Tobe K. Burgering B.M. Th. Coffer P.J. Komuro I. Akanuma Y. Yazaki Y. Kadowaki T. J. Biol. Chem. 1998; 273: 5315-5322Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). There is evidence that p70S6 kinase contributes to activation of glycogen synthase in 3T3-L1 adipocytes (31Shepherd P.R. Navé B.T. Siddle K. Biochem. J. 1995; 305: 25-28Crossref PubMed Scopus (230) Google Scholar). Consistent with the similar stimulations of glycogen synthesis mediated by TIR and TIGR, we found no difference between the chimeras with respect to the activation of p70S6 kinase.It seems clear, therefore, that the contribution of different signaling pathways to end point responses may vary not only with respect to different responses in a given cell but also with respect to a given response in different cell types. Against this background, it is difficult to predict relative efficiency of IR and IGFR in mediating metabolic effects in other tissues based on the present data for glucose transport in 3T3-L1 adipocytes. Interestingly, in hepatocytes, as in 3T3-L1 fibroblasts, glycogen synthesis was more effectively stimulated via IR than IGFR, although both receptors mediated similar activation of PKB (52Park B.-C. Kido Y. Accili D. Biochem. 1999; 38: 7517-7523Crossref PubMed Scopus (43) Google Scholar). The insulin-specific stimulation of glycogen synthesis in hepatocytes appeared to involve a rapamycin-sensitive pathway.It is not meaningful to study classical mitogenic responses (proliferation/DNA synthesis) in a terminally differentiated and nondividing cell line such as the 3T3-L1 adipocyte. However, we examined various components of the MAP kinase pathway that have been implicated in transcriptional and mitogenic responses in other cells (7Denton R.M. Tavaré J.M. Eur. J. Biochem. 1995; 227: 597-611Crossref PubMed Scopus (129) Google Scholar). TIGR cells were more responsive than TIR with regard to stimulation of Shc phosphorylation, association of Shc with Grb2, and activation of MAP kinase (Figs. 4 and 8). No difference was observed in the induction of Fra1 mRNA in TIR and TIGR cells stimulated by a near-maximal concentration of NT-3, although it is possible that relatively modest activation of MAP kinase is sufficient for maximal Fra1 induction and that a differential effect of TIGR and TIR on Fra1 mRNA might have been observed at lower NT-3 concentrations. Because TIGR was expressed at a slightly higher level than TIR in the cell lines studied, it cannot be definitely concluded that the IGFR is more efficient than the IR in phosphorylating Shc. However, the relative Shc responses do provide excellent controls for the IRS-1 phosphorylation measured in the same cells, and the ratio of IRS/Shc phosphorylation was approximately 4-fold higher in TIR cells than in TIGR cells across the full range of NT-3 concentrations tested.Various mechanisms might contribute to relative specificity in substrate phosphorylation by the IR and IGFR, including differences in the intrinsic specificity of the tyrosine kinases themselves (53Xu B. Bird V.G. Miller W.T. J. Biol. Chem. 1995; 270: 29825-29830Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 54Krüger M.S. Klein H. Siddle K. Gammeltoft S. Exp. Clin. Endocrinol. Diabetes. 1996; 104 Suppl. 2: 38Google Scholar), differences in the association of substrates with receptors via PTB or other domains (55Wolf G. Trub T. Ottinger E. Groninga L. Lynch A. White M.F. Miyazaki M. Lee J. Shoelson S.E. J. Biol. Chem. 1995; 270: 27407-27410Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 56Sawka-Verhelle D. Tartare-Deckert S. White M.F. VanObberghen E. J. Biol. Chem. 1996; 271: 5980-5983Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 57He W. Craparo A. Zhu Y. O'Neill T.J. Wang L.-M. Pierce J.H. Gustafson T.A. J. Biol. Chem. 1996; 271: 11641-11645Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 58Rother L.I. Imai Y. Caruso M. Beguinot F. Formisano P. Accili D. J. Biol. Chem. 1998; 273: 17491-17497Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), the modulation of such interactions by receptor-specific adaptor proteins, such as Grb10 (59Laviola L. Giorgino F. Chow J.C. Baquero J.A. Hansen H. Ooi J. Zhu J. Riedel H. Smith R.J. J. Clin. Invest. 1997; 99: 830-837Crossref PubMed Scopus (87) Google Scholar, 60He W. Rose D.W. Olefsky J.M. Gustafson T.A. J. Biol. Chem. 1998; 273: 6860-6867Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), or differences in endocytosis and subsequent trafficking of receptors (61Chow J.C. Condorelli G. Smith R.J. J. Biol. Chem. 1998; 273: 4672-4680Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar,62Ceresa B.P. Kao A.W. Santeler S.R. Pessin J.E. Mol. Cell. Biol. 1998; 18: 3862-3870Crossref PubMed Scopus (196) Google Scholar). No major differences between the two receptor kinases were apparent in the phosphorylation of recombinant IRS-1 in vitro (39Myers M.G. Sun X.-J. Cheatham B. Jachna B.R. Glasheen E.M. Backer J.M. White M.F. Endocrinology. 1993; 132: 1421-1430Crossref PubMed Scopus (227) Google Scholar), although this result does not exclude possible differences in activity of receptors toward IRS-1 in intact cells, where receptors and IRS-1 may be subject to regulatory phosphorylation on serine residues or modulation by other proteins. The carboxyl-terminal domains of the IR and IGFR have been implicated in signaling specificity (18Faria T.N. Blakesley V.A. Kato H. Stannard B. LeRoith D. Roberts Jr., C.T. J. Biol. Chem. 1994; 269: 13922-13928Abstract Full Text PDF PubMed Google Scholar, 19Tartare S. Mothe I. Kowalski-Chauvel A. Breittmayer J.P. Balloti R. VanObberghen E. J. Biol. Chem. 1994; 269: 11449-11455Abstract Full Text PDF PubMed Google Scholar, 20Esposito D.L. Blakesley V.A. Koval A.P. Scrimgeour A.G. Roith D.L. Endocrinology. 1997; 138: 2979-2988Crossref PubMed Google Scholar), although the influence of this region must be relatively subtle, as carboxyl-terminally truncated IR and IGFR signal normally in at least some cell types (63Myers M.G. Backer J.M. Siddle K. White M.F. J. Biol. Chem. 1991; 266: 10616-10623Abstract Full Text PDF PubMed Google Scholar, 64Grønborg M. Wulff B.S. Rasmussen J.S. K
Referência(s)