Differences in Signaling Properties of the Cytoplasmic Domains of the Insulin Receptor and Insulin-like Growth Factor Receptor in 3T3-L1 Adipocytes

Abstract

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 mitogenactivated 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)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 ␣-subunits responsible for ligand binding and transmembrane ␤-subunits possessing protein-tyrosine kinase activity, in a disulphide linked ␤-␣-␣-␤ configuration (1-3). 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 Grb2⅐Sos (4, 5). These signal transducers in turn promote activation of proteinserine kinase cascades involving phosphoinositide-dependent kinase/PKB and MAPK/Erk kinase/MAPK, respectively, which modulate the activity of glucose transporters, enzymes, and transcription factors (6, 7).Given the similarity in structu...

Arachidonic Acid Stimulates Glucose Uptake in 3T3-L1 Adipocytes by Increasing GLUT1 and GLUT4 Levels at the Plasma Membrane

“…To examine whether AA might have an effect on levels of GLUT1 or GLUT4 at the plasma membrane the plasma membrane lawn assay was used. Insulin treatment of 3T3-L1 cells produced an ϳ2-fold increase in GLUT1 and a 5-fold increase in GLUT4 plasma membrane expression, results which are consistent with previous published studies (33). AA increased levels of GLUT1 at the plasma membrane in the absence and presence of insulin by 2-(p Ͻ 0.05) and 1.3-fold (p Ͻ 0.001), respectively, and increased levels of GLUT4 at the plasma membrane in the absence and presence of insulin by 1.5-fold (p Ͻ 0.01) and 1.4-fold (p Ͻ 0.05), respectively (Fig.…”

“…For the 1-h time point, cells were starved for 1 h prior to incubation in the AA medium. Glucose uptake assays were performed as described previously (33).…”

Microarray Analysis of Insulin and Insulin-like Growth Factor-1 (IGF-1) Receptor Signaling Reveals the Selective Up-regulation of the Mitogen Heparin-binding EGF-like Growth Factor by IGF-1

“…Fewer mRNAs (11) were differentially regulated compared with those similarly regulated between the two cell lines, consistent with the broadly overlapping effects of insulin and IGF-1 (Table II). Of the 11 differentially regulated transcripts, 1 was an EST that was suppressed to a greater extent by the TIR.…”

“…We further demonstrated that MAPK is needed for TIGR signaling to HB-EGF, a requirement also observed in the IGF-1R-selective stimulation of Twist and VEGF transcription (28,29). In our study, MAPK was capable of being activated by both chimeric receptors, whereas other studies (11) 2 showed greater phosphorylation of MAPK in response to TIGR signaling. However, the relative differences in MAPK phosphorylation seen in those studies are unlikely to be sufficient to account for the all-or-nothing response we have 2 A. Parmar and K. Siddle, unpublished data.…”

“…In 3T3-L1 adipocytes, the IR chimera (TIR) stimulated GLUT4 translocation and glucose uptake to a greater extent than the IGF-1R chimera (TIGR). Differences were also seen in signaling immediately downstream of the receptors in adipocytes, with the IRS-1 signaling pathway being more effectively stimulated by the TIR chimera, and the Shc/MAPK signaling pathway being more stimulated by the TIGR chimera (11). Thus, it seems that intrinsic signaling differences between the two receptors do exist and are at least in part attributable to sequence differences in the intracellular domains of the IR and IGF-1R.…”