Differential Signaling to Glycogen Synthesis by the Intracellular Domain of the Insulin versus the Insulin-like Growth Factor-1 Receptor

Abstract

Insulin and insulin-like growth factor-1 (IGF-1) have similar cell-surface receptors yet subserve different physiological functions. To examine whether these differences relate to intrinsic signaling properties of the intracellular domains of their respective receptors, chimeric receptors were constructed using the extracellular domain of the neurotrophin-3 (NT-3) receptor, TrkC, and the intracellular domain of either the insulin receptor or the IGF-1 receptor. TrkC-IR (TIR) and TrkC-IGF-1R (TIGR) were stably expressed in 3T3-L1 cells. While TIR and TIGR cell lines expressing similar numbers of chimeric receptors showed a similar dose-response relationship in NT-3 stimulated DNA synthesis, NT-3 stimulated glycogen synthesis was greater in TIR than in TIGR cells (maximum TIGR response was 35% of maximum TIR response). Additionally, the concentration of NT-3 at which significant stimulation of glycogen synthesis was seen was 0.1 ng/ml in TIR and 1 ng/ml in TIGR cells. Basal levels of thymidine incorporation but not glycogen synthesis were consistently higher in TIR than in TIGR expressing cells. No detectable basal autophosphorylation of chimeric receptors was seen in any of the cell lines. However, exposure of cell lines to the phosphatase inhibitor bisperoxovanadate resulted in greater basal autophosphorylation of the TIR and endogenous murine IR than the TIGR and endogenous murine IGF-1R. Thus, in this system, the intracellular domain of the IR appears to couple more effectively to glycogen synthesis than that of the IGF-1R, whereas the intracellular domains of both receptors have a similar capacity to stimulate DNA synthesis.Insulin and insulin-like growth factor-1 (IGF-1) 1 are homologous peptides that are essential for normal metabolism and growth regulation. In the intact organism these hormones have significantly different physiological roles (for review, see Refs. 1-3). Insulin primarily functions as a regulator of carbohydrate, fat, and protein metabolism. In contrast, IGF-1 is largely a mediator of cell growth and differentiation primarily stimulating DNA synthesis and mitogenic events (4 -6). Despite these different functions, the cell surface receptors for insulin and IGF-1 (IR and IGF-1R, respectively) are highly homologous. They are both Type II tyrosine kinase receptors with the same disulfide-linked heterotetrameric structure consisting of two extracellular ␣-subunits containing the ligand-binding domain and two transmembrane ␤-subunits with the ligand-sensitive tyrosine kinase activity (7). The greatest homology (over 80%) is found in the tyrosine kinase domain while differences in the ligand-binding domain (overall homology 44 -60%) account for ligand specificity. The greatest sequence divergence between the two receptors is found in their C termini (44% identity) (8).In addition to the similarity of receptor structure the intracellular signaling events which result from ligand-induced receptor activation are remarkably similar (9). Both receptors phosphorylate insulin-receptor substrates IRS-1 ...

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

“…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 (12). 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 (14). 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 (16 -20), although the results have not been entirely consistent (21).…”

“…NT-3 was radioiodinated as described (14). The cells were incubated with 125 I-NT-3 (30,000 dpm) and increasing concentrations of unlabeled NT-3 in binding buffer (100 mM Hepes, 120 mM NaCl, 1 mM EDTA, 15 mM NaC 2 H 4 O 2 ) overnight at 4°C, washed twice in cold phosphate-buffered saline, solubilized in 0.1 M NaOH and counted in a NE1600 gamma counter.…”

“…Construction of Chimeras-Chimeras containing the extracellular and transmembrane portions of the neurotrophin receptor TrkC together with the intracellular portion of the human IR (TIR) or IGFR (TIGR) were constructed as described previously (14). The chimeras were expressed under the regulation of the elongation factor 1␣ promoter (a kind gift from Dr R. E. Lewis (Eppley Institute for Research in Cancer, University of Nebraska)) in the vector pcDNA3 (Invitrogen).…”

“…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 (12)(13)(14)(15). 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 (12).…”

“…Third, in cells that express both IR and IGFR a substantial fraction of receptors assemble as hybrid structures capable of binding both ligands (11). 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 (12)(13)(14)(15). 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 (12).…”