Overexpressing Insulin Receptors Research Articles

An IL-4 receptor region containing an insulin receptor motif is important for IL-4-mediated IRS-1 phosphorylation and cell growth

Interleukin-4 (IL-4) treatment of 32D cells overexpressing insulin receptor substrate 1 (IRS-1) causes prompt tyrosine phosphorylation of IRS-1. Transfection of truncation mutants of the human IL-4 (huIL-4) receptor into 32D-IRS-1 cells demonstrated that the region from amino acid 437–557 is important for IL-4 signaling. This region of the IL-4 receptor (IL-4R) contains the motif 488PL-X4-NPXYXSXSD502 (insulin/IL-4R [I4R]) found in the insulin and insulin-like growth factor 1 receptors. Mutation of Y497 to F yielded receptors that caused little or no IRS-1 phosphorylation in response to huIL-4 when expressed in 32D-IRS-1 cells. Most cell lines expressing Y497F also failed to proliferate in response to huIL-4. Furthermore, a glutathione-S-transferase fusion protein containing the I4R motif-bound IRS-1, tyrosine kinase(s), and other unidentified phosphoproteins with molecular sizes of 140, 80, and 55 kd. Thus, the central tyrosine of the I4R motif has a major role in IL-4-mediated signal transduction in 32D cells.

Insulin treatment stimulates the tyrosine phosphorylation of the alpha-type 85-kDa subunit of phosphatidylinositol 3-kinase in vivo.

After adding insulin to cells overexpressing the insulin receptor, the activity of phosphatidylinositol (PI) 3-kinase in the anti-phosphotyrosine immunoprecipitates was rapidly and greatly increased. This enzyme may therefore be a substrate for the insulin receptor tyrosine kinase and may be one of the mediators of insulin signal transduction. However, it is unclear whether or not activated tyrosine kinase of the insulin receptor directly phosphorylates PI 3-kinase at tyrosine residue(s) and whether insulin stimulates the specific activity of PI 3-kinase. We reported previously that the 85-kDa subunit of purified PI 3-kinase was phosphorylated at tyrosine residue(s) by the insulin receptor in vitro. To examine the tyrosine phosphorylation of PI 3-kinase and change of its activity by insulin treatment in vivo, we used a specific antibody to the 85-kDa subunit of PI 3-kinase. The activity of PI 3-kinase in immunoprecipitates with the antibody against the p85 subunit of PI 3-kinase was increased about 3-fold by insulin treatment of cells overexpressing insulin receptors. Insulin treatment also stimulated the tyrosine, serine, and threonine phosphorylation of the alpha-type 85-kDa subunit of PI 3-kinase in vivo. Phosphatase treatment of the immunoprecipitates abolished the increase in PI 3-kinase activity. The phosphorylation(s) of the kinase itself, tyrosine phosphorylation(s) of associated protein(s), or the complex formation of the phosphorylated PI 3-kinase with associated proteins may increase the activity of PI 3-kinase.

Relation between the insulin receptor number in cells, autophosphorylation and insulin-stimulated Ras.GTP formation.

We showed previously that upon insulin stimulation of an insulin receptor overexpressing cell line, most of the p21ras was rapidly converted into the GTP bound state (Burgering, B. M. T., Medema, R. H., Maassen, J. A., Van de Wetering, M. L., Van der Eb, A. J., McCormick, F., and Bos, J. L. (1991) EMBO J. 10, 1103-1109). To determine whether this process also occurs in cells expressing physiologically relevant numbers of insulin receptors, insulin stimulated Ras.GTP formation was quantitated in Chinese hamster ovary (CHO)-derived cell lines expressing varying numbers of insulin receptors. In the parental CHO9 cells, expressing only 5.10(3) insulin receptors, insulin stimulation for 3 min increased Ras.GTP levels with 10%. Upon increasing the number of insulin receptors in these cells, Ras.GTP levels increased almost proportionally until a plateau value of 60% is reached at high receptor numbers. These data show that receptor overexpression is not a prerequisite for insulin-stimulated Ras.GTP formation. The yield of Ras.GTP generated is 0.2-1.0 mol/mol autophosphorylated insulin receptor in CHO9- and NIH3T3-derived cell lines, respectively. These values argue against signal-amplifying processes between the insulin receptor and p21ras. To determine whether receptor autophosphorylation is required for Ras.GTP formation, NIH3T3 cells overexpressing insulin receptors were stimulated with a monoclonal antibody which activates the receptor and subsequent glucose transport without inducing detectable autophosphorylation. Also, CHO cells expressing the mutant Ser1200 receptor, which has markedly impaired tyrosyl autophosphorylation but is capable of mediating insulin-stimulated metabolic effects in CHO cells, were used. In both cases, no Ras.GTP formation was observed. Furthermore, Rat-1-derived cell lines expressing mutant p21ras, which is permanently in the active GTP-bound form, still responded to insulin by increasing the glucose uptake. These results support our hypothesis that Ras.GTP formation is activated by the tyrosyl-phosphorylated insulin receptor and suggest that an active Ras.GTP complex does not mediate metabolic signaling.

Insulin activation of protein kinase C: a reassessment

Although insulin is known to activate several protein serine/threonine protein kinases, its ability to activate protein kinase C remains controversial. We reinvestigated this question, taking advantage of several technical advances such as the development of fibroblast cell lines that overexpress normal human insulin receptors, and the development of antibodies to and expression vectors for the myristoylated, alanine-rich C kinase substrate (MARCKS) protein, a major cellular substrate for protein kinase C. In HIR 3.5 cells, a mouse 3T3 cell derivative that expresses about 6 x 10(6) human insulin receptors/cell, insulin (70 nM for 10 min) stimulated phosphorylation of the MARCKS protein by approximately 2-fold (p less than 0.005). This phosphorylation was not further increased by different times of insulin exposure, different insulin concentrations, or longer periods of serum deprivation. The insulin stimulation represented about 14% of the response to phorbol 12-myristate 13-acetate and about 17% of the response to 10% fetal calf serum. No significant stimulation of MARCKS protein phosphorylation was seen in four other insulin-sensitive cell lines, in which insulin is known to activate other protein serine/threonine kinases: HIRC-B, BC3H-1, 3T3-L1 adipocytes, and H35 rat hepatoma cells made to stably express the MARCKS protein. In these four cell lines, serum and/or phorbol 12-myristate 13-acetate exerted a large stimulatory effect on MARCKS protein phosphorylation. We conclude that insulin may activate protein kinase C to a minor extent in certain cell types that vastly overexpress insulin receptors; however, we believe that this effect of insulin is unlikely to be of physiological importance.

High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells.

In order to develop a simple, efficient system for the high-level expression of human insulin receptors in eukaryotic cells, a full-length human kidney insulin receptor cDNA was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH 3T3 cells with this construct, seven cell lines expressing insulin receptors were isolated; two cell lines had more than 10(6) receptors per cell. The cell line with the highest insulin binding (NIH 3T3 HIR3.5) had 6 X 10(6) receptors with a Kd of 10(-9) M. This level was not dependent on exposure to metals but could be increased further to 2 X 10(7) receptors per cell by addition of sodium butyrate to the culture medium. The alpha and beta subunits had apparent molecular weights of 147,000 and 105,000, respectively (compared to 135,000 and 95,000 in IM-9 human lymphocytes), values identical to those of the alpha and beta subunits of the insulin receptors of nontransformed NIH 3T3 cells. This size difference was due to altered carbohydrate composition, as N-glycanase digestion reduced the apparent receptor subunit size of the transfected cells and IM-9 lymphocytes to identical values. The alteration in N-linked oligosaccharide composition could not be ascribed to differences in the kinetics of posttranslational processing of the insulin receptors, which was comparable to that of other cells studied. The basal rate of glycogen synthesis in the cells overexpressing insulin receptors was increased 4- to 5-fold compared with controls. Low levels of added insulin (0.1 nM) caused a 50% increase in the rate of glycogen synthesis.