CHO-T Cells Research Articles

The triphenylmethane dye brilliant blue G is only moderately effective at inhibiting amyloid formation by human amylin or at disaggregating amylin amyloid fibrils, but interferes with amyloid assays; Implications for inhibitor design.

The development of inhibitors of islet amyloid formation is important as pancreatic amyloid deposition contributes to type-2 diabetes and islet transplant failure. The Alzheimer’s Aβ peptide and human amylin (h-amylin), the polypeptide responsible for amyloid formation in type-2 diabetes, share common physio-chemical features and some inhibitors of Aβ also inhibit amyloid formation by h-amylin and vice versa. Thus, a popular and potentially useful strategy to find lead compounds for anti-amylin amyloid agents is to examine compounds that have effects on Aβ amyloid formation. The triphenylmethane dye, brilliant blue G (BBG, Sodium;3-[[4-[(E)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-N-ethyl-3-methylanilino]methyl]benzenesulfonate) has been shown to modulate Aβ amyloid formation and inhibit Aβ induced toxicity. However, the effects of BBG on h-amylin have not been examined, although other triphenylmethane derivatives inhibit h-amylin amyloid formation. The compound has only a modest impact on h-amylin amyloid formation unless it is added in significant excess. BBG also remodels preformed h-amylin amyloid fibrils if added in excess, however BBG has no significant effect on h-amylin induced toxicity towards cultured β-cells or cultured CHO-T cells except at high concentrations. BBG is shown to interfere with standard thioflavin-T assays of h-amylin amyloid formation and disaggregation, highlighting the difficulty of interpreting such experiments in the absence of other measurements. BBG also interferes with ANS based assays of h-amylin amyloid formation. The work highlights the differences between inhibition of Aβ and h-amylin amyloid formation, illustrates the limitation of using Aβ inhibitors as leads for h-amylin amyloid inhibitors, and reinforces the difficulties in interpreting dye binding assays of amyloid formation.

Direct Imaging of Mobile Nanodomains in the Live Cell Plasma Membrane by using a Two-Color Photobleaching Approach

We recently developed a method termed TOCCSL (Moertelmaier,APL-2005) (‘Thinning out Clusters while Conserving Stoichiometry of Labeling’) which allowed for the first time the direct imaging of nanoscopic stable platforms with raft-like properties diffusing in the live cell plasma membrane (Brameshuber,JBC-2010). Our method sensed these platforms by their property to assemble the putative raft markers glycosylphosphatidylinositol-anchored monomeric GFP (mGFP-GPI) and GM1-Bodipy on a time-scale of seconds on the cell membrane of living CHO and Jurkat T-cells. In order to resolve platforms we used a special photobleaching protocol to reduce the surface density of labeled mobile platforms down to the level of well-isolated diffraction-limited spots, without altering the single spot brightness. The statistical distribution of probe molecules per platform was determined by single-molecule brightness analysis. To further validate our method we extended TOCCSL by utilizing two-color co-localization in combination with stoichiometric photobleaching (Ruprecht,SoftMatter-2010). Glycosylphosphatidylinositol-anchored SNAP-tag (GPI-SNAP) was stably expressed by CHO cells and labeled with SNAP-AF488 and SNAP-AF647 at an equimolar ratio. For both colors we were able to observe a homogeneous surface staining with a density of 300-1000 molecules/μm². To reduce the surface density we applied TOCCSL parallel to both color channels. An appropriate recovery time yielded a stoichiometrically reduced surface density enabling the localization of single fluorescent GPI-SNAPs in both color channels with a resolution down to a few ten nanometers. After correction of chromatic aberration we used an automatic algorithm to detect pairs of co-localized GPI-SNAPs within a diffraction limited distance. The number of co-localized GPI-SNAPs and their stability was found to be comparable to our previous measured mGFP-GPI data: around 30% of GPI-SNAPs homo-associated with a lifetime of seconds and disappeared after cholesterol-oxidase treatment - thus supporting the lipid raft concept.

Techniques for Direct Imaging of Nanoplatforms in the Live Cell Plasma Membrane

We recently developed a method termed TOCCSL (‘Thinning out Clusters while Conserving Stoichiometry of Labeling') which allows for the first time the direct imaging of nanoscopic stable platforms with raft-like properties diffusing in the live cell plasma membrane. Our method senses these platforms by their property to assemble a characteristic set of fluorescent marker-proteins/lipids on a time-scale of seconds. A special photobleaching protocol was used to reduce the surface density of labeled mobile platforms down to the level of well-isolated diffraction-limited spots, without altering the single spot brightness. The statistical distribution of probe molecules per platform was determined by single molecule brightness analysis. For demonstration, we used the consensus raft marker glycosylphosphatidylinositol-anchored monomeric GFP and the fluorescent lipid analogue Bodipy-GM1 which preferentially partitions into liquid ordered phases. For both markers we found cholesterol-dependent homo-association in the plasma membrane of living CHO and Jurkat T-cells in the resting state, thereby demonstrating the existence of small, mobile, stable platforms containing these probes.To further validate our method we extended TOCCSL by utilizing two-color co-localization and photo-activation. While two-color TOCCSL allows for direct imaging of mobile nanoplatforms containing different probe molecules and thus supporting the lipid raft concept, photo-activation based TOCCSL addresses an additional population of observed probes. Since TOCCSL is suitable for characterizing the mobile fraction of marker-proteins/lipids, the information about slowly diffusing or immobile nanoplatforms is not accessible. This can be circumvented by substituting the fluorescent marker by a photo-activatable protein linked to the molecule of interest. By irreversibly switching a small fraction of markers from a dark into the fluorescent state single molecule brightness and diffusion analysis after activation will add to the characterization of nanodomains.Key Publication: Brameshuber et al., JBC 2010, 285(53): 411765-71

Functional dissociation between PIKfyve-synthesized PtdIns5P and PtdIns(3,5)P2 by means of the PIKfyve inhibitor YM201636

PIKfyve is an essential mammalian lipid kinase with pleiotropic cellular functions whose genetic knockout in mice leads to preimplantation lethality. Despite several reports for PIKfyve-catalyzed synthesis of phosphatidylinositol 5-phosphate (PtdIns5P) along with phosphatidylinositol-3,5-biphosphate [PtdIns(3,5)P(2)] in vitro and in vivo, the role of the PIKfyve pathway in intracellular PtdIns5P production remains underappreciated and the function of the PIKfyve-synthesized PtdIns5P pool poorly characterized. Hence, the recently discovered potent PIKfyve-selective inhibitor, the YM201636 compound, has been solely tested for inhibiting PtdIns(3,5)P(2) synthesis. Here, we have compared the in vitro and in vivo inhibitory potency of YM201636 toward PtdIns5P and PtdIns(3,5)P(2). Unexpectedly, we observed that at low doses (10-25 nM), YM201636 inhibited preferentially PtdIns5P rather than PtdIns(3,5)P(2) production in vitro, whereas at higher doses, the two products were similarly inhibited. In cellular contexts, YM201636 at 160 nM inhibited PtdIns5P synthesis twice more effectively compared with PtdIns(3,5)P(2) synthesis. In 3T3L1 adipocytes, human embryonic kidney 293 and Chinese hamster ovary (CHO-T) cells, levels of PtdIns5P dropped by 62-71% of the corresponding untreated controls, whereas those of PtdIns(3,5)P(2) fell by only 28-46%. The preferential inhibition of PtdIns5P versus PtdIns(3,5)P(2) at low doses of YM201636 was explored to probe contributions of the PIKfyve-catalyzed PtdIns5P pool to insulin-induced actin stress fiber disassembly in CHO-T cells, GLUT4 translocation in 3T3L1 adipocytes, and induction of aberrant cellular vacuolation in these or other cell types. The results provide the first experimental evidence that the principal pathway for PtdIns5P intracellular production is through PIKfyve and that insulin effect on actin stress fiber disassembly is mediated entirely by the PIKfyve-produced PtdIns5P pool.

Identification of elongation factor 1α as a potential associated binding partner for Akt2

Akt protein kinase has been shown to play a pivotal role in diverse cell functions, including motility, apoptosis, growth and metabolism. How it differentially regulates these diverse functions is of significant interest. Three isoforms have been well characterized, Akt1, 2, and 3, encoded by separate genes, but showing high homology over the entire coding sequence (> 80%). An area of variability between the three isoforms is the C-terminal tail. To find potentially regulating binding partners of Akt2, the isoform implicated in metabolic control, we used a glutathione-S-transferase (GST) fusion protein expressing the C-terminal 75 residues of Akt2 (GST-Akt2 tail) to screen for proteins that specifically bound the Akt2 tail. Elongation factor 1alpha (EF1alpha) and beta-tubulin were identified as binding partners for the Akt2 tail by peptide mass fingerprinting. These two proteins have themselves been previously identified as interacting partners (Nakazawa et al.FEBS Lett. 453,29-34, 1999). Using CHOT cells that overexpress insulin receptors and HA-tagged Akt2, we showed that EF1alpha co-immunoprecipitated with HA-tagged Akt2. It is thus possible that these proteins colocalise as part of a regulatory signaling complex with the cytoskeleton directing them to sites of cell activity.

Epi‐CHO, an episomal expression system for recombinant protein production in CHO cells

This study describes the development of a transient expression system for CHO cells based on autonomous replication and retention of transfected plasmid DNA. A transient expression system that allows extrachromosomal amplification of plasmids permits more plasmid copies to persist in the transfected cell throughout the production phase leading to a significant increase in transgene expression. The expression system, named Epi-CHO comprises (1) a CHO-K1 cell line stably transfected with the Polyomavirus (Py) large T (LT) antigen gene (PyLT) and (2) a DNA expression vector, pPyEBV encoding the Py origin (PyOri) for autonomous plasmid amplification and encoding Epstein-Barr Virus (EBV) nuclear antigen-1 (EBNA-1) and OriP for plasmid retention. The CHO-K1 cell line expressing PyLT, named CHO-T was adapted to suspension growth in serum-free media to facilitate large-scale transient transfection and recombinant gene expression. Enhanced green fluorescent protein (EGFP) and human growth hormone (hGH) were used as reporter proteins to demonstrate transgene expression and productivity. Transfection of suspension-growing CHO-T cells with the vector pPyEBV encoding hGH resulted in a final concentration of 75 mg L(-1) of hGH in culture supernatants 11 days following transfection.

Role for a novel signaling intermediate, phosphatidylinositol 5-phosphate, in insulin-regulated F-actin stress fiber breakdown and GLUT4 translocation.

The cellular functions and regulation of phosphatidylinositol (PtdIns) 5-phosphate (5-P), the newest addition to the family of phosphoinositides (PIs), are still elusive. Here we have examined a plausible role of PtdIns 5-P as a signaling intermediate in acute insulin action. A wortmannin-insensitive transient increase of PtdIns 5-P mass levels that peaked at 10 min, and declined 20-30 min after insulin stimulation, was observed in both Chinese hamster ovary (CHO)-T cells stably expressing the insulin receptor and 3T3-L1 adipocytes. Similarly to insulin, found to induce a rapid disassembly of Texas-Red phalloidin-labeled actin stress fibers in CHO-T cells, microinjected PtdIns 5-P, but not other PIs, decreased the number and length of F-actin stress fibers in this cell type to a magnitude seen in response to insulin. Likewise, increases of PtdIns 5-P by ectopic expression of the PtdIns 5-P-producing enzyme PIKfyve yielded a similar effect. As with insulin, the PtdIns 5-P-induced loss of actin stress fibers was independent of PI 3-kinase activation. Furthermore, sequestration of functional PtdIns 5-P, either by ectopic expression of 3xPHD domains that bind selectively PtdIns 5-P or by microinjecting the GST-3xPHD fusion peptide, abrogated insulin-induced F-actin stress fiber disassembly in CHO-T cells. In 3T3-L1 adipocytes, microinjected PtdIns 5-P, but not other PIs, partially mimicked insulin's effect of translocating enhanced green fluorescent protein-GLUT4 to the cell surface. Conversely, insulin-induced myc-GLUT4 vesicle dynamics was arrested in the presence of coexpressed enhanced green fluorescent protein-3xPHD. Involvement of PIKfyve membrane recruitment, but not activation, and/or a decrease in PtdIns 4,5-bisphosphate levels are likely to be among the mechanisms underlying the insulin-induced PtdIns 5-P increase. Together, these results identify PtdIns 5-P as a novel key intermediate for insulin signaling in F-actin remodeling and GLUT4 translocation.

Insulin stimulates movement of sorting nexin 9 between cellular compartments: a putative role mediating cell surface receptor expression and insulin action.

SNX9 (sorting nexin 9) is one member of a family of proteins implicated in protein trafficking. This family is characterized by a unique PX (Phox homology) domain that includes a proline-rich sequence and an upstream phospholipid binding domain. Many sorting nexins, including SNX9, also have a C-terminal coiled region. SNX9 additionally has an N-terminal SH3 (Src homology 3) domain. Here we have investigated the cellular localization of SNX9 and the potential role it plays in insulin action. SNX9 had a cytosolic and punctate distribution, consistent with endosomal and cytosolic localization, in 3T3L1 adipocytes. It was excluded from the nucleus. The SH3 domain was responsible, at least in part, for the membrane localization of SNX9, since expression of an SH3-domain-deleted GFP (green fluorescent protein)-SNX9 fusion protein in HEK293T cells rendered the protein cytosolic. Membrane localization may also be attributed in part to the PX domain, since in vitro phospholipid binding studies demonstrated SNX9 binding to polyphosphoinositides. Insulin induced movement of SNX9 to membrane fractions from the cytosol. A GST (glutathione S-transferase)-SNX9 fusion protein was associated with IGF1 (insulin-like growth factor 1) and insulin receptors in vitro. A GFP-SNX9 fusion protein, overexpressed in 3T3L1 adipocytes, co-immunoprecipitated with insulin receptors. Furthermore, overexpression of this GFP-SNX9 fusion protein in CHOT cells decreased insulin binding, consistent with a role for SNX9 in the trafficking of insulin receptors. Microinjection of 3T3L1 cells with an antibody against SNX9 inhibited stimulation by insulin of GLUT4 translocation. These results support the involvement of SNX9 in insulin action, via an influence on the processing/trafficking of insulin receptors. A secondary role in regulation of the cellular processing, transport and/or subcellular localization of GLUT4 is also suggested.

Extracellular Matrix Proteins Modulate Endocytosis of the Insulin Receptor

Internalization of the insulin receptor (IR) is a highly regulated multi-step process whose underlying molecular basis is not fully understood. Here we undertook to study the role of extracellular matrix (ECM) proteins in the modulation of IR internalization. Employing Chinese hamster ovary cells that overexpress IR (CHO-T cells), our results indicate that IR internalization proceeds unaffected even when Tyr phosphorylation of IR substrates, such as IRS-1, is impaired (e.g. in CHO-T cells overexpressing IRS-1 whose pleckstrin-homology domain has been deleted or in CHO-T cells that overexpress the PH/PTB domain of IRS-1). In contrast, IR internalization is affected by the context of the ECM proteins to which the cells adhere. Hence, IR internalization was inhibited 40-60% in CHO-T cells adherent onto galectin-8 (an ECM protein and an integrin ligand of the galectin family) when compared with cells adherent onto fibronectin, collagen, or laminin. Cells adherent to galectin-8 manifested a unique cytoskeletal organization, which involved formation of cortical actin and generation of F-actin microspikes that contrasted with the prominent stress-fibers formed when cells adhered to fibronectin. To better establish a role for actin filament organization in IR endocytosis, this process was assayed in CHO-T cells (adherent onto fibronectin), whose actin filaments were disrupted upon treatment with latrunculin B. Latrunculin B did not affect insulin-induced Tyr phosphorylation of IR or its ability to phosphorylate its substrates; still, a 30-50% reduction in the rate of IR internalization was observed in cells treated with latrunculin B. Treatment of cells with nocodazole, which disrupts formation of microtubules, did not affect IR internalization. These results indicate that proper actin, but not microtubular, organization is a critical requirement for IR internalization and suggest that integrin-mediated signaling pathways emitted upon cell adhesion to different extracellular matrices and the altered cytoskeletal organizations generated thereof affect the itinerary of the insulin receptor.

Demonstration of glycated insulin in human diabetic plasma and decreased biological activity assessed by euglycemic-hyperinsulinemic clamp technique in humans.

The presence and biological significance of circulating glycated insulin has been evaluated by high-pressure liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS), radioimmunoassay (RIA), receptor binding, and hyperinsulinemic-euglycemic clamp techniques. ESI-MS analysis of an HPLC-purified plasma pool from four male type 2 diabetic subjects (HbA(1c) 8.1 +/- 0.2%, plasma glucose 8.7 +/- 1.3 mmol/l [means +/- SE]) revealed two major insulin-like peaks with retention times of 14-16 min. After spectral averaging, the peak with retention time of 14.32 min exhibited a prominent triply charged (M+3H)(3+) species at 1,991.1 m/z, representing monoglycated insulin with an intact M(r) of 5,970.3 Da. The second peak (retention time 15.70 min) corresponded to native insulin (M(r) 5,807.6 Da), with the difference between the two peptides (162.7 Da) representing a single glucitol adduct (theoretical 164 Da). Measurement of glycated insulin in plasma of type 2 diabetic subjects by specific RIA gave circulating levels of 10.1 +/- 2.3 pmol/l, corresponding to approximately 9% total insulin. Biological activity of pure synthetic monoglycated insulin (insulin B-chain Phe(1)-glucitol adduct) was evaluated in seven overnight-fasted healthy nonobese male volunteers using two-step euglycemic-hyperinsulinemic clamps (2 h at 16.6 micro g x kg(-1) x min(-1), followed by 2 h at 83.0 micro g x kg(-1) x min(-1); corresponding to 0.4 and 2.0 mU x kg(-1) x min(-1)). At the lower dose, the exogenous glucose infusion rates required to maintain euglycemia during steady state were significantly lower with glycated insulin (P < 0.01) and approximately 70% more glycated insulin was required to induce a similar rate of insulin-mediated glucose uptake. Maximal responses at the higher rates of infusion were similar for glycated and control insulin. Inhibitory effects on endogenous glucose production, insulin secretion, and lipolysis, as indicated by measurements of C-peptide, nonesterified free fatty acids, and glycerol, were also similar. Receptor binding to CHO-T cells transfected with human insulin receptor and in vivo metabolic clearance revealed no differences between glycated and native insulin, suggesting that impaired biological activity is due to a postreceptor effect. The present demonstration of glycated insulin in human plasma and related impairment of physiological insulin-mediated glucose uptake suggests a role for glycated insulin in glucose toxicity and impaired insulin action in type 2 diabetes.

Insulin and secretagogues differentially regulate fluid-phase pinocytosis in insulin-secreting beta-cells.

The physiological role of the beta-cell insulin receptor is unknown. To evaluate a candidate function, the insulin regulation of fluid-phase pinocytosis was investigated in a clonal insulinoma cell line (beta TC6-F7) and, for comparison, also in Chinese hamster ovary cells transfected with the human insulin receptor (CHO-T cells). In CHO-T cells, the net rate of fluid-phase pinocytosis was rapidly increased 3-4-fold over the basal rate by 100 nM insulin, with half-maximal stimulation at 2 nM insulin, as assayed by cellular uptake of horseradish peroxidase from the medium. Wortmannin, an inhibitor of phosphatidylinositol (PI)-3-kinase, blocked insulin-stimulated pinocytosis with an IC50 of 7.5 nM without affecting the basal rate of pinocytosis. In insulin-secreting beta TC6-F7 cells, the secretagogues glucose and carbachol (at maximally effective concentrations of 15 mM and 0.5 mM respectively) augmented fluid-phase pinocytosis 1.65-fold over the basal rate. Wortmannin also inhibited secretagogue-stimulated pinocytosis in these beta-cells with an IC50 of 7 nM but did not affect the basal rate of pinocytosis measured in the absence of secretagogues. Wortmannin did not influence either basal or secretagogue-induced insulin secretion. Although these beta TC6-F7 cells have cell-surface insulin receptors, adding exogenous insulin or insulin-like growth factor 1 did not affect their rate of fluid-phase pinocytosis, either in the absence or presence of secretagogues. From these observations, we conclude that: (1) in both insulin-secreting beta-cells and in conventional, insulin-responsive CHO-T cells, a common, wortmannin-sensitive reaction, which probably involves PI-3-kinase, regulates fluid-phase pinocytosis; (2) the insulin-receptor signal transduction pathway is dissociated from the regulation of fluid-phase pinocytosis in the insulin-secreting beta-cell line we studied; and (3) the enhancement of fluid-phase pinocytosis associated with secretagogue-induced insulin release in beta TC6-F7 cells is not attributable to autocrine activation of beta-cell surface insulin receptors.

Insulin Regulation of Membrane-associated Insulin Receptor Substrate 1

Insulin stimulation of 3T3-L1 adipocytes results in rapid activation of the insulin receptor tyrosine kinase followed by autophosphorylation of the receptor and phosphorylation of insulin receptor substrate 1 (IRS-1), its major substrate. The insulin receptor resides mostly at the cell surface of 3T3-L1 adipocytes under basal conditions, while about two-thirds of IRS-1 fractionates with intracellular membranes and one-third fractionates with cytosol. To test whether insulin receptor internalization is required for optimal tyrosine phosphorylation of IRS-1, 3T3-L1 adipocytes and CHO-T cells were incubated at 4 degrees C which inhibits receptor endocytosis but not its tyrosine kinase activity. Under these conditions, tyrosine phosphorylation of IRS-1 in the low density microsome fraction in response to insulin was as intense as that observed at 37 degrees C, indicating that endocytosis of insulin receptors is not necessary for tyrosine phosphorylation of IRS-1 to occur. Surprisingly, at 37 degrees C, insulin action on 3T3-L1 adipocytes progressively decreased the amount of IRS-1 protein associated with the low density microsome fraction and increased that in the cytosol. This redistribution of IRS-1 from the low density microsome fraction to the cytosol in response to insulin was accompanied by decreased electrophoretic mobility of IRS-1 on SDS-polyacrylamide gel electrophoresis. Incubation of adipocytes at 4 degrees C blocked the appearance of tyrosine-phosphorylated IRS-1 in the cytosol. Taken together, these data indicate that insulin receptors phosphorylate IRS-1 at the cell surface, perhaps in coated pits which are included in the low density microsome fraction. The results also suggest a desensitization mechanism in which the tyrosine-phosphorylated membrane-bound IRS-1, associated with signaling molecules such as phosphatidylinositol 3-kinase, is released into the cytoplasm in concert with its serine/threonine phosphorylation.

Regulation by insulin of phosphatidylinositol 3'-kinase bound to alpha- and beta-isoforms of p85 regulatory subunit.

The roles of the alpha- and beta-isoforms of phosphatidylinositol (PI) 3'-kinase p85 regulatory subunit were studied with isoform-specific antisera in three model systems in which the insulin receptor mediates rapid phosphorylation of insulin receptor substrate-1 (IRS-1). Insulin receptor signaling stimulated the association of IRS-1 with p85 alpha protein, and p85 alpha-associated PI 3-kinase activity in 3T3-L1 adipocytes, and in transfected Chinese hamster ovary cells (CHO-T) and COS-1 cells expressing high levels of human insulin receptors. While not detectable in 3T3-L1 adipocytes, the p85 beta isoform was also found to associate with IRS-1 in response to insulin receptor activation in COS-1 and CHO-T cells. However, selective immunoprecipitation of p85 beta from unstimulated COS-1 or CHO-T cell lysates was accompanied by higher levels of PI 3-kinase activity than that associated with p85 alpha. Remarkably, the large stimulation of PI 3-kinase activity associated with p85 alpha (7.8 +/- 2.0-fold, n = 6) in insulin-treated CHO-T cells was not observed in p85 beta immunoprecipitates (1.8 +/- 0.6-fold, n = 6), and in COS-1 cells p85 beta-associated PI 3-kinase activity was completely insensitive to stimulation by the insulin receptor. These data suggest the novel hypothesis that binding of p85 beta to IRS-1 complexes in COS-1 and CHO-T cells does not mediate marked activation of PI 3-kinase activity as does p85 alpha.

The pathway mediating insulin's effects on pyruvate dehydrogenase bypasses the insulin receptor tyrosine kinase

The effect of insulin on pyruvate dehydrogenase activity was examined in two different cell types that over expressed either normal or defective human insulin receptors, RAT 1 embryonic fibroblasts and Chinese hamster ovary (CHO) cells. Insulin stimulated pyruvate dehydrogenase activity in cells that expressed normal insulin receptors (RAT 1 HIRc, and CHO-WT and CHO-T cells), or receptors in which lysine 1018 in the ATP-binding site of the tyrosine kinase domain was exchanged for alanine (RAT 1 A/K1018 and CHO-mut cells). For both rat and hamster cell lines, the insulin dose-response curves from cells that expressed the mutant receptors were identical to those from the appropriate controls that over expressed the normal insulin receptors. Insulin failed to stimulate pyruvate dehydrogenase activity in CHO-delta cells, which expressed a mutant human insulin receptor that was truncated by 112 amino acids at the carboxyl terminal of the beta chain. Control studies verified that all the cells used in this study exhibited the expected phenotypes with respect to the number of insulin receptors which they expressed, insulin-stimulated tyrosine kinase activity, and the biological consequences of inactivating the insulin receptor tyrosine kinase. These findings show that the insulin receptor tyrosine kinase does not play an obligatory role in the insulin signaling pathway that stimulates pyruvate dehydrogenase activity.

Expression of Atypical and Classical Insulin Receptors in Chinese Hamster Ovary Cells Transfected with Cloned cDNA for the Human Insulin Receptor*

Human placenta and IM-9 lymphocytes contain subpopulations of atypical insulin receptors which differ from classical insulin receptors in their higher binding affinity for insulin-like growth factors I and II (IGF-I and IGF-II). Both types of insulin receptors may be derived from different but related genes, or may represent alternative post-translational modifications of the same gene product. To test these possibilities, we have examined the IGF binding characteristics of the human insulin receptors expressed in Chinese hamster ovary (CHO) cells which had been stably transfected with cloned human insulin receptor cDNA (CHO-T cells). The parent CHO cells contained 3 x 10(3) rodent insulin receptors/cell, and the CHO-T cells, 2.0 x 10(6) human insulin receptors/cell. Competition binding studies showed that the binding of [125I]IGF-I and [125I]multiplication stimulating activity (MSA/rat IGF-II) to parent CHO cells was primarily to type I and II IGF receptors, which cross-react poorly or not at all with insulin. However, competition binding studies with CHO-T cells showed that [125I]IGF-I binding was displaced 60-70%, and [125I]MSA binding, 50-55%, by low concentrations of insulin (20 ng/ml) and no further by higher concentrations of insulin (500 ng/ml). The insulin-insensitive IGF binding sites corresponded to the rodent type I and II IGF receptors; the insulin-sensitive IGF binding sensitive sites resembled the human atypical insulin receptors in that they bound IGF-I and MSA with moderately high affinity and reacted with insulin, MSA, and IGF-I in that order of potency. Atypical insulin receptors were also demonstrated by insulin-sensitive [125I]IGF-I and [125I]MSA binding to solubilized CHO-T proteins adsorbed to microtiter wells coated with monoclonal antibodies specific for the human insulin receptor. These results suggest that atypical human insulin receptors are generated by differential post-translational processing of the same gene product as classical human insulin receptors.