Insulin and IGF-I action on insulin receptors, IGF-I receptors, and hybrid insulin/IGF-I receptors in vascular smooth muscle cells

Insulin and insulin-like growth factor I (IGF-I) are known to affect cardiovascular disease. We have investigated ligand binding and the dose-response relationship for insulin and IGF-I on vascular smooth muscle cells (VSMCs) at the receptor level. VSMCs from rat thoracic aorta were serum starved, stimulated with IGF-I or insulin, lysed, immunoprecipitated, and analyzed by Western blot. d-[U-(14)C]Glucose accumulation and [6-(3)H]thymidine incorporation into DNA were also measured. Specific binding of both insulin and IGF-I was demonstrated, being higher for IGF-I. Both IGF-I receptor (IGF-IR) and insulin receptor (IR) beta-subunits were detected and coprecipitated after immunoprecipitation (IP) against either of the two. No coprecipitation was found after reduction of disulphide bonds with dithiotreitol before IP. After stimulation with 10(-10)-10(-9) M IGF-I, IP of the IGF-IR, or IR beta-subunit and immunoblot with anti-phosphotyrosine antibody, we found two distinct bands indicating phosphorylation of both the IGF-IR and the IR beta-subunit. Stimulation with 10(-10)-10(-9) M insulin and IP against the IGF-IR did not show phosphorylation of either beta-subunit, whereas after IP of the IR we found phosphorylation of the IR beta-subunit. [(14)C]Glucose accumulation and [(3)H]thymidine incorporation were elevated in cells stimulated with IGF-I at 10(-10)-10(-7) M, reaching maximum by 10(-9) M. Insulin stimulation showed measurable effects only at supraphysiological concentrations, 10(-8)-10(-7) M. In conclusion, coprecipitation of both the IGF-IR and the IR beta-subunit indicates the presence of hybrid insulin/IGF-I receptors in VSMC. At a physiological concentration, insulin activates the IR but does not affect either glucose metabolism or DNA synthesis, whereas IGF-I both activates the receptor and elicits biological effect.     

Insulin stimulates phosphorylation of serine residues in soluble insulin receptors

Using lectin affinity-purified receptor preparations from human hepatoma cells, insulin (10(-7)M) specifically stimulated phosphorylation of the 95,000 dalton (beta) subunit of its own receptor. Phospho-amino acid analysis of the receptor subunit revealed that insulin increased at least 2.5-fold the content of phosphoserine and of phosphotyrosine. In intact cells, the major effect of insulin is to increase the phosphoserine content of its receptor. These findings are the first demonstration of an insulin-stimulated serine kinase in a cell-free system.     

Evidence for hybrid rodent and human insulin receptors in transfected cells.

Chinese hamster ovary cells and NIH 3T3 cells overexpressing mutant human insulin receptors were examined for the presence of hybrid receptors composed of human and rodent insulin receptors. In the present studies, most of the endogenous rodent receptors were found to be immunoprecipitated from the transfected cells but not the parental cells with a monoclonal antibody specific for human receptor. These data indicate that in these transfected cells, most of the endogenous rodent receptors are in a hybrid complex with the overexpressed human receptor. These results together with the in vitro studies of Treadway et al. (Treadway, J.L., Morrison, B.D., Soos, M.A., Siddle, K., Olefsky, J., Ullrich, A., McClain, D.A., and Pessin, J.E. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 214-218) showing that hybrid receptors exhibit transdominant inhibition explain the prior finding indicating that overexpression of defective insulin receptors interferes with the normal signaling of endogenous receptors.     

Differential phosphorylation of IRS-1 and IRS-2 by insulin and IGF-I receptors

The specific contribution of insulin and IGF-I receptors to IRS-protein activation remains elusive. We studied the signalling properties of AspB10-insulin, an analog with enhanced affinity for the IGF-I receptor, in comparison to native insulin using primary human skeletal muscle cells. In myoblasts regular insulin and AspB10-insulin were equipotent in stimulating the IRS cascade, whereas this analog induced a significantly higher Shc phosphorylation. Phosphorylation of IRS-1 in response to insulin was inhibited equally by blocking either the insulin or the IGF-I receptor. IRS-1 activation by AspB10-insulin was only inhibited by blocking the IGF-I receptor. IRS-2 phosphorylation induced by both insulin and AspB10-insulin was nearly insensitive to blocking the insulin receptor, being predominantly mediated by the IGF-I receptor. We conclude that in myoblasts IRS-2, but not IRS-1, functions as preferred substrate for the IGF-I receptor. These data suggest a specific role for IRS-2 in growth and differentiation of human skeletal muscle.     

Multisite and hierarchal protein phosphorylation.

Multisite phosphorylation is a prevalent form of protein modification whose full implications are just beginning to be understood. Multiple protein modifications expand the repertoire of structural changes that can be elicited in proteins and permit more intricate regulatory circuits to operate.     

Differential roles of the insulin and insulin-like growth factor-I (IGF-I) receptors in response to insulin and IGF-I

Insulin and insulin-like growth factor-I (IGF-I) receptors are highly homologous tyrosine kinase receptors that share many common steps in their signaling pathways and have ligands that can bind to either receptor with differing affinities. To define precisely the signaling specific to the insulin receptor (IR) or the IGF-I receptor, we have generated brown preadipocyte cell lines that lack either receptor (insulin receptor knockout (IRKO) or insulin-like growth factor receptor knockout (IGFRKO)). Control preadipocytes expressed fewer insulin receptors than IGF-I receptors (20,000 versus 60,000), but during differentiation, insulin receptor levels increased so that mature adipocytes expressed slightly more insulin receptors than IGF-I receptors (120,000 versus 100,000). In these cells, insulin stimulated IR homodimer phosphorylation, whereas IGF-I activated both IGF-I receptor homodimers and hybrid receptors. Insulin-stimulated IRS-1 phosphorylation was significantly impaired in IRKO cells but was surprisingly elevated in IGFRKO cells. IRS-2 phosphorylation was unchanged in either cell line upon insulin stimulation. IGF-I-dependent phosphorylation of IRS-1 and IRS-2 was ablated in IGFRKO cells but not in IRKO cells. In control cells, both insulin and IGF-I produced a dose-dependent increase in phosphorylated Akt and MAPK, although IGF-I elicited a stronger response at an equivalent dose. In IRKO cells, the insulin-dependent increase in phospho-Akt was completely abolished at the lowest dose and reached only 20% of the control stimulation at 10 nm. Most interestingly, the response to IGF-I was also impaired at low doses, suggesting that IR is required for both insulin- and IGF-I-dependent phosphorylation of Akt. Most surprisingly, insulin- or IGF-I-dependent phosphorylation of MAPK was unaltered in either receptor-deficient cell line. Taken together, these results indicate that the insulin and IGF-I receptors contribute distinct signals to common downstream components in response to both insulin and IGF-I.     

Multisite phosphorylation of the epidermal growth factor receptor. Use of site-directed mutagenesis to examine the role of serine/threonine phosphorylation

The major sites of serine and threonine phosphorylation of the human epidermal growth factor (EGF) receptor observed in intact cells are Thr654, Thr669, Ser1046, and Ser1047. Phosphorylation of the EGF receptor is increased at these sites in cells treated with platelet-derived growth factor or phorbol ester. This increase in EGF receptor phosphorylation is associated with an inhibition of the high affinity binding of EGF to cell surface receptors and an inhibition of the receptor tyrosine protein kinase activity. In order to test the hypothesis that the phosphorylation of the EGF receptor is mechanistically related to the modulation of EGF receptor function, we replaced the major sites of serine and threonine phosphorylation with alanine residues. EGF receptors containing single point mutations or multiple mutations were expressed in Chinese hamster ovary cells. Analysis of the regulation of the EGF receptor tyrosine protein kinase activity demonstrated that phorbol ester caused an inhibition of the tyrosine phosphorylation of wild-type receptors and receptors lacking Thr669, Ser1046, or Ser1047. In contrast, the inhibition of EGF receptor tyrosine phosphorylation caused by phorbol ester was not observed for any of the mutated EGF receptors that lacked Thr654. These data are consistent with the hypothesis that the phosphorylation of the EGF receptor at Thr654 is required for the inhibition of the receptor tyrosine protein kinase activity caused by phorbol ester. Investigation of the apparent affinity of the EGF receptor demonstrated that treatment with phorbol ester caused an inhibition of the high affinity binding of 125I-EGF to cells expressing wild-type EGF receptors and each of the mutated EGF receptors examined. We conclude that the regulation of the apparent affinity of the EGF receptor is independent of the major sites of serine and threonine phosphorylation of the EGF receptor.     

The role of insulin receptors and IGF-I receptors in cancer and other diseases

There is evidence, both in vitro and in vivo, that receptor tyrosine kinases play a key role in the formation and progression of human cancer. In particular, the insulin-like growth factor receptor (IGF-IR), a tyrosine kinase receptor for IGF-I and IGF-II, has been well documented in cell culture, animal studies, and humans to play a role in malignant transformation, progression, protection from apoptosis, and metastasis. In addition, the hormone insulin (which is very closely related to the IGFs) and its tyrosine kinase receptor (the IR, which is very closely related to the IGR-IR) have been documented both in vitro and in vivo to play a key role in cancer biology. Indeed, several epidemiological studies have shown that insulin resistance status, characterized by hyperinsulinaemia, is associated with an increased risk for a number of malignancies, including carcinomas of the breast, prostate, colon and kidney. Recent data have elucidated some molecular mechanisms by which IR is involved in cancer. IR is over-expressed in several human malignancies. Interestingly, one of the two IR isoform (IR-A) is especially over-expressed in cancer. IR-A is the IR foetal isoform and has the peculiar characteristic to bind not only insulin but also IGF-II. In addition, the IR contributes to formation of hybrid receptors with the IGF-IR (HR). By binding to hybrid receptors, insulin may stimulate specific IGF-IR signalling pathways. Over-expression of IR-A is, therefore, a major mechanism of IGF system over-activation in cancer. In this respect, IR-A isoform and hybrid receptors should be regarded as potential molecular targets, in addition to IGF-IR, for novel anti-cancer therapy. These findings may have important implications for both the prevention and treatment of common human malignancies. They underline the concept that hyperinsulinaemia, associated with insulin resistance and obesity, should be treated by changes in life style and/or pharmacological approaches to avoid an increased risk for cancer. Moreover, native insulin and insulin analogue administration should be carefully evaluated in terms of the possible increase in cancer risk.     

Multisite phosphorylation and the countdown to S phase.

Remarkably, SCF(Cdc4) ubiquitin ligase binds and ubiquitinates Sic1 decorated with six, but not five, phosphates. This numerical wizardry suggests how analog inputs can be rectified to digital outputs. Unraveling the counting mechanism promises to generate new insights into the architecture of protein nanoprocessors.     

Interaction between heterologous receptor tyrosine kinases. Hormone-stimulated insulin receptors activate unoccupied IGF-I receptors.

To determine whether heterologous receptor tyrosine kinases interact with each other we have investigated the ability of insulin receptors to transphosphorylate and transactivate IGF-I receptors. Using partially purified receptors we show that hormone-stimulated insulin receptors induced a 40% increase in IGF-I receptor phosphorylation. Remarkably, this transphosphorylation of IGF-I receptors by insulin receptors resulted in a 2.5-fold augmentation of the IGF-I receptor tyrosine kinase activity for substrates. Our findings demonstrate that transphosphorylation with transactivation can occur between insulin and IGF-I receptors. We would like to propose that such a phenomenon participates in the insulin-induced pleiotropic program by mediating the growth promoting effects of the hormone.     

Secretion of soluble functional insulin receptors by transfected NIH3T3 cells

In order to determine whether the human insulin receptor ectodomain can be expressed as a functional protein, the coding regions for the transmembrane and cytoplasmic domain of a full-length human insulin receptor cDNA were deleted by site-directed mutagenesis, and the resultant construct was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH3T3 cells, a cell line secreting an insulin binding protein was isolated. The insulin binding alpha subunit had an Mr of 138,000 and a beta subunit of Mr 48,000 (compared to 147,000 and 105,000 for the full-length human insulin receptor expressed in NIH3T3 cells). This difference in size of the alpha subunit was due to a difference in glycosylation as N-glycanase digestion reduced the apparent size of the alpha subunits of secreted and normal membrane-bound receptors to identical values. The secreted receptor formed disulfide-linked heterotetrameric structures with an Mr of 280,000. It was synthesized as an Mr 160,000 precursor which was cleaved into mature subunits with a t1/2 of 3 h. Increasing expression of the cDNA by induction with sodium butyrate lead to the appearance of an Mr 180,000 protein in the medium as well as the mature alpha and beta subunits. A Scatchard plot of insulin binding to the secreted receptor was curvilinear with a Kd of 7 X 10(-10) M for the high affinity sites and 10(-7) M for the low affinity site (compared to Kd values of 1.1 X 10(-9) M and 10(-7) M, respectively, for human insulin receptors expressed in these cells.     

Aspirin inhibits serine phosphorylation of insulin receptor substrate 1 in tumor necrosis factor-treated cells through targeting multiple serine kinases

The hypoglycemic effects of high dose salicylates in the treatment of diabetes were documented before the advent of insulin. However, the molecular mechanisms by which salicylates exert these anti-diabetic effects are not well understood. In this study, we analyzed the effects of aspirin (acetylsalicylic acid) on serine phosphorylation of insulin receptor substrate 1 (IRS-1) in cells treated with tumor necrosis factor (TNF)-alpha. Phosphorylation of IRS-1 at Ser307, Ser267, and Ser612 was monitored by immunoblotting with phospho-specific IRS-1 antibodies. In 3T3-L1 and Hep G2 cells, phosphorylation of IRS-1 at Ser307 in response to TNF-alpha treatment correlated with phosphorylation of JNK, c-Jun, and degradation of IkappaBalpha. Moreover, phosphorylation of IRS-1 at Ser307 in embryo fibroblasts derived from either JNK or IKK knockout mice was reduced when compared with that in the wild-type controls. Taken together, these data suggest that serine phosphorylation of IRS-1 in response to TNF-alpha is mediated, in part, by JNK and IKK. Interestingly, aspirin treatment inhibited the phosphorylation of IRS-1 at Ser307 as well as the phosphorylation of JNK, c-Jun, and degradation of IkappaBalpha. Furthermore, other serine kinases including Akt, extracellular regulated kinase, mammalian target of rapamycin, and PKCzeta were also activated by TNF-alpha (as assessed by phospho-specific antibodies). Phosphorylation of IRS-1 at Ser267 and Ser612 correlated with the activation of these kinases. Phosphorylation of Akt and the mammalian target of rapamycin (but not extracellular regulated kinase or PKCzeta) in response to TNF-alpha was inhibited by aspirin treatment. Finally, aspirin rescued insulin-induced glucose uptake in 3T3-L1 adipocytes pretreated with TNF-alpha. We conclude that aspirin may enhance insulin sensitivity by protecting IRS proteins from serine phosphorylation catalyzed by multiple kinases.     

Mitogenic effect of the insulin analogue glargine in malignant cells in comparison with insulin and IGF-I.

The aim of the study was to investigate if the insulin analogue glargine, with an increased affinity for the IGF-I receptor (IGF-IR), affects the cell growth to a larger extent than human insulin in malignant cells expressing IGF-IRs. The breast cancer cell lines MCF-7 and SKBR-3, and the osteosarcoma cell line SaOS-2 were used. Gene expression was determined by real-time RT-PCR and receptor protein quantified by ELISAs. Receptor phosphorylation was assessed by immunoprecipitation and Western blot. Mitogenic effect was determined as (3)H-thymidine incorporation into DNA. The gene expression of insulin receptor (IR) varied between 4.3-7.5 x 10(-3) and the expression of IGF-IR between 7.7-147.7 x 10(-3) in relation to GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Insulin receptor and IGF-IR protein varied between 2.0-4.1 ng/mg protein and 2.0-40.4 ng/mg protein, respectively. The IGF-IR was phosphorylated by IGF-I at a concentration of 10(-10)-10(-9) M. All three polypeptides stimulated DNA synthesis in MCF-7, SKBR-3, and SaOS-2 cells. SaOS-2 cells were more sensitive to IGF-I than to insulin and glargine. MCF-7 cells were more sensitive to des(1-3)IGF-I than to IGF-I. In SKBR-3 and SaOS-2 cells, glargine tended to be more potent than human insulin to stimulate DNA synthesis. Our results suggest that glargine, compared to human insulin, has little or no increased mitogenic effect in malignant cells expressing IGF-IRs.     

Immobilization and internalization of mutated IgE receptors in transfected cells.

Earlier studies have shown that the mast cell receptor IgE (Fc epsilon RI) for is expressed on COS-7 cells transfected with the cDNA for each of the three types of subunits that form the tetrameric, alpha beta gamma 2, receptor. Although such transfected COS cells fail to exhibit some of the early biochemical perturbations initiated by aggregation of the receptor on normal mast cells and related tumor lines, we show here that other characteristics of the endogenous Fc epsilon RI are retained. Thus, the unaggregated transfected wild-type receptors were found to have a restricted translational diffusion similar to that observed for endogenous receptors on mast cells as assessed by fluorescence photobleaching and recovery. Similarly, as with endogenous receptors the mobility of transfected receptors was sharply reduced when the receptors were aggregated by reaction with small oligomers of IgE. In addition, aggregation of the transfected Fc epsilon RI caused them to be internalized by the COS cells by a cytochalasin-sensitive mechanism, albeit at a considerably slower rate than was seen with endogenous receptors on mast cells or with transfected receptors in a line of receptor-deficient mast cells. We also examined the mobility and internalization before and after aggregation, of some 13 different combinations of receptor subunit mutants in which one or more of the five cytoplasmic domains of the receptor had been truncated. Our results show that whatever interactions between the receptor and cellular components may account for the phenomena we studied, such interactions do not critically depend upon the bulk of the cytoplasmic domains of the receptor.     

Receptors for insulin and insulin-like growth factor-I can form hybrid dimers. Characterisation of hybrid receptors in transfected cells.

We have demonstrated the formation of hybrid insulin/insulin-like growth factor-I(IGF-I) receptors in transfected rodent fibroblasts, which overexpress human receptors, by examining reactivity with species- and receptor-specific monoclonal antibodies. In NIH 3T3 and Rat 1 fibroblasts, endogenous IGF-I receptors were unreactive with anti-(human insulin receptor)monoclonal antibodies (47-9, 25-49, 83-14, 83-7, 18-44). However, in transfected cells expressing high levels of insulin receptors, 60-80% of high-affinity IGF-I receptors reacted with these antibodies, as assessed either by inhibition of ligand binding in intact cells or by precipitation of solubilized receptors. Conversely, endogenous insulin receptors in NIH 3T3 cells were unreactive with anti-(IGF-I receptor) antibodies alpha IR-3 and 16-13. However, approx. 50% of high-affinity insulin receptors reacted with these antibodies in cells expressing high levels of human IGF-I receptors. The hybrid receptors in transfected cells bound insulin or IGF-I with high affinity. However, responses to these ligands were asymmetrical, in that binding of IGF-I inhibited subsequent binding of insulin, but prior binding of insulin did not affect the affinity for IGF-I. The existence of hybrid receptors in normal tissues could have important implications for metabolic regulation by insulin and IGF-I.     

Dissection of the growth versus metabolic effects of insulin and insulin-like growth factor-I in transfected cells expressing kinase-defective human insulin receptors

We have recently reported that the expression of an in vitro mutated, kinase-defective insulin receptor (A/K1018) leads to cellular insulin resistance when expressed in Rat 1 fibroblasts. That is, despite the presence of normal numbers of activatable native insulin receptors in the host cell, the A/K1018 receptors prevent the normal receptors from phosphorylating endogenous substrates and from signalling insulin action, perhaps by competing for limiting amounts of these substrates. We report here that insulin-like growth factor I-stimulated phosphorylation of two endogenous substrate proteins, pp220 and pp170, is also inhibited in cells expressing A/K1018 receptors. Because insulin-like growth factor I stimulation of glucose uptake is not inhibited in cells with A/K1018 receptors while pp220 and pp170 phosphorylation is inhibited, it is unlikely that either pp220 or pp170 are involved in mediating the stimulation of glucose transport. In contrast, insulin-like growth factor I-mediated stimulation of mitogenesis is inhibited in cells with A/K1018 receptors. Thus, pp170 or pp220 could be involved in mitogenic signalling. We also report that both H2O2 and tetradecanoylphorbolacetate stimulate glucose transport normally in cells with A/K1018 receptors. Phorbol esters also lead to the phosphorylation of both normal and A/K1018 receptors on serine and/or threonine. This argues that phorbol esters or H2O2 bypass the normal proximal steps in signalling insulin action.     

Role of tyrosine sulfation and serine phosphorylation in the processing of procholecystokinin to amidated cholecystokinin and its secretion in transfected AtT-20 cells

Mammalian procholecystokinin (pro-CCK) is known to have three sulfated tyrosine residues, one of which is present in the CCK 8 moiety and two additional residues present in the carboxyl-terminal extension. In the present study, inhibition of tyrosine sulfation by sodium chlorate decreased the secretion of processed CCK 8 in CCK-expressing endocrine cells in culture. It was then demonstrated that when each of these tyrosines individually, as well as all three together, was mutated to phenylalanine and expressed in endocrine cells, CCK was still processed and secreted. However, the amount of CCK secreted varied with the type of mutation. Substitution of Phe to Tyr in CCK 8 reduced the quantity of secreted CCK 8 by 50%, and when all the sulfated Tyr were mutated to Phe the quantity of secreted CCK was reduced by about 70%, similar to what is observed with chlorate treatment. Changing of the putative phosphorylation site serine to alanine does not affect the processing. Serine phosphorylation at this site may play a functional role in regulatory events. Our results demonstrate that tyrosine sulfation alters the amount of secretion but is not an absolute requirement for the processing and secretion of CCK in this cell line. Tyrosine sulfation of CCK may still be important for its solubility, stabilization, and/or functional interaction.     

Activation of delta-, kappa-, and mu-opioid receptors induces phosphorylation of tuberin in transfected HEK 293 cells and native cells

A number of G protein-coupled receptors have been shown to stimulate tuberin phosphorylation, which is critical for the regulation of translation and is apparently involved in neurotrophin-promoted survival of serum-deprived cells. Here, in HEK 293 cells transiently expressing the delta-, kappa-, or mu-opioid receptors, Western blotting analysis using a phosphospecific anti-tuberin antibody revealed a dose- and time-dependent increase in tuberin phosphorylation upon stimulation by specific opioid agonists. In NG108-15, PC12, and SH-SY5Y cells that endogenously express delta-, kappa-, and mu-opioid receptors, respectively, specific opioid agonists also stimulated tuberin phosphorylation in a dose- and time-dependent manner. Pretreatment of cells with pertussis toxin or PI3K inhibitor wortmannin blocked the opioid-stimulated tuberin phosphorylation, implicating the possible involvement of the G(i/o) proteins and the phosphatidylinositol-3 kinase/Akt pathway in opioid-induced tuberin phosphorylation. This is the first study that demonstrates the regulatory role of opioid receptors on tuberin.     

Binding kinetics of mutated insulin receptors in transfected cells grown in suspension culture: application to the Tyr----Phe 960 insulin receptor mutant

Site-directed mutagenesis of the insulin receptor cDNA is now widely used to elucidate the role of various domains and residues of the receptor, particularly in order to examine the functional importance of the beta chain-associated tyrosine kinase. However, little has been done to correlate the functional repercussions of such mutations with alterations in the complex insulin binding kinetics. This is due in part to the difficulty of conducting large scale experiments using transfected cells on culture dishes. In an effort to overcome this problem, we have developed a method for culturing Chinese hamster ovary (CHO) cells in suspension culture, which provides a large number of cells and obviates the need for enzymatic or mechanical detachment of cells. The feasibility of this approach is demonstrated in a detailed study of the kinetics of insulin binding to the Tyr----Phe 960 insulin receptor mutant.