Biomedical subjects

C R Kahn

Publications and source records attributed to C R Kahn.

At least 181 records · Page 10Linked to original sources

PubMed1992

Effect of phospholipase treatment on insulin receptor signal transduction.

To study the role of membrane lipids in signal transduction by the insulin receptor, we have studied the effect of phospholipase C (Clostridium perfringens) and a phosphatidylinositol-specific phospholipase (Staphylococcus aureus) on insulin binding, a function of the alpha-subunit, and tyrosine kinase activity, a function of the beta-subunit in IM-9 lymphocytes and NIH 3T3 fibroblasts transfected with the human insulin receptor. Treatment of the cells with phospholipase C at concentrations up to 3.4 U/ml did not affect specific insulin binding, but reduced insulin-stimulated receptor phosphorylation by 50%. This effect of phospholipase C was observed within 10 min of treatment and occurred with no change in the basal level of phosphorylation. Pre-treatment of cells with insulin for 5 min prior to enzyme addition prevented any change in kinase activity. Insulin-stimulated phosphorylation of pp 185, the presumed endogenous substrate for the insulin receptor kinase, was also reduced following phospholipase C treatment, with an almost complete loss of insulin stimulation after exposure of cells to enzyme at concentrations as low as 0.6 U/ml. In contrast to these effects of phospholipase C on intact cells, receptor autophosphorylation was not affected in insulin receptors purified on wheat germ agglutinin-agarose from phospholipase C treated cells. Likewise, the phospholipase C effect was reduced by the addition of phosphatidylcholine, but not by the addition of the protease inhibitors, aprotinin and phenylmethylsulfonyl fluoride, to the incubation indicating its dependence on phospholipid hydrolysis. Treatment of cells with the phosphatidylinositol-specific phospholipase C did not affect any of the parameters studied.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗

PubMed1992

Divergent regulation of the Glut 1 and Glut 4 glucose transporters in isolated adipocytes from Zucker rats.

We have studied the relationship between glucose uptake rate and Glut 1 and Glut 4 protein and mRNA levels per fat cell in lean (FA/FA) and obese (fa/fa) Zucker rats at 5, 10, and 20 wk of age, and after induction of acute diabetes with streptozotocin. 5 wk obese rats exhibit insulin hyperresponsive glucose uptake, whereas 20 wk obese rats show insulin resistant glucose uptake. The relative abundance of Glut 1 and Glut 4 mRNA and protein per equal amount of total RNA and total membrane protein, respectively, is lower in adipocytes from obese rats. However, at all ages the enlargement of fat cells from obese rats is accompanied by a severalfold increase in total RNA and total membrane protein per cell. Thus, on a cellular basis, mRNA and protein levels of Glut 4 increases in young obese rats and gradually declines as a function of age. Basal glucose uptake is increased severalfold in fat cells from obese rats, and in parallel Glut 1 expression per cell in obese rats is two- to threefold increased over lean rats at all ages. Acute diabetes in 20 wk obese rats causes a profound downregulation of glucose uptake and a concomitant reduction of both Glut 1 and Glut 4 protein levels. Thus, changes in Glut 4 expression are a major cause of alteration in insulin-stimulated glucose uptake of adipocytes during evolution of obesity and diabetes in Zucker rats.

Adipose Tissue↗

PubMed1992

Regulation of insulin receptor substrate-1 in liver and muscle of animal models of insulin resistance.

Insulin rapidly stimulates tyrosine phosphorylation of a protein of approximately 185 kD in most cell types. This protein, termed insulin receptor substrate-1 (IRS-1), has been implicated in insulin signal transmission based on studies with insulin receptor mutants. In the present study we have examined the levels of IRS-1 and the phosphorylation state of insulin receptor and IRS-1 in liver and muscle after insulin stimulation in vivo in two rat models of insulin resistance, i.e., insulinopenic diabetes and fasting, and a mouse model of non-insulin-dependent diabetes mellitus (ob/ob) by immunoblotting with anti-peptide antibodies to IRS-1 and anti-phosphotyrosine antibodies. As previously described, there was an increase in insulin binding and a parallel increase in insulin-stimulated receptor phosphorylation in muscle of fasting and streptozotocin-induced (STZ) diabetic rats. There was also a modest increase in overall receptor phosphorylation in liver in these two models, but when normalized for the increase in binding, receptor phosphorylation was decreased, in liver and muscle of STZ diabetes and in liver of 72 h fasted rats. In the hyperinsulinemic ob/ob mouse there was a decrease in insulin binding and receptor phosphorylation in both liver and muscle. The tyrosyl phosphorylation of IRS-1 after insulin stimulation reflected an amplification of the receptor phosphorylation in liver and muscle of hypoinsulinemic animals (fasting and STZ diabetes) with a twofold increase, and showed a significant reduction (approximately 50%) in liver and muscle of ob/ob mouse. By contrast, the levels of IRS-1 protein showed a tissue specific regulation with a decreased level in muscle and an increased level in liver in hypoinsulinemic states of insulin resistance, and decreased levels in liver in the hyperinsulinemic ob/ob mouse. These data indicate that: (a) IRS-1 protein levels are differentially regulated in liver and muscle; (b) insulin levels may play a role in this differential regulation of IRS-1; (c) IRS-1 phosphorylation depends more on insulin receptor kinase activity than IRS-1 protein levels; and (d) reduced IRS-1 phosphorylation in liver and muscle may play a role in insulin-resistant states, especially of the ob/ob mice.

Animals↗

PubMed1992

Alterations in insulin receptor and substrate phosphorylation in hypertensive rats.

Insulin stimulates tyrosine phosphorylation of the insulin receptor and of an endogenous substrate of approximately 185 kd (insulin receptor substrate 1 or IRS-1) in most cell types. Tyrosine phosphorylation of insulin receptor and of IRS-1 have been implicated in insulin signal transmission based on studies with insulin receptor mutants. In the study presented here, the levels and phosphorylation state of the insulin receptor and IRS-1 in liver and muscle after insulin stimulation in vivo have been examined in spontaneously hypertensive rats (SHR) by immunoblotting with antipeptide antibodies to insulin receptor and IRS-1 and antiphosphotyrosine antibodies. It was found that the levels of insulin receptor and IRS-1 protein in liver and muscle are similar in controls (Wistar-Kyoto rats) and SHR. By contrast, there is a decrease in autophosphorylation in the liver and muscle of SHR and a parallel decrease in phosphorylation of IRS-1. These data indicate that reduced insulin receptor kinase activity and reduced substrate phosphorylation may play an important role in the impaired insulin action in the hypertensive rat.

Animals↗

PubMed1991

Pertussis toxin inhibits autophosphorylation and activation of the insulin receptor kinase.

Pertussis toxin is an ADP-ribosyltransferase which alters the function of some of the GTP-binding proteins and inhibits some actions of insulin. In vivo, pertussis toxin (2 micrograms/ml/2h) inhibited insulin-stimulated tyrosyl autophosphorylation of the insulin receptor by 50% in FaO cells, and nearly completely inhibited phosphorylation of the cellular insulin receptor substrate pp185. Similarly, insulin-stimulated autophosphorylation and kinase activity of the insulin receptor purified on wheat germ agglutinin-agarose from pertussis toxin-treated FaO cells was diminished 50%; however, treatment of cells with the catalytically inactive B-oligomer of the toxin had no effect on receptor tyrosine kinase activity in vitro. Pertussis toxin did not alter insulin binding or the cellular levels of ATP, cAMP, and cGMP. Furthermore, immunoprecipitation of the insulin receptor from intact cells with anti-insulin receptor antibodies showed that pertussis toxin did not increase the phosphorylation of serine or threonine residues in the insulin receptor. These results suggest that pertussis toxin can modulate signal transduction of insulin at the level of the insulin receptor kinase.

Animals↗

PubMed1991

Autophosphorylation within insulin receptor beta-subunits can occur as an intramolecular process.

The insulin receptor is a complex membrane-spanning glycoprotein composed of two alpha-subunits and two beta-subunits connected to form an alpha 2 beta 2 holoreceptor. Insulin binding to the extracellular alpha-subunits activates intracellular beta-subunit autophosphorylation and substrate kinase activity. The current study was designed to differentiate mechanisms of transmembrane signaling by the insulin receptor, specifically whether individual beta-subunits undergo cis- or trans-phosphorylation. We compared relative kinase activities of trypsin-truncated receptors, alpha beta-half receptors, and alpha 2 beta 2 holoreceptors under conditions that allowed us to differentiate intermolecular and intramolecular events. Compared to the insulin-stimulated holoreceptors, the trypsin-truncated receptor undergoes autophosphorylation at similar tyrosine residues and catalyzes substrate phosphorylation in the absence of insulin at a comparable rate. The truncated receptor sediments on a sucrose gradient at a position consistent with a structure comprising a single beta-subunit attached to a fragment of the alpha-subunit and undergoes autophosphorylation in this form in the absence of insulin. Autophosphorylation of the truncated insulin receptor is independent of receptor concentration, and immobilization of the truncated receptor on a matrix composed of an anti-receptor antibody bound to protein A-Sepharose diminishes neither autophosphorylation nor receptor-catalyzed substrate phosphorylation. Therefore, true intramolecular (cis) phosphorylations, which occur within individual beta-subunits derived from trypsin-truncated receptors, lead to kinase activation. However, insulin-stimulated autophosphorylation of insulin receptor alpha beta heterodimers is concentration-dependent, and both autophosphorylation and kinase activity are markedly reduced following immobilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein.

Since the discovery of insulin nearly 70 years ago, there has been no problem more fundamental to diabetes research than understanding how insulin works at the cellular level. Insulin binds to the alpha subunit of the insulin receptor which activates the tyrosine kinase in the beta subunit, but the molecular events linking the receptor kinase to insulin-sensitive enzymes and transport processes are unknown. Our discovery that insulin stimulates tyrosine phosphorylation of a protein of relative molecular mass between 165,000 and 185,000, collectively called pp185, showed that the insulin receptor kinase has specific cellular substrates. The pp185 is a minor cytoplasmic phosphoprotein found in most cells and tissues; its phosphorylation is decreased in cells expressing mutant receptors defective in signalling. We have now cloned IRS-1, which encodes a component of the pp185 band. IRS-1 contains over ten potential tyrosine phosphorylation sites, six of which are in Tyr-Met-X-Met motifs. During insulin stimulation, the IRS-1 protein undergoes tyrosine phosphorylation and binds phosphatidylinositol 3-kinase, suggesting that IRS-1 acts as a multisite 'docking' protein to bind signal-transducing molecules containing Src-homology 2 and Src-homology-3 domains. Thus IRS-1 may link the insulin receptor kinase and enzymes regulating cellular growth and metabolism.

Animals↗

PubMed1991

Purification and partial sequence analysis of pp185, the major cellular substrate of the insulin receptor tyrosine kinase.

Insulin stimulates the tyrosine phosphorylation of a 185-kDa putative cytosolic substrate protein (pp185) in diverse cell types. After intravenous insulin infusion into the live intact rat, pp185 and the 95-kDa insulin receptor beta-subunit were the major proteins that tyrosine phosphorylated in liver, skeletal muscle, and adipose tissue. Both proteins were maximally phosphorylated within 30 s, and both increased in phosphotyrosine content in parallel with increasing insulin dose. However, pp185 tyrosine phosphorylation was transient, with almost complete dephosphorylation within 2-3 min despite continued insulin stimulation. To identify pp185 directly, we purified pp185 from insulin-stimulated rat liver, using a denaturation-based extraction procedure that blocks endogenous protein phosphatases and thus allows a high yield, single step isolation of phosphotyrosyl proteins by anti-phosphotyrosine antibody immunoaffinity absorption. From 50 rat livers, 50-100 pmol of pp185 was isolated. Edman degradation of seven internal tryptic peptide fragments of pp185 yielded novel amino acid sequences, indicating that pp185 is a new protein. Antipeptide antibodies were raised which specifically recognize a single, 185-kDa insulin-stimulated phosphotyrosyl protein in liver, skeletal muscle, adipose tissue, and several cultured cell lines. These results indicate that pp185 is expressed in a variety of insulin-responsive tissues, is the major protein rapidly tyrosine phosphorylated under physiological conditions in the intact animal, and also provide a route for cloning the pp185 gene and elucidating the function of pp185 in insulin signal transduction.

Amino Acid Sequence↗

PubMed1991

The 90-kDa heat shock protein (hsp-90) possesses an ATP binding site and autophosphorylating activity.

The 90-kDa heat shock protein (hsp-90) is an abundant cytosolic protein believed to play a role in maintenance of protein trafficking and closely associated with several steroid hormone receptors. Incubation of highly purified hsp-90 with [gamma-32P]ATP results in its autophosphorylation on serine residues. There are several lines of evidence which suggest that this activity is due to a kinase intrinsic to hsp-90 rather than some closely associated protein kinases: 1) the phosphorylation persists after the removal of casein kinase II by heparin chromatography and after immunoprecipitation of hsp-90 with anti-hsp-90 antibodies. 2) The approximate kM for ATP of the reaction is 0.16 mM, higher than that of many other protein kinases. 3) Phosphorylation is not affected by a number of activators and inhibitors of other known kinases which might associate with hsp-90. 4) The phosphorylation displays a unique cation dependence being most active in the presence of Ca2+ and practically inactive with Mg2+, although the autophosphorylation in the presence of Mg2+ is activated by histones and polyamines. 5) The activity is remarkably heat-stable; incubation of hsp-90 for 20 min at 95 degrees C results in only a 60% decrease in autophosphorylation. 6) Finally, and most importantly, purified hsp-90 can be labeled with azido-ATP and it is able to bind to ATP-agarose. The binding shows similar cation dependence to the autophosphorylation. These data are in agreement with the presence of a consensus sequence for ATP binding sites in the primary structure of the protein similar to that observed in the 70-kDa heat-shock proteins. Our data suggest the 90-kDa heat shock protein possesses an enzymatic activity analogous in many respects to the similar activity of the 70-kDa heat shock proteins. This may represent an important, previously unrecognized function of hsp-90.

Adenosine Triphosphate↗

PubMed1991

Vanadate normalizes hyperglycemia in two mouse models of non-insulin-dependent diabetes mellitus.

We have studied the effects of oral administration of vanadate, an insulinometic agent and a potent inhibitor of phosphotyrosyl protein phosphatase (PTPase) in vitro, on blood glucose and PTPase action, in two hyperinsulinemic rodent models of non-insulin-dependent diabetes mellitus (NIDDM). Oral administration of vanadate (0.25 mg/ml in the drinking water) to ob/ob mice for 3 wk lowered blood glucose level from 236 +/- 4 to 143 +/- 2 mg/dl without effect on body weight. Administration of vanadate to db/db mice produced a similar effect. Electron microscopic examination revealed no signs of hepatotoxicity after 47 d of treatment. There was a slight reduction in insulin receptor autophosphorylation when tested by immunoblotting with antiphosphotyrosine antibody after in vivo stimulation, and the phosphorylation of the endogenous substrate of the insulin receptor, pp185, was markedly decreased in the ob/ob mice. Both cytosolic and particulate PTPase activities in liver of ob/ob mice measured by dephosphorylation of a 32P-labeled peptide corresponding to the major site of insulin receptor autophosphorylation were decreased by approximately 50% (P less than 0.01). In db/db diabetic mice, PTPase activity in the cytosolic fraction was decreased to 53% of control values (P less than 0.02) with no significant difference in the particulate PTPase activity. Treatment with vanadate did not alter hepatic PTPase activity as assayed in vitro, or receptor and substrate phosphorylation as assayed in vivo, in ob/ob mice despite its substantial effect on blood glucose. These data indicate that vanadate is an effective oral hypoglycemic treatment in NIDDM states and suggest that its major effects occurs distal to the insulin receptor tyrosine kinase.

Animals↗

PubMed1991

High fat feeding causes insulin resistance and a marked decrease in the expression of glucose transporters (Glut 4) in fat cells of rats.

With the identification of two different glucose transporter species in adipose cells it is crucial to determine the role of these transporters in the alterations in glucose transport activity associated with different metabolic and nutritional states. In the present study we assess levels of expression of Glut 1 and Glut 4 transporters and basal and insulin-stimulated glucose transport activity in adipocytes from Sprague-Dawley rats fed standard chow (control), combined liquid diet and standard chow (overfed), high fat diet, or energy-restricted diet for 7 weeks. High fat feeding was associated with relative postprandial hypoglycemia (P less than 0.05) and hypoinsulinemia (P less than 0.05). Although the high fat fed animals had lower body weights (P less than 0.05) than control rats, their body compositions showed obesity, with 36% heavier epididymal fat pads (P less than 0.05) and a 47% increase in adipocyte volume (P less than 0.05). Fat feeding caused a 78% reduction in insulin-stimulated glucose transport per adipocyte (P less than 0.05). In parallel we found 92% and 94% reductions in Glut 4 protein and mRNA per adipocyte, respectively, (P less than 0.01) in fat-fed rats. Substantial reductions were also seen in Glut 1 protein and mRNA per fat cell in the same rats (62% and 76%, respectively; P less than 0.05). However, the changes in Glut 1 expression were of the same magnitude as changes in the cytoskeletal protein beta-actin, reflecting a decreased expression of several proteins in this nutritional state. Even though overfeeding and energy restriction brought about opposite changes in adiposity, no significant alterations were demonstrated in glucose transport rate or glucose transporter expression. The impaired insulin-stimulated glucose transport in adipose cells from high fat-fed rats occurs in the presence of a dramatic decrease in the expression of the major insulin-responsive glucose transporter (Glut 4). The reduced gene expression may be caused by chronic hypoinsulinemia and may contribute to the insulin resistance observed in this state.

Adipose Tissue↗

PubMed1991

Insulin-like growth factor-mediated phosphorylation and protooncogene induction in Madin-Darby canine kidney cells.

We have characterized the role of tyrosine phosphorylation in protooncogene induction mediated by insulin-like growth factors I and II (IGF-I and IGF-II) in the Madin-Darby canine kidney (MDCK) cell line. These cells possess few, if any, insulin receptors, thus allowing determination of the effects of these growth factors in the absence of any secondary signal mediated through the insulin receptor. We found that IGF-I produced a specific stimulation of tyrosine kinase activity of the 97-kDa beta-subunit of the IGF-I receptor, resulting in autophosphorylation of the receptor and an increase in kinase activity toward a synthetic peptide substrate. This was associated with a gradual decrease in the level of phosphorylation of pp120, the major constitutive phosphotyrosine-containing protein of MDCK cells, and an increase in the ratio of serine to tyrosine phosphorylation. This was followed by a rapid, but transient, induction of c-fos gene expression, with no change in the levels of c-myc mRNA. Cycloheximide treatment resulted in a superinduction of both c-fos and c-myc and prevented any further stimulation by IGF-I. IGF-II did not stimulate tyrosine phosphorylation of its own receptor, but was 25% as active as IGF-I in stimulating phosphorylation of the IGF-I receptor. Despite this, IGF-II did not significantly enhance the expression of either nuclear protooncogene. Insulin also produced a delayed stimulation of IGF-I receptor phosphorylation, but was unable to stimulate biological effects in these cells. Under these conditions neither of the IGFs nor insulin produced any significant stimulation of thymidine incorporation into DNA. These data indicate that the IGF-I receptor can be activated upon binding of IGF-I, and to a lesser extent IGF-II, in intact cells to mediate cellular events. The nature of the signal generated by the IGF-I receptor appears to vary depending on the ligand that occupies it.

Animals↗

PubMed1991

Heterogeneity of expression and secretion of native and mutant [AspB10]insulin in AtT20 cells.

AtT20 (pituitary corticotroph) cells were transfected with either the native or a mutant [AspB10]rat insulin II gene, using a plasmid containing the insulin gene and a neomycin resistance gene under the control of independent constitutive promoters. The cellular immunoreactive insulin (IRI) content ranged from 0.8-440 ng/10(6) cells, with the highest value similar to that found for a rat insulinoma cell line (RIN) and corresponding to approximately 1% that of native pancreatic B-cells. There was a direct correlation between insulin mRNA levels and IRI content and no correlation between mRNA levels and rat insulin II gene copy number. Furthermore, in some lines the insulin II transgene was lost even though the gene encoding neomycin resistance was retained. IRI release was stimulated up to 4-fold by isobutylmethylxanthine in all lines transfected with the native rat insulin II gene, and HPLC analysis showed most IRI as fully processed insulin, with less than 5% as proinsulin. These cells, thus, directed most proinsulin to secretory granules for conversion and regulated release regardless of the absolute amount of IRI expressed. One of the lines transfected with the AspB10 mutant gene (line AA9) released nearly 50% of IRI as proinsulin under basal conditions, with stimulation of insulin, but not proinsulin, release by isobutylmethylxanthine. This confirmed our previous finding of partial diversion of this mutant proinsulin from the regulated to the constitutive pathway. A second line (IC6) expressing the same mutant gene at much higher levels appeared to direct all mutant proinsulin to the regulated pathway, suggesting that for this particular mutant proinsulin, the secretory pathway employed by the transfected cells can be affected by the amount of proinsulin synthesized.

Animals↗

PubMed1991

Coordinate phosphorylation of insulin-receptor kinase and its 175,000-Mr endogenous substrate in rat hepatocytes.

To investigate the early events in insulin signal transmission in liver, isolated rat hepatocytes were labeled with 32P, and proteins phosphorylated in response to insulin were detected by immunoprecipitation with anti-phosphotyrosine and anti-receptor antibodies and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and autoradiography. In these cells, insulin rapidly stimulated tyrosine phosphorylation of the 95,000-Mr beta-subunit of the insulin receptor and a 175,000-Mr phosphoprotein (pp175). Both proteins were precipitated by anti-phosphotyrosine antibody, whereas only the insulin receptor was recognized with anti-insulin-receptor antibody. In the insulin-stimulated state, both pp175 and the receptor beta-subunit were found to be phosphorylated on tyrosine and serine residues. Based on precipitation by the two antibodies, receptor phosphorylation was biphasic with an initial increase in tyrosine phosphorylation followed by a more gradual increase in serine phosphorylation over the first 30 min of stimulation. The time course of phosphorylation of pp175 was rapid and paralleled that of the beta-subunit of the insulin receptor. The pp175 was clearly distinguished from the insulin receptor, because it was detected only when boiling SDS was used to extract cellular phosphoproteins, whereas the insulin receptor was extracted with either Triton X-100 or SDS. In addition, the tryptic peptide maps of the two proteins were distinct. The dose-response curve for insulin stimulation was shifted slightly to the left of the insulin receptor, suggesting some signal amplification at this step. These data suggest that pp175 is a major endogenous substrate of the insulin receptor in liver and may be a cytoskeletal-associated protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1990

Slow glucose removal rate and hyperinsulinemia precede the development of type II diabetes in the offspring of diabetic parents.

OBJECTIVE: To determine whether insulin resistance or insulin deficiency is primary in the pathogenesis of type II diabetes. DESIGN: Cohort analytic study of persons with normal glucose tolerance but with a high risk for developing type II diabetes (average follow-up time, 13 years). SETTING: Outpatients had an intravenous glucose tolerance test and were contacted periodically to ascertain diagnoses of diabetes. PARTICIPANTS: One hundred and fifty-five normal offspring, ranging in age from 16 to 60 years, of two parents with type II diabetes and 186 normal control subjects in the same age range who had no family history of diabetes. MEASUREMENTS AND MAIN RESULTS: Two phenotypic characteristics distinguished the offspring of diabetic parents from control subjects. They had slower glucose removal rates (Kg) (P less than 0.01) and higher insulin levels (fasting and during the second phase of insulin response to intravenous glucose; P less than 0.0001) than did control subjects, even after adjustment for differences in obesity. Sixteen percent of the offspring developed type II diabetes. Mean Kg at baseline was 1.7%/min among offspring who subsequently developed diabetes, 2.2%/min among offspring who remained nondiabetic, and 2.3%/min among control subjects. Corresponding means for first-phase insulin were 498, 354, and 373 pM, respectively, whereas second-phase insulin means were 329, 117, and 87 pM, respectively. In multivariate analysis, low Kg and high serum insulin levels independently increased the risk for developing diabetes among the offspring of diabetic parents. CONCLUSIONS: One to two decades before type II diabetes is diagnosed, reduced glucose clearance is already present. This reduced clearance is accompanied by compensatory hyperinsulinemia, not hypoinsulinemia, suggesting that the primary defect is in peripheral tissue response to insulin and glucose, not in the pancreatic beta cell.

Administration, Oral↗

PubMed1990

Epidermal growth factor stimulated phosphorylation of a 120-kilodalton endogenous substrate protein in rat hepatocytes.

Endogenous substrates of the EGF receptor have been described in transformed cells; however, little is known about substrates in normal tissue. To characterize epidermal growth factor (EGF) receptor phosphorylation and search for endogenous substrates in normal rat hepatocytes, cells were labeled with [32P]orthophosphate, and phosphotyrosine-containing proteins were sought by using a high-affinity, specific anti-phosphotyrosine antibody. Exposure of 32P-labeled freshly isolated hepatocytes to 1 microgram/mL EGF caused phosphorylation of several proteins of Mr 185K, 160K, and 120K. The 185- and 160-kDa proteins (pp185 and pp160) were identified as the intact and proteolyzed forms of the EGF receptor by virtue of their immunoprecipitation with anti-EGF receptor antibody. This antibody failed to recognize the 120-kDa phosphoprotein (pp120). The phosphopeptide map derived from pp120 was by trypsinization and HPLC separation different from that of pp185, further indicating that pp120 is distinct from the EGF receptor. This pp120 was also immunologically distinct from the pp120 substrate of the insulin receptor kinase and from ATP-citrate lyase. Phosphoamino acid analysis revealed pp120 to be phosphorylated on both tyrosine and serine residues. Autophosphorylation of EGF receptor and phosphorylation of pp120 were almost maximal within 1 min of EGF stimulation. The dose-response curves for phosphorylation of the EGF receptor and pp120 were identical (ED50 = 30 ng/mL) and were superimposable with the fractional occupancy of the EGF receptor. In A431 cells, a transformed cell line whose growth is inhibited by EGF, EGF produced a decrease in pp120 phosphorylation. These data suggest that pp120 is an endogenous substrate for the EGF receptor in hepatocytes whose phosphorylation may be closely related to EGF stimulation of cell growth.

Animals↗

PubMed1990

Receptor-mediated internalization of insulin requires a 12-amino acid sequence in the juxtamembrane region of the insulin receptor beta-subunit.

The juxtamembrane region of the insulin receptor (IR) beta-subunit contains an unphosphorylated tyrosyl residue (Tyr960) that is essential for insulin-stimulated tyrosyl phosphorylation of some endogenous substrates and certain biological responses (White, M.F., Livingston, J.N., Backer, J.M., Lauris, V., Dull, T.J., Ullrich, A., and Kahn, C.R. (1988) Cell 54, 641-649). Tyrosyl residues in the juxtamembrane region of some plasma membrane receptors have been shown to be required for their internalization. In addition, a juxtamembrane tyrosine in the context of the sequence NPXY [corrected] is required for the coated pit-mediated internalization of the low density lipoprotein receptor. To examine the role of the juxtamembrane region of the insulin receptor during receptor-mediated endocytosis, we have studied the internalization of insulin by Chinese hamster ovary (CHO) cells expressing two mutant receptors: IRF960, in which Tyr960 has been substituted with phenylalanine, and IR delta 960, in which 12 amino acids (Ala954-Asp965), including the putative consensus sequence NPXY [corrected], were deleted. Although the in vivo autophosphorylation of IRF960 and IR delta 960 was similar to wild type, neither mutant could phosphorylate the endogenous substrate pp185. CHO/IRF960 cells internalized insulin normally whereas the intracellular accumulation of insulin by CHO/IR delta 960 cells was 20-30% of wild-type. However, insulin internalization in the CHO/IR delta 960 cells was consistently more rapid than that occurring in CHO cells expressing kinase-deficient receptors (CHO/IRA1018). The degradation of insulin was equally impaired in CHO/IR delta 960 and CHO/IRA1018 cells. These data show that the juxtamembrane region of the insulin receptor contains residues essential for insulin-stimulated internalization and suggest that the sequence NPXY [corrected] may play a general role in directing the internalization of cell surface receptors.

Amino Acid Sequence↗

PubMed1990

The dissociation and degradation of internalized insulin occur in the endosomes of rat hepatoma cells.

We have studied the intracellular processing of insulin in the rat hepatoma cell line Fao. Fao cells internalized cohorts of surface-bound 125I-insulin or 125I-insulin-like growth factor II within 3-5 min. Degraded 125I-insulin-like growth factor II did not appear in the medium until 20-30 min after uptake, consistent with a time course of lysosomal delivery. In contrast, internalized insulin was completely degraded within 7-10 min. The half-times for dissociation and degradation of internalized insulin were identical at 37 degrees C (3 min), suggesting that the two processes occurred in the same compartment. Subcellular fractionation of Fao cells showed that a pulse of internalized insulin was largely intact after 3 min and associated with a light membrane fraction devoid of lysosomal markers. After an additional 4 min, the amount of insulin in this compartment decreased by 40%, with an increase in degraded insulin in the cytosol; no transfer of intact insulin to lysosomes or the cytosol was detected. The relationship between insulin-receptor dissociation and insulin degradation was further studied with inhibitors of insulin processing. Monensin blocked both dissociation and degradation of internalized insulin, as did incubation of the cells at 20 degrees C, suggesting that both endosomal acidification and endosomal fusion were required for insulin processing. At 25 degrees C, dissociation (+ t 1/2 = 12.9 min) preceded degradation (+ t 1/2 = 15.8 min). Inhibitors of lysosomal proteases were without effect on the half-time for either process. In contrast, bacitracin, an inhibitor of insulin degradation, caused a 2-fold increase in the half-times for both dissociation and degradation. Thus, intracellular insulin dissociation and degradation are tightly coupled endosomal processes in Fao cells, and insulin degradation facilitates the dissociation of insulin from its receptor inside the cell.

Animals↗