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P F Pilch

Publications and source records attributed to P F Pilch.

At least 91 records · Page 5Linked to original sources
PubMed1987

Purification of insulin-dependent exocytic vesicles containing the glucose transporter.

In muscle and fat, insulin causes the cellular redistribution of glucose transporters and insulin-like growth factor II receptors from an intracellular pool of membranes (low density microsomes) to the plasma membrane. This translocation is a major mechanism by which insulin stimulates cellular glucose uptake. Our aim was to purify and characterize the insulin-regulatable exocytic intracellular membranes that are enriched in glucose transporter. Low density microsome and plasma membrane fractions were isolated from basal and insulin-stimulated rat adipocytes by differential centrifugation. In cells exposed to insulin, glucose transporters were decreased in the low density microsomes and correspondingly increased in the plasma membranes as determined by immunoblotting and cytochalasin B binding. Low density microsomes were further fractionated by sucrose density gradient centrifugation. Membranes containing glucose transporters were separated from the major protein-containing peaks and from plasma membranes, Golgi, and endoplasmic reticulum. Further fractionation was achieved by agarose gel electrophoresis. Overall, the intracellular membranes enriched in transporter were purified 9-fold compared to low density microsomes. These purified membranes had the following characteristics: 1) uniformly sized vesicles, diameter 60-100 nm; 2) insulin-regulatable protein composition, one constituent being an Mr 43,000 protein that co-migrated with immunoblotted glucose transporters; 3) enrichment in insulin-like growth factor II receptors, but of a lesser degree than the enrichment in transporters. Thus, using a three-step procedure, insulin-sensitive translocatable vesicles from adipocytes have been highly purified. These are similar in size and density to endosomes, and the glucose transporter is a major constituent of this distinct vesicle population.

Adipose Tissue↗
PubMed1987

The insulin receptor. Structural basis for high affinity ligand binding.

Treatment of the soluble insulin receptor from human placenta with 1.25 mM dithiothreitol and 75 mM Tris at pH 8.5 results in complete reduction of interhalf disulfide bonds (class 1 disulfides) and dissociation of the tetrameric receptor into the dimeric alpha beta form. The alpha beta receptor halves exhibit a reduced affinity for insulin binding (Böni-Schnetzler, M., Rubin, J. B., and Pilch, P. F. (1986) J. Biol. Chem. 261, 15281-15287). Kinetic experiments reveal that reduction of class 1 disulfides is a faster process than the loss of affinity for ligand, indicating that events subsequent to reduction of interhalf disulfides are responsible for the affinity change. We show that a third class of alpha subunit intrachain disulfides is more susceptible to reduction at pH 7.6 than at pH 8.5 and appears to form part of the ligand binding domain. Reduction of the intrachain disulfide bonds in this part of the alpha subunit leads to a loss of insulin binding. Modification of this putative binding domain by dithiothreitol can be minimized if reduction is carried out at pH 8.5. When the insulin receptor in placental membranes is reduced at pH 8.5, the receptor's affinity for insulin is not changed when binding is measured in the membrane. However, the Kd for insulin binding is reduced 10-fold when alpha beta receptor halves are subsequently solubilized. Scatchard analysis of insulin binding to reduced or intact receptors in the membrane and in soluble form together with sucrose density gradient analysis of soluble receptors suggests that alpha beta receptor halves remain associated in the membrane after reduction, but they are dissociated upon solubilization. We interpret these results to mean that the association of two ligand binding domains, 2 alpha beta receptor halves, is required for the formation of an insulin receptor with high affinity for ligand.

Cell Membrane↗
PubMed1987

Internalization of insulin receptors and HLA antigens in human hepatoma cells.

Human HepG2 hepatoma cells express a high number of insulin receptors. Growing cells exhibit 70% of their insulin receptors on the plasma membrane. Moreover, cell-surface insulin receptors form molecular complexes with class I major histocompatibility antigens, as determined by co-immunoprecipitation of the receptors by anti-class I monoclonal antibodies. On exposure to saturating concentrations of insulin, the hormone is rapidly internalized into a Pronase-resistant compartment. Internalization of insulin is accompanied by a rapid (t1/2 = 2-3 min) redistribution of insulin receptors from the cell surface to an intracellular compartment. On removal of insulin from the medium, functional receptors recycle back to the plasma membrane, where they can bind insulin again. With chronic exposure of HepG2 cells to insulin, the initial redistribution of receptors is followed by a slow (t1/2 = 9 h) down-regulation of the receptors. Finally, notwithstanding their interaction at the cell surface, insulin receptors and class I major histocompatibility antigens are internalized at different rates and with independent regulation.

Carcinoma, Hepatocellular↗
PubMed1987

Mechanism of epidermal growth factor receptor autophosphorylation and high-affinity binding.

Epidermal growth factor (EGF) receptor monomers and noncovalently associated dimers were isolated by sucrose density gradient centrifugation, and their respective binding and autophosphorylation activities were determined. We find that monomers are low-affinity receptors and dimers are high-affinity receptors. In the absence of EGF, dimers exhibit a 4-fold higher autophosphorylation activity than do monomers. Addition of EGF increases autophosphorylation on monomers an average of 4.8-fold but has a minimal effect on autophosphorylation of dimers. Furthermore, EGF binding shifts the receptor monomer-dimer equilibrium to the dimer form. We conclude that EGF stimulates in vitro receptor autophosphorylation by inducing kinase-inactive receptor monomers to associate and form receptor dimers, in which conformation the autophosphorylation activity is enhanced.

Cells, Cultured↗
PubMed1986

Intrinsic differences of insulin receptor kinase activity in red and white muscle.

The sensitivity and responsiveness of glucose uptake and glycogen synthesis to insulin are 3-4-fold greater in red than in white skeletal muscle (James, D. E., Jenkins, A. B., and Kraegen, E. W. (1985) Am. J. Physiol. 248, E567-E574). In the present study, the insulin receptor tyrosine kinase activity has been examined in red and white muscle of rats. Partially purified insulin receptors were obtained from muscle following solubilization in detergent, ultracentrifugation, and lectin affinity chromatography. Total insulin receptor number per gram of tissue was slightly higher in red (30%) than in white muscle. In contrast, basal and insulin-stimulated autophosphorylation, normalized for receptor number, were 2.3-fold higher in red muscle. A similar difference was observed in the ability of partially purified receptors to phosphorylate the exogenous substrate polyglutamate/tyrosine. The integrity of the insulin receptor preparation in the two fiber types was identical as determined by affinity cross-linking of [125I-TyrB26]insulin to the receptor. Mixing partially purified receptors from red and white muscle resulted in an additive response for exogenous substrate phosphorylation, suggesting that the difference in tyrosine kinase activity was not due to the presence of an inhibitor or activator. The results suggest that there are differences in the insulin receptors of red and white muscles that lead to discordance in their basal and insulin-stimulated intrinsic tyrosine kinase activity. The correlation between these differences and insulin action in red and white muscle supports the concept that the insulin receptor tyrosine kinase activity is involved in the initiation of insulin action.

Animals↗
PubMed1986

Structural requirements for the transmembrane activation of the insulin receptor kinase.

Tetrameric insulin holoreceptor (alpha 2 beta 2) was reduced with dithiothreitol into alpha beta dimers such that they maintain up to 50% of insulin binding at tracer ligand concentrations. Scatchard analysis of insulin binding to dimers revealed that they had a reduced affinity for ligand by a factor of 3-6 compared to holoreceptor, whereas the maximum number of high affinity binding sites was not affected. The alpha beta dimers can be separated from holoreceptor by sucrose density gradient centrifugation, and hence, they are not associated by noncovalent interactions. Insulin-dependent autophosphorylation of alpha beta dimers isolated from low ionic strength sucrose density gradients was minimal and was always accompanied by reoxidation of dimers to the tetrameric holoreceptor. The reformed tetramer exhibited a strong insulin-dependent autophosphorylation reaction. Reoxidation was prevented by isolating alpha beta dimers in sucrose density gradients containing 0.15 M NaCl. Under these conditions, no insulin-dependent autophosphorylation was observed. When insulin receptor was first autophosphorylated and then reduced, receptor kinase activity, as assayed by histone phosphorylation, was not affected. Also, the insulin-independent, basal autophosphorylation was maintained after reduction into alpha beta dimers. We conclude that alpha beta-alpha beta interaction is not necessary for the maintenance of basal kinase activity or for insulin-activated kinase activity once autophosphorylation occurs. However, dimer-dimer interaction appears critical for the insulin-dependent activation of the receptor's intrinsic kinase activity.

Cell Membrane↗
PubMed1986

Dipeptide metalloendoprotease substrates are glucose transport inhibitors and membrane structure perturbants.

Dipeptide substrates for metalloendoproteases have previously been shown to block biological processes requiring membrane fusion. Thus, we employed such compounds as potential inhibitors of the insulin-dependent activation of glucose transport in fat cells. This event is thought to involve vesicle movement from an intracellular site to the cell surface and would therefore require membrane fusion during the activation step. We find that synthetic dipeptides which are metalloendoprotease substrates rapidly and reversibly inhibit insulin-activated glucose oxidation in a dose-dependent manner but exhibit essentially no effect on basal levels. A similar result is obtained when glucose transport is measured directly in intact fat cells, in metabolically poisoned cells, and in isolated membrane vesicles derived from insulin-activated or untreated fat cells. That is, the dipeptide substrates inhibit insulin-activated glucose uptake to a greater extent than basal transport, and they do so even when vesicle translocation and fusion have already taken place as in ATP-depleted cells and isolated vesicles. Onset of transport inhibition after dipeptide addition is rapid, but not instantaneous, with a t 1/2 of 15-30 s. The metalloendoprotease substrates also inhibit glucose uptake and cytochalasin B binding in human erythrocytes but not in human placental microsomes. Finally, light microscopic examination of substrate-treated red cells reveals marked cupping and/or echinolation of the cell membrane. We conclude the following from these observations: Metalloendoprotease substrates are inhibitors of adipocyte glucose transport.(ABSTRACT TRUNCATED AT 250 WORDS)

3-O-Methylglucose↗
PubMed1986

The ligand binding subunit of the insulin-like growth factor 1 receptor has properties of a peripheral membrane protein.

125I-insulin-like growth factor 1 was cross-linked to its receptor in human placenta microsomal membranes. The microsomes were treated with urea, with dithiothreitol or with both reagents prior to centrifugation at 100,000 X g. We found that greater than 80% of the label was membrane-associated following separate treatment with urea or dithiothreitol, but greater than 80% of the radioactivity remained in the supernatant after simultaneous exposure to both reagents. In identical experiments employing 125I-epidermal growth factor, no condition led to the release of greater than 10% of label from the membrane. We conclude that the ligand binding subunit of the insulin-like growth factor 1 receptor, like peripheral membrane proteins, lacks a membrane anchoring domain.

Dithiothreitol↗
PubMed1984

Characterization and solubilization of the cytochalasin B binding component from human placental microsomes.

Human placental microsomes exhibit uptake of D-[3H]glucose which is sensitive to inhibition by cytochalasin B (apparent Ki = 0.78 microM). Characterization of [3H]cytochalasin B binding to these membranes reveals a glucose-sensitive site, inhibited by D-glucose with an ED50 = 40 mM. The glucose-sensitive cytochalasin B binding site is found to have a Kd = 0.15 microM by analysis according to Scatchard. Solubilization with octylglucoside extracts 60-70% of the glucose-sensitive binding component. Equilibrium dialysis binding of [3H]cytochalasin B to the soluble protein displays a pattern of inhibition by D-glucose similar to that observed for intact membranes, and the measurement of an ED50 = 37.5 mM D-glucose confirms the presence of the cytochalasin B binding component, putatively assigned as the glucose transporter. Further evidence is attained by photoaffinity labelling; ultraviolet-sensitive [3H]cytochalasin B incorporation into soluble protein (Mr range 42000-68000) is prevented by the presence of D-glucose. An identical photolabelling pattern is observed for incorporation of [3H]cytochalasin B into intact membrane protein, confirming the usefulness of this approach as a means of identifying the presence of the glucose transport protein under several conditions.

Affinity Labels↗
PubMed1984

Identification of a protein kinase as an intrinsic component of rat liver coated vesicles.

Purified rat liver coated vesicles phosphorylate two peptides, Mr 53 000 and Mr 51 000, in the presence of [gamma-32P]ATP. Incorporation of phosphate into these peptides is not stimulated by cAMP, Ca2+, or Ca2+ plus calmodulin and occurs principally on a threonine residue. Mild conditions that result in removal of coat proteins from coated vesicles remove most of the protein kinase activity, suggesting the enzyme(s) is (are) not an integral membrane protein. Photolabeling of coated vesicles with 8-azido-[alpha-32P]ATP results in specific labeling of only the Mr 53 000 and Mr 51 000 peptides. Preincubation with 10 mM N-ethylmaleimide inhibits kinase activity and concomitantly reduces photolabeling of the two peptides. Thus, the data are consistent with the hypothesis that protein kinase activity resides with these two coated vesicle proteins and that they are catalyzing an autophosphorylation reaction.

Adenosine Triphosphate↗
PubMed1983

The insulin receptor protein kinase. Physicochemical requirements for activity.

We determined that the rate of insulin-stimulated autophosphorylation of the insulin receptor is independent of receptor concentration and thus proceeds via an intramolecular process. This result is consistent with the possibility that ligand-dependent autophosphorylation may be a means by which cells can distinguish occupied from unoccupied receptors. We employed dithiothreitol to dissociate tetrameric receptor into alpha beta halves in order to further elucidate the structural requirements for the receptor-mediated kinase activity. Dithiothreitol had a complex biphasic effect on insulin-stimulated receptor kinase activity. Marked stimulation of kinase activity was observed at 1-2 mM dithiothreitol when the receptor was predominantly tetrameric and kinase activity diminished when dimeric alpha beta receptor halves predominate (greater than 2 mM dithiothreitol). N-Ethylmaleimide inhibits insulin-stimulated receptor kinase activity. We suggest that the tetrameric holoreceptor is the most active kinase structure and this structure requires for maximal activity, a reduced sulfhydryl group at or near the active site. We treated receptor preparations with elastase to generate receptor proteolytically "nicked" in the beta subunit. This treatment completely abolishes insulin-dependent autophosphorylation and histone phosphorylation with essentially no effects on insulin binding as determined by affinity labeling of the receptor alpha subunit. We suggest such treatment functionally uncouples insulin binding from insulin-stimulated receptor kinase activity. The possible physiological significance of these findings is discussed.

Dithiothreitol↗
PubMed1983

Unique cytochalasin B binding characteristics of the hepatic glucose carrier.

Cytochalasin B is shown to inhibit uptake of 3-O-methylglucose into isolated rat hepatocytes with a Ki = 1.9 microM. The nature of this inhibition was characterized by studies of [3H]cytochalasin B binding to liver plasma membranes. Scatchard analysis of [3H]cytochalasin B binding reveals a complex curvilinear binding pattern. This pattern can be resolved into three components: (1) a high-affinity (ca. 10(-8) M) cytochalasin E sensitive site unrelated to glucose uptake, (2) a glucose-sensitive site, and (3) a low-affinity site. When 5 microM cytochalasin E is employed to mask the high-affinity site, glucose displaces 40-60% of the remaining [3H]cytochalasin B binding. Analysis of this glucose-sensitive cytochalasin B binding according to Scatchard reveals a Kd = 1.7 microM, indistinguishable from the concentration of cytochalasin B which half-maximally inhibits hepatic glucose uptake. These data identify a glucose-sensitive cytochalasin B binding site in liver plasma membranes which corresponds to the glucose carrier in the intact hepatocyte. The Ki of 1.9 microM for inhibition of hepatic glucose uptake by cytochalasin B and the Kd of 1.7 microM for [3H]cytochalasin B binding to liver plasma membranes are values 1 order of magnitude higher than values for the same parameters determined in all previous studies of facilitated hexose diffusion systems. The hepatic hexose carrier is therefore unique, and this uniqueness may be of regulatory significance with regard to glucose homeostasis.

Animals↗
PubMed1983

The beta subunit of the insulin receptor is an insulin-activated protein kinase.

In the presence of adenosine 5'-[gamma-32P]triphosphate ([gamma-32P]ATP) and a partially purified human placental insulin receptor preparation, insulin stimulates the phosphorylation of an Mr 94000 protein in a time- and dose-dependent manner. Half-maximal stimulation of 32P incorporation occurs at (2-3) X 10(-9) M insulin, a concentration identical with the Kd for insulin binding in this preparation. Immunoprecipitations with monoclonal anti-insulin receptor antibody demonstrate that the Mr 94000 protein kinase substrate is a component of the insulin receptor, the beta subunit. If the partially purified, soluble placental receptor preparation is immunoprecipitated and then exposed to [gamma-32P]ATP and insulin, phosphorylation of the Mr 94000 protein is maintained. The photoincorporation of 8-azido[alpha-32P]ATP into placental insulin receptor preparations was carried out to identify the ATP binding site responsible for the protein kinase activity. Photoincorporation into numerous proteins was observed, including both subunits of the insulin receptor. However, when photolabeling was performed in the presence of excess adenosine 5'-(beta, gamma-imidotriphosphate), a nonhydrolyzable ATP derivative, the beta subunit of the insulin receptor was the only species protected from label incorporation. These data indicate that the beta subunit of the insulin receptor has insulin-dependent protein kinase activity. Phosphotyrosine formation is the primary result of this activity in placental insulin receptor preparations.

Female↗
PubMed1983

Coated vesicles participate in the receptor-mediated endocytosis of insulin.

We have purified coated vesicles from rat liver by differential ultracentrifugation. Electron micrographs of these preparations reveal only the polyhedral structures typical of coated vesicles. SDS PAGE of the coated vesicle preparation followed by Coomassie Blue staining of proteins reveals a protein composition also typical of coated vesicles. We determined that these rat liver coated vesicles possess a latent insulin binding capability. That is, little if any specific binding of 125I-insulin to coated vesicles is observed in the absence of detergent. However, coated vesicles treated with the detergent octyl glucoside exhibit a substantial specific 125I-insulin binding capacity. We visualized the insulin binding structure of coated vesicles by cross-linking 125I-insulin to detergent-solubilized coated vesicles using the bifunctional reagent disuccinimidyl suberate followed by electrophoresis and autoradiography. The receptor structure thus identified is identical to that of the high-affinity insulin receptor present in a variety of tissues. We isolated liver coated vesicles from rats which had received injections of 125I-insulin in the hepatic portal vein. We found that insulin administered in this fashion was rapidly and specifically taken up by liver coated vesicles. Taken together, these data are compatible with a functional role for coated vesicles in the receptor-mediated endocytosis of insulin.

Animals↗
PubMed1982

Modification of the insulin receptor by diethyl pyrocarbonate: effect on insulin binding and action.

Insulin binding to rat liver plasma membranes is inhibited in a time- and dose-dependent fashion by prior treatment of membranes with the histidine-specific reagent diethyl pyrocarbonate. If all receptors are occupied by unlabeled hormone during diethyl pyrocarbonate treatment, no inhibition of 125I-labeled insulin binding is observed folowing washout of unlabeled hormone and unreacted reagent. Scatchard analysis of the binding inhibtion due to diethyl pyrocarbonate reveals a loss in receptor number rather than a change in receptor affinity for hormone. Fat cells treated with diethyl pyrocarbonate exhibit a rightward shift in the dose-response relationship for insulin-stimulated glucose oxidation consistent with a loss in receptor number due to the reagent. The pH profile for inhibition of insulin binding by diethyl pyrocarbonate and the partial reversibility of this inhibition by hydroxylamine are consistent with modification of a histidine residue. These results suggest that a histidine residue at or near the receptor binding site is required for formation of the biologically relevant insulin - receptor complex.

Adipose Tissue↗