H. Joseph Goren

Binding and structural properties of oxytocin receptors in isolated rat epididymal adipocytes

Oxytocin initiates its insulin-like action in adipocytes through oxytocin-specific receptors. We have studied binding and structural properties of these receptors with the radioligand [3H]oxytocin. Steady-state binding was reached after 45 min, at 21 degrees C, and 10 min at 37 degrees C. Scatchard analyses of equilibrium binding data indicated a single class of oxytocin binding sites at 21 degrees C (KD = 3.3 nM, RT = 6 X 10(4) sites/cell) and 2 binding sites at 37 degrees C (KD = 1.5 nM, RT = 6 X 10(4) sites/cell; and KD = 20 nM, RT = 30 X 10(4) sites/cell). Insulin, insulin-like growth factor I, and epidermal growth factor increased oxytocin binding (approximately 20-40%), whereas adenosine, a regulator of oxytocin action, did not affect oxytocin binding. Binding activity of oxytocin was impaired by pretreatment of the hormone or adipocytes with dithiothreitol. Dithiothreitol treatment of adipocytes preferentially inactivated high-affinity binding sites. N-ethyl maleimide inhibited oxytocin binding in adipocytes more than dithiothreitol. In contrast to the inhibitory effects of dithiothreitol and N-ethyl maleimide, proteases (trypsin, chymotrypsin and papain) were not able to inhibit fat cell binding activity. These results suggested that in isolated adipocytes: there are high-affinity and low-affinity receptors, but the low-affinity receptors are absent at 21 degrees C; the binding of oxytocin can be regulated by insulin, and growth factors; and the oxytocin receptors contain disulfide bridges and free thiols that are essential for the maintenance of oxytocin binding.

The 180000 molecular weight plasma membrane insulin receptor substrate is a protein tyrosine phosphatase and is elevated in diabetic plasma membranes

Wheat germ agglutinin-purified non-diabetic and diabetic human placenta membranes were or were not depleted of EGF receptor with monoclonal anti-EGF receptor antibody B1D8, and subsequently phosphorylated. Phosphorylated insulin receptor beta-subunit was lower and pp180 was higher in diabetic placenta membranes than in non-diabetic membranes. Phosphorylated-beta-subunit was also lower in diabetic (streptozotocin-induced) rat liver whereas the amount of pp180 was dependent on membrane protein concentration. When rat liver tyrosine-phosphorylated proteins were incubated 30 min, 4 degrees C with EDTA-terminated 32P-phosphorylation reaction mixtures of wheat germ agglutinin-purified rat liver proteins, less phosphorylated proteins were immunoprecipitated with antiphosphotyrosine. The decrease in tyrosine-phosphorylated products suggested that pp180 was a protein tyrosine phosphatase. Taken together, the results suggest that diabetic plasma membranes contain more tyrosine phosphatase than non-diabetic membranes.

Adenosine modulation of fat cell responsiveness to insulin and oxytocin

We have investigated the effects of adenosine on the stimulation of glucose oxidation and lipogenesis by oxytocin and insulin in rat epididymal adipocytes. The addition of adenosine deaminase (1 U/ml) to the assay medium reduced the maximal oxytocin response (glucose oxidation and lipogenesis) to between 25 and 50% of the maximum response in control cells. The maximal response to insulin was not appreciably affected under these conditions. The addition of adenosine (10 or 30 microM) increased the cell sensitivity to oxytocin by elevating the maximum rate of oxytocin-stimulated glucose metabolism. Adenosine also increased the cell sensitivity to insulin by decreasing its ED50. A change in ED50, however, was observed only when control or adenosine-treated cells were compared to adenosine deaminase-treated cells; but not when control and adenosine-treated cells were compared. On its own, adenosine also caused an appreciable increase in both glucose oxidation and lipogenesis (ED50 approximately equal to 3 microM adenosine). The difference in the effect of adenosine on oxytocin action, compared with the effect on insulin action, points to differences in the mechanisms by which insulin and oxytocin stimulate glucose metabolism in adipocytes.

Phosphorylation and nuclear processing of the androgen receptor

Although transformed androgen receptor (AR) complexes derived from cytosol and nuclear AR complexes have been shown to bind with high affinity to nuclei and DNA, we have shown that the binding characteristics of the two receptor populations to rat ventral prostate nuclei are different. To account for these differences, we investigated the possibility that the two receptor populations differed in phosphorylation status. Significantly, an anti-phosphotyrosine antibody immunoprecipitated androgen binding from the nuclear AR preparation but not from the transformed cytosolic receptor preparation. These studies suggest that (i) further processing of the AR complex takes place after it has become transformed, and (ii) phosphorylation of the complex is one modification which occurs during the processing of the nuclear receptor.

Ligand-Receptor Interactions at the Cell Surface

Over the past 20 years, studies of pharmacological receptors have undergone a veritable biochemical metamorphosis. The first thorough study of the binding of a pharmacologically active compound (atropine) to its receptor (muscarinic cholinergic: Paton and Rang, 1965) was soon followed by a multitude of studies of the binding of a variety of ligands to their putative receptors. Now, it is realized that receptor structures are dynamic cellular elements that display considerable structural and functional complexities. Thus, in order to measure the interaction of a ligand with its cell surface receptor, it is essential to have a grasp of the many variables that can affect such measurements. In this chapter, we attempt to provide a suitable context in which the measurements of ligand-receptor interactions can be interpreted, and we provide several models of receptor structure and function that have emerged over the past 10 years.

In vitro, insulin receptor catalyses phosphorylation of clathrin heavy chain and a plasma membrane 180,000 molecular weight protein

Insulin receptor mutation studies indicate that the receptor tyrosine kinase activity is necessary for receptor endocytosis, and several insulin receptor-containing tissues have a plasma membrane-associated protein (Mr congruent to 180,000, p180) whose tyrosine phosphorylation is receptor catalysed. Since clathrin heavy chain (Mr congruent to 180,000 in dodecyl sulphate gel electrophoresis) is a major component of coated vesicles, the latter functioning in receptor endocytosis, we investigated whether insulin receptors can catalyse clathrin phosphorylation and whether p180 is clathrin. Bovine brain triskelion or coated vesicles and 32P-ATP were added to prephosphorylated insulin receptor preparations (wheat germ agglutinin-purified human placenta membrane proteins). Antiphosphotyrosine immunoprecipitated a phosphorylated 180,000 molecular weight protein. Insulin (10(-7) M) increased the rate of phosphorylation. Monoclonal anti-clathrin antibody immunoprecipitated the phosphorylated 180,000 molecular weight protein, whereas monoclonal anti-insulin receptor antibodies (alpha-IR1, MA10) immunoprecipitated both insulin receptors and the phosphorylated 180,000 molecular weight protein. In the absence of added clathrin, anticlathrin immunoprecipitated no proteins, and alpha-IR1 immunoprecipitated only the insulin receptor. Density gradient (glycerol 7.5-30%, w/v) centrifugation separated human placenta microsomal membrane proteins into endosomal, plasma membrane, cytoplasmic and coated vesicle fractions. Antiphosphotyrosine immunoprecipitated phosphorylated-microsomal proteins that centrifugated into endosomal and plasma membrane fractions. Addition of glycerol gradient fractions to a prephosphorylated insulin receptor preparation, however, gave a tyrosine-phosphorylated 180,000 molecular weight protein when cytoplasmic and coated vesicle fractions were added. Taken together these results suggest: (1) that, in vitro, human placenta insulin receptors can phosphorylate bovine brain and human placenta clathrin heavy chain; (2) that both assembled and unassembled clathrin can be phosphorylated; and (3) that p180, the plasma membrane-associated insulin receptor substrate, is not clathrin heavy chain.

A 180,000 molecular weight glycoprotein substrate of the insulin receptor tyrosine kinase is present in human placenta and in rat liver, muscle, heart and brain plasma membrane preparations

Cell signalling for insulin may include insulin receptor tyrosine kinase catalysing the phosphorylation of one or more cell proteins. Since temporally the insulin receptor will encounter plasma membrane proteins first, we have studied the in vitro phosphorylation of purified plasma membrane preparations. Two proteins were immunoprecipitated with anti-phosphotyrosine antibody from rat liver, muscle, heart and brain membranes and from human placenta membranes: the insulin receptor (detected as a phosphorylated-beta-subunit) and a 180,000 molecular weight protein (pp180). pp180 is a monomeric glycoprotein that in the absence of dithiothreitol migrated in denaturing gels like a 150,000 molecular weight protein. pp180 was a substrate for the insulin receptor: (i) receptor and pp180 phosphorylation followed a similar insulin dose-response, although fold-stimulation of autophosphorylation was greater; and (ii) removal of insulin receptors with monoclonal antibodies prevented subsequent pp180 phosphorylation. Insulin-activated receptors increased the extent, but not the rate, of pp180 phosphorylation; the increased phosphate was incorporated into tyrosine and appeared to do so in three or four of pp180s 12 tryptic phosphopeptides. Some data suggest that pp180 is the same protein in each of the tested tissues. The occurrence of pp180, an insulin receptor substrate, in plasma membranes of several insulin responsive tissues suggests that it has a role in insulin signalling.

Determination of dependence of binding parameters on receptor occupancy

Measurements of the binding of ligand to receptors that are macromolecules, either free or components of biomembranes, often show deviation from what is expected of a simple reaction described by an association and a dissociation rate constant. A more versatile model and more discriminating experiments are required for a satisfactory explanation. This paper is based on a general model of the binding reaction in which the rate constants and equilibrium constant are dependent upon occupancy of receptors. The analysis of the model leads to three kinds of experiments: (1) equilibrium measurements which permit quantitative determination of a dissociation equilibrium parameter as a function of receptor occupancy; (2) measurements prior to equilibrium which yield the same information; and (3) measurements prior to equilibrium which reveal quantitatively the dependence of both association and dissociation rate parameters separately, on occupancy.

Glucose oxidation, glucose transport and insulin binding in isolated rat epididymal adipocytes in the presence of anesthetics

Abstract Insulin binding and glucose oxidation were measured in isolated rat adipocytes in the presence of several anesthetics; ethanol, n-octanol, pentobarbital, chlorpromazine and tetracaine. Ethanol and chlorpromazine, at anesthetic and pentobarbitol at sub-anesthetic concentrations are inhibitory to both basal and insulin stimulated rates of glucose oxidation. At all concentrations of ethanol, pentobarbital or chlorpromazine tested binding of insulin is not affected. Since anesthetics may alter membrane fluidity, it is suggested that an anesthetic-induced increase in membrane fluidity beyond that which occurs at 37°C is detrimental to glucose oxidation. Of the 5 anesthetics examined, only chlorpromazine (10 μM or less) and tetracaine (500 μM) stimulate glucose oxidation. These two agents are known to bind to a cells cytoskeletal system; the binding of chlorpromazine to microtubules is entropy driven. The temperature and concentration dependence of chlorpromazine stimulation of glucose oxidation (transport) are consistent with this form of binding. It is proposed that chlorpromazine binds to the cytoskeletal system of the adipocyte and that this system is normally restrictive to the motion of membrane proteins. Disruption of the cytoskeletal system by chlorpromazine or tetracaine would increase the frequency of insulin-receptor and glucose-carrier contact. Activation of glucose transport could ensue.

In the absence of antibody IgGsorb precipitates human placenta phosphotyrosine-containing proteins

Lectin-purified human placenta plasma membrane proteins were phosphorylated in vitro. Mixing the reaction mixture with IgGsorb and incubation of the resultant pellet with p-nitrophenyl phosphate demonstrated the presence of phosphorylated-insulin receptor beta-subunit and a phosphorylated-180 kDa protein in acrylamide gel electrophoresis. The same two proteins were detected in the electrophoretic analyses of anti-phosphotyrosine immunoprecipitated phosphorylation reaction mixtures. In the absence of antibody, the amount of phosphorprotein in the IgGsorb pellet was dependent on the amount of IgGsorb added. IgGsorb did not precipitate 125I-labeled lectin-purified human placenta protein. Further, 10 mM O-phosphotyrosine completely inhibited the precipitation of phosphorylated human placenta proteins. These data suggest that IgGsorb specifically bound and precipitated phosphotyrosine-containing proteins in soluble human placenta plasma membranes.