Insulin down-regulates insulin receptor number and up-regulates insulin receptor affinity in cells expressing a tyrosine kinase-defective insulin receptor

We examined the effect of insulin treatment on HTC cells transfected with large numbers of either normal insulin receptors (HTC-IR) or insulin receptors defective in tyrosine kinase (HTC-IR/M-1030). In both HTC-IR and HTC-IR/M-1030 cells, 20 h of insulin treatment (1 microM) at 37 degrees C resulted in a 65% decrease in the number of binding sites with a reciprocal 6-fold increase in affinity. In contrast, treatment with 10 nM insulin (20 h, 37 degrees C) also increased receptor affinity but had a smaller effect on the number of binding sites. 125I-Insulin binding to soluble receptors from HTC-IR and HTC-IR/M-1030 cells pretreated with insulin showed results similar to those obtained in intact cells. In both HTC-IR and HTC-IR/M-1030 cells, insulin enhanced insulin receptor degradation. In HTC-IR/M-1030 cells a 1-h incubation with insulin did not change receptor number and had only a small effect on receptor affinity; also there was no effect of insulin after a 20-h incubation at 15 degrees C. Inhibiting protein synthesis by pretreatment with cycloheximide (100 microM) did not block either the decrease in receptor number or the increase in receptor affinity. Both HTC-IR and HTC-IR/M-1030 cells exhibited a very slow rate of insulin and insulin receptor internalization and no differences were seen in this parameter when HTC-IR cells were compared to HTC-IR/M-1030 cells. These studies indicate, therefore, that in cells expressing kinase-defective insulin receptors, insulin down-regulates insulin receptor number via enhanced receptor degradation, and up-regulates receptor affinity. These effects were time- and temperature-dependent, but not dependent on new protein synthesis, and suggest that activation of tyrosine kinase may not be a prerequisite for certain mechanisms whereby insulin regulates its receptor.     

Regulation of insulin receptor internalization in vascular endothelial cells by insulin and phorbol ester

Phorbol 12-myristate 13-acetate (PMA) was used to examine the role of insulin receptor phosphorylation in the regulation of insulin receptor internalization in vascular endothelial cells. Association of 125I-insulin in rat capillary and bovine aortic endothelial cells preincubated with PMA was increased by 80 and 64% over control, respectively. The increase was due to enhanced 125I-insulin internalization as opposed to an effect on surface-bound hormone. PMA had no significant effect on 125I-insulin degradation or on release of internalized insulin from the cells. Internalization of 125I-labeled insulin receptor was determined by the resistance of labeled receptor to trypsinization. At 10 degrees C, nearly all of the labeled receptor was sensitive to removal by trypsin, indicating that it was exposed on the cell surface. Exposure of labeled cells to insulin (100 nM) at 37 degrees C resulted in the rapid appearance of trypsin-resistant insulin receptor, indicating receptor internalization. Steady state for receptor internalization was attained at 10-15 min. When surfaced-labeled cells were preincubated with PMA at 37 degrees C, the rate of insulin receptor internalization was increased by 3.6 +/- 0.2-fold and 2.1 +/- 0.5-fold at 1 and 5 min of insulin exposure, respectively (ED50 at 16 nM PMA). This effect of PMA was associated with an increase in serine phosphorylation of the insulin receptor. Thus, PMA increased insulin internalization in the endothelial cells by modulating the insulin-induced internalization of the receptor. The additive effects of PMA and insulin on insulin receptor phosphorylation suggest that the phorbol ester and insulin act via independent signaling mechanisms.     

Kinetics of insulin binding and kinase activity of the partially purified insulin receptor from human skeletal muscle

The kinetics of insulin binding and kinase activity of soluble, partially purified insulin receptors from human skeletal muscle are considered. An equilibrium for insulin binding was obtained within 2 h at 37 degrees C. At lower temperatures the equilibrium for insulin binding was less clearly defined. Dissociation of 125I-labelled insulin was incomplete unless an excess amount of unlabelled insulin was added. Insulin-stimulatable autophosphorylation of the 95 kDa subunit was verified by gel electrophoresis. The kinase activity was measured with the synthetic polypeptide poly(Glu-Tyr(4:1] as a phosphoacceptor. The insulin receptor kinase activity correlated significantly (r = 0.92, P less than 0.0001) to the concentration of high-affinity insulin binding sites in the eluate. Autophosphorylation of the insulin receptor was necessary for the activation of the receptor kinase. When activated the receptor kinase activity was stable for at least 60 min at 21 degrees C with a pH optimum of approx. 7.8, similar to the pH optimum for insulin binding. The non-ionic detergent Triton X-100 inhibited the sensitivity of the receptor kinase to insulin. Insulin stimulated the Vmax of the kinase reaction about 3-fold, decreased the Km for ATP from 35 +/- 5 microM (mean +/- S.E.) to 8 +/- 1 microM (P less than 0.02) and induced a positive cooperativity to ATP with an increase in the Hill coefficient from 1.00 +/- 0.02 to 1.37 +/- 0.07 (P less than 0.05). According to the Hill plots, insulin itself showed no cooperativity with respect to receptor binding or kinase activation.     

Insulin receptor cycling and insulin action in the rat adipocyte

The possibility that the insulin receptor of adipocytes undergoes cycling was examined by a method involving pronase digestion at 12 degrees C, followed by insulin binding studies to determine receptor location and quantity. In the absence of insulin treatment, the amount of internal receptors (i.e. protected from pronase) was 10% of total receptor content. Following a 30-min insulin treatment (0.1 microM) at 37 degrees C, the internal receptor content increased 2-fold (206 +/- 12% of control, 100%). This effect was rapid, and maximum internalization was approached by 5 min of insulin treatment. Warming pronase-digested cells to 37 degrees C allowed the internal receptors to move to the cell surface. This movement was rapid also, and expansion of the internal pool by insulin pretreatment provided a 2.4-fold increase in the reinsertion of cell-surface receptors (238 +/- 28% of nontreated cells, 100%). Insulin-pretreated and nontreated cells had approximately 13 and 6%, respectively, of their original cell-surface receptor content, i.e. their content before pronase digestion. These receptors appeared intact after the cycling process, as judged by affinity labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the receptor and its binding subunit. The ability of the recycled receptor to respond to insulin was examined by studies of glucose incorporation into lipids and the inhibition of isoproterenol-stimulated lipolysis. Cells pretreated with insulin and allowed to recycle (e.g. 13% of normal receptor content) were 2-3-fold more responsive and 7-fold more sensitive to subsequent insulin stimulation than nontreated cells (e.g. 6% of normal receptor content), indicating that the recycled receptors are biologically active and coupled to cellular effector systems.     

Characterization of specific insulin binding sites on chromaffin cells from bovine adrenal medulla

1. Insulin receptors were investigated in isolated chromaffin cells from bovine adrenal medulla. 2. The cells were incubated with [125I]insulin in HEPES buffer, pH 7.8 at 15 degrees C for 180 min to obtain steady state binding. Specific binding was linearly related to the number of cells in the range 0.5-10 x 10(6) cells/ml. Insulin and proinsulin caused half maximal displacement of specifically bound tracer in concentrations of 0.18 and 2.46 nM, respectively. 3. Computer analysis of the binding data gave a linear Scatchard plot, consistent with a single class of non-interacting receptors with an affinity constant of 5.6 nM-1, the total number of receptors per cell being 1700. 4. The apparent MW of the insulin binding subunit of the receptor was 135,000, determined by affinity crosslinking and SDS gel electrophoresis under reducing conditions.     

Biochemical and morphological evidence that the insulin receptor is internalized with insulin in hepatocytes

There is morphological and biochemical evidence that insulin is internalized in hepatocytes. The present study was designed to investigate the fate of the insulin receptor itself, subsequently to the initial binding step of the hormone to the hepatocyte plasma membrane. The insulin receptor was labeled with a 125I-photoreactive insulin analogue (B2[2-nitro,4-azidophenylacetyl]des-PheB1-insulin). This photoprobe was covalently coupled to the receptor by UV irradiation of hepatocytes after an initial binding step of 2-4 h at 15 degrees C. At this temperature, only limited (approximately 20%) internalization of the ligand occurred. In a second step, hepatocytes were resuspended in insulin-free buffer and further incubated for 2-4 h at 37 degrees C. After h at 37 degrees C, no significant radioactivity could be detected in non-UV-irradiated cells, whereas 12-15 % of the radioactivity initially bound remained associated to UV-irradiated cells. Morphological analysis after electron microscopy revealed that approximately 70% of this radioactivity was internalized and preferentially associated with lysosomal structures. SDS PAGE analysis under reducing conditions revealed that most of the radioactivity was associated with a 130,000-dalton band, previously identified as the major subunit of the insulin receptor in a variety of tissues. Internalization of the labeled insulin-receptor complex at the end of the 37 degrees C incubation was further demonstrated by its inaccessibility to trypsin. Conversely, at the end of the association step, the receptor (also characterized as a predominant 130,000-dalton species) was localized on the cell surface since it was cleaved by trypsin. We conclude that in hepatocytes the insulin receptor is internalized with insulin.     

Homogeneous functional insulin receptor from 3T3-L1 adipocytes. Purification using N alpha B1-(biotinyl-epsilon-aminocaproyl)insulin and avidin-sepharose

Insulin receptor was purified 10,000-fold from cultured mouse 3T3-L1 adipocytes in 35% overall yield. The specific activities of 125I-insulin binding and autophosphorylation increased in parallel, following the initial Triton X-100 extraction of membranes. The isolation protocol, performed entirely at pH 8.45, entailed adsorption by avidin-Sepharose CL-4B of a complex formed between Triton X-100-solubilized insulin receptor and N alpha B1-(biotinyl-epsilon-aminocaproyl)insulin, and the specific elution of the complex with biotin. The avidin-Sepharose CL-4B was a partially denatured preparation, showing estimated dissociation constants of 0.2 microM for biotin and approximately 1 microM for the bifunctional ligand at, pH 7, 4 degrees C. The bifunctional ligand was characterized by 70% competency in binding to avidin, 100% competency in binding to solubilized insulin receptor, full stimulation of autophosphorylation of the isolated receptor, and maximal stimulation of hexose uptake by intact 3T3-L1 adipocytes. The insulin binding properties of the insulin receptor were uniform throughout this purification procedure. At pH 8.45, 4 degrees C, an average Kd = 0.72 nM was determined for a single class of noninteracting insulin binding sites. The apparent autophosphorylation of the beta-subunit was also unchanged following affinity chromatography. A single oligomeric structure was established for the purified receptor, composed only of 135,000- and 95,000-Da subunits, whose association was lost by denaturation in the presence of reducing agent. This single structure occurred in the initial Triton X-100 extract. The purified insulin receptor was capable of autophosphorylating the beta-subunit and catalyzed phosphorylation of protein substrates.     

Mouse glomerular endothelial cells have an insulin receptor

An insulin receptor was found on the surface of cloned mouse glomerular endothelial cells in vitro. Total specific binding was 2.5 +/- 0.3%/10(6) cells at 90 min and 22 degrees C. Analysis according to Scatchard resulted in a curvilinear plot, with a kd for the high and low affinity sites estimated at 1.41 x 10(-10) and 8.2 x 10(-8) respectively. Insulin binding decreased following 12 hour exposure to 50 ng/ml of insulin suggesting that down regulation of the receptor had occurred, an effect which was reversible. Covalent crosslinking of the receptor to 125I insulin revealed one band at Mr 125,000 by SDS-PAGE which disappeared following preincubation with excess unlabeled insulin. Insulin was also able to stimulate phosphorylation of the beta subunit. The characteristics of this insulin receptor appear very similar to that of endothelial cell types from other microvascular beds.     

Inability of insulin and insulinlike growth factor-1 to stimulate sugar or amino acid transport and thymidine incorporation in cultured myeloma cells.

NS-1 mouse plasmacytoma cells were examined for their insulin and insulinlike growth factor-1 (IGF-1) binding characteristics and ability to produce peptide-dependent cellular effects. At concentrations of labelled insulin (i.e., 1.7 x 10(-10) M) or IGF-1 (i.e., 1.5 x 10(-10) M), NS-1 cells specifically bind 0.2 +/- 0.06 fmol insulin per 10(6) cells (n = 7), where little, if any, IGF-1 specific binding was observed (0.02 +/- 0.01 fmol/10(6) cells) (n = 3). Additionally, the data indicate that the total number of insulin binding sites per cell was 3200 +/- 390 (n = 3). Insulin was employed at various concentrations (6.7-667 nM) and failed to stimulate either sugar or amino acid transport. Insulin at low concentrations (i.e., 6.7 or 67 nM) did not stimulate DNA synthesis, yet a small but significant increase was observed at a concentration of 667 nM insulin. IGF-1 did not stimulate DNA synthesis at all concentrations employed (1.4-143 nM). In summary, there exists a small but significant number of insulin receptors, little insulin-stimulated DNA synthesis, and no apparent insulin stimulation of sugar or amino acid transport. Also, since there is no significant IGF-1 binding and no IGF-1 stimulation of DNA synthesis, these findings indicate that this cell line might be a good candidate for the study of insulin receptor function as a transfection recipient of insulin receptor genes.     

Insulin elicits a redistribution of transferrin receptors in 3T3-L1 adipocytes through an increase in the rate constant for receptor externalization

Incubation of 3T3-L1 adipocytes with insulin at 37 degrees C resulted in a 2-fold increase in specific binding of transferrin to cell-surface receptors, as measured by a subsequent incubation of cells at 4 degrees C with 125I-transferrin. The insulin concentration required for half-maximal effect was 10 nM, and the half-time for insulin action was 40 s. By comparison, insulin stimulated hexose transport in 3T3-L1 adipocytes with a half-maximal effect at 8 nM and a half-time of 105 s. Scatchard analysis of 125I-transferrin binding to cells at 4 degrees C showed that the insulin-induced increase in transferrin receptor binding was due to an increase in the number of surface transferrin receptors. When cells were incubated for 2 h at 37 degrees C with 125I-transferrin to achieve steady-state binding and then exposed to insulin, there was a 1.7-fold increase in surface-bound transferrin (acid-sensitive) and a corresponding decrease in intracellularly bound transferrin (acid-insensitive). Thus, insulin elicits translocation of intracellular transferrin receptors to the plasma membrane. Concomitant with the 2-fold increase in surface receptors in response to insulin, there was a 2-fold increase in the rate of 59Fe3+ uptake from 59Fe3+-loaded transferrin. The rate of externalization of the intracellular 125I-transferrin-receptor complex at 37 degrees C was determined for basal and insulin-treated cells. Insulin increased the first-order rate constant for this process 1.7-fold. The effect of insulin on the rate of externalization is sufficient to account for the increase in surface transferrin receptors.     

Thyrotropin releasing hormone action in pituitary cells. Protein kinase C-mediated effects on the epidermal growth factor receptor

Thyrotropin releasing hormone (TRH) causes phosphatidylinositol bisphosphate hydrolysis to form inositol trisphosphate and diacylglycerol. Since diacylglycerol activates protein kinase C (Ca2+/phospholipid-dependent enzyme), this enzyme may be involved in mediating the physiological response to TRH. Activation of protein kinase C leads to phosphorylation of receptors for epidermal growth factor (EGF) and decreased EGF affinity. The present study examined the effect of TRH on EGF binding to intact GH4C1 rat pituitary tumor cells to test whether TRH activates protein kinase C. Cells were incubated with TRH at 37 degrees C and specific 125I-EGF binding was then measured at 4 degrees C. 125I-EGF binding was decreased by a 10-min treatment with 0.1-100 nM TRH to 30-40% of control in a dose-dependent manner. 125I-EGF binding was not altered if cells were incubated at 4 degrees C, although TRH receptors were saturated or in a variant pituitary cell line without TRH receptors. TRH (10 min at 37 degrees C) decreased EGF receptor affinity but caused little change in receptor density, 125I-EGF internalization, or degradation. When cells were incubated continuously with TRH, there was a recovery of 125I-EGF binding after 24 h. Incubation with the protein kinase C activating phorbol ester TPA caused an immediate (less than 10 min) profound (greater than 85%) decrease in 125I-EGF binding followed by partial recovery at 24 h. Maximally effective doses of TRH and TPA decreased EGF receptor affinity with half-times of 3 min. EGF treatment (5 min) caused an increase in the tyrosine phosphate content of several proteins; prior incubation with TRH resulted in a small decline in the EGF response. GH4C1 cells were incubated with 500 nM TPA for 24 h in order to down-regulate protein kinase C. Protein kinase C depletion was confirmed by immunoblots and the effects of TRH and TPA on 125I-EGF binding were tested. TRH and TPA were both much less effective in cells pretreated with phorbol esters. TRH increased cytoplasmic pH measured with an intracellularly trapped pH sensitive dye after mild acidification with nigericin. This TRH response is presumed to be the result of protein kinase C-mediated activation of the amiloride-sensitive Na+/H+ exchanger and was blunted in protein kinase C-depleted cells. All of these results are consistent with the view that TRH acts rapidly in the intact cell to activate protein kinase C and that a consequence of this activation is EGF receptor phosphorylation and Na+/H+ exchanger activation.     

Distribution of insulin receptors between plasmalemma and intracellular compartments: effect of amino acids

Insulin receptor regulation was studied in the rat erythroblastic leukemic (EBL) cell in primary culture. After 1-2-hr incubations in medium containing 12 essential amino acids, glutamine, and serine, EBL cell protein synthesis and insulin receptor concentrations were increased compared to cells incubated without serine. Deficiency of medium isoleucine in the presence of serine rapidly decreased protein synthesis and insulin binding to intact cells. Supplementation of deficient media with serine or isoleucine had no effect on total insulin receptor numbers measured in solubilized cell preparations. Increased insulin binding following serine exposure was seen with binding assays at both 4 and 37 degrees C. Dissociation experiments to quantitate intracellular ligand after 37 degrees C binding assays showed increased in both surface binding and intracellular [125I]insulin accumulation. These data combined with previous observations suggest that amino acids essential for this cell are required for the rapid synthesis of a labile regulatory protein which facilitates the redistribution and/or recycling of insulin receptors.     

Insulin Binding and Effects on Pyrimidine Nucleoside Uptake and Incorporation in Cultured Mouse Astrocytes

Binding of 125I-insulin to primary cultures of differentiated mouse astrocytes was time-dependent, reaching equilibrium after 2 h at 22 degrees C, with equilibrium binding corresponding to 20.79 fmol/mg of protein, representing approximately 5,000 occupied binding sites/cell. The half-life of 125I-insulin dissociation at 22 degrees C was 2 min, with an initial dissociation rate constant of 4.12 X 10(-2) s-1. Dissociation of bound 125I-insulin was not accelerated significantly in the presence of unlabeled insulin (16.7 microM). Porcine and desoctapeptide insulins competed for specific 125I-insulin binding in a dose-dependent manner, whereas growth hormone, glucagon, and somatostatin did not. For porcine insulin, Scatchard analysis suggested multiple-affinity binding sites (high-affinity Ka = 4.92 X 10(8) M-1; low-affinity Ka = 0.95 X 10(7) M-1). After incubation with insulin (0.5 microM) for 2 h at 37 degrees C, increases above basal values of 254 +/- 23 and 189 +/- 34% for [3H]uridine uptake and incorporation, respectively, were observed. After incubation with insulin (0.5 microM) for 24 h at 37 degrees C, there were increases of 145 +/- 6% for [3H]thymidine uptake and 166 +/- 11% for thymidine incorporation. Basal and stimulated uridine and thymidine uptake and incorporation were inhibited by 50 microM dipyridamole. These studies confirm that mouse astrocytes in vitro possess specific insulin receptors and demonstrate an effect of insulin on pyrimidine nucleoside uptake and incorporation.     

Evidence that insulin plus ATP may induce a conformational change in the beta subunit of the insulin receptor without inducing receptor autophosphorylation.

The effect of insulin and ATP on insulin receptor beta subunit conformation was studied in vitro with radioiodinated monoclonal antibodies directed at several regions of the receptor beta subunit. Insulin plus ATP inhibited their binding to the receptor. The greatest inhibitory effect of insulin and ATP was seen with antibody 17A3 which recognizes a domain of the beta subunit that is near the major tyrosine autophosphorylation sites at residues 1158, 1162, and 1163. ATP alone inhibited 17A3 binding with a one-half maximal ATP inhibitory concentration of 186 +/- 7 microM. Insulin at concentrations as low as 100 pM potentiated the effect of ATP; at 100 nM where insulin had its maximal effect, insulin lowered the one-half maximal inhibitory concentration of ATP to 16 +/- 6 microM. At 1 mM CTP, GTP, ITP, TTP, and AMP were without effect in either the presence or absence of insulin; in contrast, ADP was inhibitory in the presence of insulin. Of major interest was adenyl-5'-yl imidodiphosphate (AMP-PNP). This nonhydrolyzable analog of ATP inhibited 17A3 binding, and the effect of AMP-PNP (like ATP) was potentiated by insulin. Two insulin receptor beta subunit mutants then were studied. Mutant receptor F3, where the major tyrosine autophosphorylation sites at residues 1158, 1162, and 1163 were changed to phenylalanines, bound to 17A3; antibody binding was inhibited by insulin and ATP in a manner similar to normal receptors. In contrast, mutant receptor M1030, where the lysine in the ATP binding site at residue 1030 was changed to methionine, bound 17A3, but unlike either normal receptors or F3 receptors, the binding of 17A3 was not inhibited by insulin and ATP. Therefore, these studies raise the possibility that, in vivo, ATP binding in the presence of insulin may induce a conformational change in the insulin receptor beta subunit which in turn signals some of the biological effects of insulin.     

Up-regulation of insulin receptors with dexamethasone in cultured human urinary bladder carcinoma cells

Cultured human urinary bladder carcinoma cells ( JTC -32) were used to investigate the regulation of insulin receptors by dexamethasone. When the cells were preincubated with dexamethasone at 37 degrees C, insulin binding sites increased up to 24 h. A large increase in insulin binding sites took place for 14 h of preincubation with dexamethasone. At lower concentrations of dexamethasone (less than 1 nM), no significant increase in insulin binding sites was observed, but the maximal increase was observed at more than 10 nM. Scatchard plots showed that dexamethasone increased the number of high affinity insulin binding sites (2.8 fold) without any change in the apparent equilibrium constant in JTC -32 cells. In addition, this steroid hormone also increased the number of low affinity insulin binding sites (1.6 fold) with a small change in the apparent equilibrium constant. Although insulin and dexamethasone did not affect the number of cells or the amount of cellular proteins per dish, dexamethasone plus insulin slightly increased them.     

Kinetics of insulin binding to rat white fat cells at 15 degrees C

The kinetics of insulin binding to isolated rat epididymal fat cells was investigated at 15 degrees C, at which temperature the system was simplified by the absence of lysosomal insulin degradation. The data were fit by maximum likelihood criteria with differential equations describing a number of models for the interaction of insulin and cells. Among those models that yielded a fit, the selection criteria were minimization of the Akaike information criterion and compatibility of the overall equilibrium constant for the system calculated from rate constants with the previously obtained experimental value. The results of the analysis indicated that insulin, I, first reversibly bound to cell surface receptors, R, whereupon this initial insulin-receptor complex, RI, reversibly altered its state or cellular location to R'I, according to the following equation. (Formula: see text) No evidence was found that insulin could either associate or dissociate from R'I directly. The association rate constant was kappa 12 = 1.6 x/divided by 1.4 X 10(5) liter mol-1 s-1, a value shown to be incompatible with diffusion control. The other rate constants were: kappa 21 = 3.4 x/divided by 1.6 X 10(-3) s-1, kappa 23 = 3.2 x/divided by 1.5 X 10(-4) s-1, and kappa 32 = 2.0 x/divided by 1.5 X 10(-4) s-1. From these rate constants, an equilibrium constant of 8.4 x/divided by 1.5 nM was calculated, in excellent agreement with the previously measured value of 8.8 x/divided by 1.3 nM (Lipkin, E. W., Teller, D. C., and de Ha?n, C. (1986) J. Biol. Chem. 260, 1694-1701). The kinetic analysis also yielded receptor numbers similar to those obtained by equilibrium binding studies. The nature of the R'I state is discussed in terms of an internalized state, in terms of insulin receptor complex in caveolae, in terms of receptor aggregates, and in terms of being a Michaelis complex between insulin bound to the receptor and cell surface-bound insulin protease.     

Modification of membrane physical properties, biological response and insulin binding in Friend cells by low serum concentration

The effect of low serum concentration on plasma membrane fluidity and lipid composition, differentiation and insulin binding was investigated in three Friend erythroleukemia clones. Both FLC (clones No. 745) and F(+) (Ostertag F4N) Friend erythroleukemia cells can be induced to differentiate and to produce hemoglobin when exposed to DMSO. Clone R(3) (Ostertag F4-D5-1) is a DMSO-resistant clone when grown under normal conditions (15% serum) but could undergo differentiation with accumulation of protoporphyrin IX upon induction with DMSO when grown in low serum concentration (2.5% serum). Electron spin resonance measurements of the order parameters (S) and S(T parallel) demonstrate that R(3) has a more fluid plasma membrane than the FLC and F(+). The order parameters of the outer hyperfine splittings S(T parallel) at 37 degrees C are 0.60 +/- 0.009, 0.62 +/- 0.008 and 0.64 +/- 0.009 for R(3), F(+) and FLC, respectively. We have used the insulin receptors as a model for a polypeptide hormone receptor associated with the plasma membrane of the Friend clones. Insulin binding studies demonstrated that the receptor of R(3) had a decreased affinity for insulin manifest as a 10-fold increase in the amount of insulin required to compete for half of the tracer binding (18 nM for R(3) vs. 2 nM for FLC and F(+)). Computer-fit Scatchard plot analysis by the negative cooperativity model reveal that R(3) possessed a similar number of sites/cell (about 70,000) as the FLC or F(+) cells, with similar high and low affinities. Growing the DMSO-resistant clone R(3) in low serum concentration caused a decrease in receptor number by 35%, and an increase in receptor affinity to that seen with the differentiable clones. Thus, the abnormal properties of the plasma membrane and insulin receptor of the DMSO-resistant clone in our earlier report (Simon et al. (1984) Biochim. Biophys. Acta 803, 39-47) were partially reversed by growing the cells in a low serum concentration, restoring the cellular response to the differentiation agent.     

The devapamil-binding site of the purified skeletal muscle receptor for organic-calcium channel blockers is modulated by micromolar and millimolar concentrations of Ca2+.

The interaction of 2,7-dimethyl-3-(3,4-dimethoxyphenyl)-3-cyan-7-aza-9-(3- methoxyphenyl) nonahydrochloride (devapamil), a stereospecific analog of (3-[2-(3,4-dimethoxyphenyl)ethyl]- methylaminopropyl-3,4-dimethoxy-(1-methylethyl)benzeneacetonitr ile (verapamil), with the purified skeletal muscle receptor for calcium channel blockers (CaCB) was studied at 4 degrees C and 30 degrees C in the absence and presence of calcium. The purified CaCB receptor bound 0.9 mol devapamil/mol calcium-channel alpha 1 subunit, with an apparent Kd of 13 +/- 2.6 nM at 4 degrees C in the presence of 0.4 microM Ca2+. The affinity, and not the density, of the devapamil-binding site was decreased by lowering the pH from 8.5-6.5, or by increasing the Ca2+ concentration from 0.4 microM to 100 mM. The same results were obtained at 30 degrees C, although the ligand-receptor complex was not stable at Ca2+ concentrations below 10 microM. These binding data were confirmed by kinetic experiments. The rate constants calculated for a pseudo-first-order and a second-order reactions were identical and yielded fourfold lower k-1/k+1 (KD) values than the equilibrium experiments performed using 1 nM and 0.4 microM Ca2+, but the same values using 1 mM Ca2+. 1 mM Ca2+ increased the k-1/k+1 (KD) by decreasing 10-fold the association rate at 4 degrees C. The dissociation rate was increased about 10-fold by 5 microM devapamil or 100 microM D-cis-diltiazem, suggesting that the high affinity site is negatively regulated allosterically by millimolar Ca2+ concentrations and by the occupation of a second low-affinity site. Incubation of the CaCB receptors in the absence of Ca2+ and devapamil at 30 degrees C, but not at 4 degrees C, resulted in an apparent loss of devapamil-binding sites. The decrease in binding sites was caused by a reduced affinity. This apparent loss of binding sites was prevented by the addition of Ca2+ with an apparent median effective concentration of 0.4 microM. The apparent half-maximal inactivation times of the devapamil-binding site were 90 s and 12 min in the presence of 1 nM and 0.4 microM Ca2+, respectively. These results show that micromolar Ca2+ concentrations stabilize the CaCB receptor in a conformation which allows high-affinity binding of phenylalkylamines. Millimolar Ca2+ concentrations induce a low-affinity state of the devapamil-binding site on a stable CaCB receptor.     

The development of insulin receptors and responses in the differentiating nonfusing muscle cell line BC3H-1

We have studied the development of high affinity insulin receptors and insulin-stimulated responses in the differentiating nonfusing muscle cell line BC3H-1. In the logarithmic growth phase, these myoblasts exhibit very low levels of insulin binding and no detectable insulin-stimulated glucose or amino acid uptake. Following the cessation of cell division and subsequent spontaneous differentiation, the resulting myocytes develop a 5-fold increase in specific 125I-insulin binding and demonstrate physiologic insulin-stimulated glucose and amino acid uptake (100% increase above baseline) with half-maximum stimulation at 1-3 nM in agreement with the known in vivo and in vitro insulin sensitivity of muscle tissue. Insulin stimulation of 2-deoxyglucose uptake is detectable within 3 min, becomes maximal within 15 min, and is mediated by a rapid increase of plasma membrane transport units, as determined by D-glucose-inhibitable cytochalasin B binding, resulting in a 2-fold increase in the Vmax for 2-deoxyglucose transport with no change in Km. Myocyte insulin binding is specific, reversible, and saturable, yielding equilibrium within 18 h at 4 degrees C. Scatchard analysis identified the high affinity insulin receptor with a Kd of 0.5 nM at 4 degrees C. The myocytes also demonstrate sensitive down-regulation of cell surface insulin receptors, with a maximum decrease of 50% in cell surface insulin binding following exposure to 20 nM insulin for 18 h at 37 degrees C. Since the differentiation of this muscle cell line from myoblasts to nonfusing myocytes is accompanied by the development of high affinity insulin receptors and physiologic insulin-stimulated glucose and alpha-methylaminoisobutyric acid uptake, this continuously cultured system provides an excellent model for the study of differentiation and mechanism of insulin action in muscle, its quantitatively most significant target tissue.     

Nuclear estradiol binding in rat adipocytes. Regional variations and regulatory influences of hormones.

The nuclear estrogen receptor was characterised in isolated rat adipocytes. The binding reaction with [3H]estradiol was performed with intact isolated rat adipocytes and the radioactivity associated with the nucleus was subsequently determined after cell lysis. The nuclear uptake of [3H]estrogen in rat adipocytes was temperature dependent and steroid specific. The steady-state binding was achieved after 30 min at 37 degrees C and was constant for several hours. Estradiol was found to bind to a homogeneous class of nuclear receptors in epididymal adipocytes with an apparent Kd of 3.1 +/- 0.76 nM and a Bmax of 7.98 +/- 1.11 fmol/10(6) cells corresponding to about 4800 receptors per nucleus. The estradiol binding exhibited regional variations in isolated adipocytes. In lean rats the highest receptor number was found in epididymal adipocytes, whereas there was a significantly lower number of nuclear binding sites in perirenal and subcutaneous adipocytes (P less than 0.05), unlike in older and more obese rats where the nuclear estradiol binding was greatest in adipocytes from the perirenal fat depot. Incubations with isoproterenol (10 microM) and dibutyryl-cAMP (2.5 mM) both reduced estradiol binding by 56% (P less than 0.005), while insulin (1 nM) enhanced the estradiol binding by 37% (P less than 0.01). In conclusion, a specific and high affinity nuclear estradiol receptor was demonstrated in rat adipocytes and regional differences in nuclear estradiol binding were detected. Furthermore, it was demonstrated that nuclear estradiol binding could be modulated by other agents known to affect adipocyte metabolism.