An analysis of the relationship between the cellular distribution and the rate of turnover for the separate classes of unoccupied, noncovalently occupied, and covalently occupied insulin receptor

To further investigate insulin's role in regulating the turnover of insulin receptor during down-regulation in 3T3-L1 adipocytes, the relationship between the cellular distribution and turnover of unoccupied, noncovalently occupied, and covalently occupied receptor was examined. At steady-state 12% of the unoccupied receptors and 46% of covalently occupied receptors are intracellular. The apparent first-order rate constant (Kapp) for turnover of the total pool of covalently occupied receptors (0.16 h-1) is 3.8-fold higher than that for unoccupied receptors (0.042 h-1). When unlabeled insulin is added, identical values for both Kapp (0.10 h-1) and distribution (26% internal) are measured for noncovalently and covalently occupied receptors. The rate constant (Kdeg), describing the relative sensitivity of internalized receptor to degradation, is identical (0.36-0.41 h-1) for unoccupied, noncovalently occupied, and permanently occupied pools of internal receptor. Mechanisms for down-regulation postulating: (a) an occupancy-dependent alteration in the conformation of internal receptor increasing receptor sensitivity to internal proteases, (b) a preferential sorting of internal occupied receptor to degradative pathways, or (c) induction of intracellular proteases by insulin, would all reflect a substantial change in Kdeg for occupied receptor and thus are unlikely mechanisms by which insulin increases the rate of receptor turnover. The turnover of insulin receptor in 3T3-L1 adipocytes is regulated primarily by its intracellular concentration and not by the state of occupancy of internalized receptor.     

Kinetics of insulin receptor transit to and removal from the plasma membrane

The heavy isotope density shift method, in combination with a procedure for labeling cell surface insulin receptors, was used to determine the rate of transit of receptor to the cell surface from their site of synthesis and to follow the net rate of receptor removal from the plasma membrane in 3T3-L1 adipocytes. To label surface receptors, 125I-insulin was bound to cells at 4 degrees C and then covalently cross-linked to the receptors with disuccinimidyl suberate. The identity of the surface-labeled product as insulin receptor was established by immunoprecipitation with antireceptor antibody and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fully differentiated 3T3-L1 adipocytes were shifted to medium containing heavy (greater than 95% 15N, 13C and 2H) amino acids. The rates of appearance of newly synthesized heavy receptor at the cell surface and the loss of previously synthesized light receptor from the cell surface were followed by resolving labeled heavy and light surface receptors in CsCl density gradients and quantitating labeled receptor subunits by gel electrophoresis. It was shown that 2.5-3.0 h are required for newly synthesized insulin receptor to reach and become functional in the plasma membrane. Insulin-induced down-regulation of cellular insulin receptor level had no effect on the time required for the newly synthesized receptors to reach the cell surface. Down-regulation, however, increased the first order rate constants for the inactivation of cell surface insulin receptors from 0.046 to 0.10 h-1. The fact that the rate constants for inactivation of cell surface and total cellular insulin receptors were identical in the up-regulated state (0.046 and 0.044 h-1, respectively) or in the down-regulated state (0.10 and 0.096 h-1, respectively) suggests that the rate-limiting step in the receptor inactivation pathway occurs at the cell surface.     

Internalization and recycling of insulin receptors in hepatoma cells. Absence of regulation by receptor occupancy.

Insulin receptors of Fao hepatoma cells were labelled with a 125I-labelled photoreactive insulin analogue or by surface iodination catalysed by lactoperoxidase. Cells were then incubated at 37 degrees C, and the cellular localization of the labelled receptors was assessed by limited exposure of intact cells to trypsin. The results show that: (1) photolabelled insulin-receptor complexes are internalized and recycled in Fao hepatoma cells; (2) the dynamics of photolabelled insulin receptors (internalization and recycling) is similar before and after down-regulation; (3) the unoccupied receptors labelled by surface iodination are internalized and recycled similarly to covalent insulin-receptor complexes; (4) insulin does not induce internalization of surface-iodinated insulin receptors. We conclude that internalization and recycling of insulin receptors are independent of receptor occupancy by insulin in Fao hepatoma cells.     

Down-regulation of transforming growth factor-beta receptors: cooperativity between the types I, II, and III receptors and modulation at the cell surface.

The types I, II, and III receptors (RI, RII, RIII) for transforming growth factor-beta (TGF-beta) become down-regulated in response to ligand, presumably via their internalization from the cell surface. This report examines the down-regulation of full-length RI, RII, and RIII in cells endogenously or transiently expressing these receptors. Down-regulation occurred rapidly (within 2 h after TGF-beta1 treatment at 37 degrees C) and showed a dose response, between 10 and 200 pM TGF-beta1, in cells expressing RI, RII, and RIII (Mv1lu and A549 cells). A comparison between Mv1Lu and mutant cell derivatives R-1B (lacking RI) or DR-26 (lacking RII) indicated that all three receptors were necessary for efficient down-regulation. Down-regulation experiments, utilizing TGF-beta-treated 293 cells transiently expressing different combinations of these receptors indicated that neither RII or RIII were down-regulated when expressed alone and that RI was required for maximal down-regulation of RII. RII and RIII were partially down-regulated when these receptors were coexpressed in the absence of RI (in R-1B and 293 cells). Surprisingly, TGF-beta receptors were partially down-regulated in Mv1Lu, A549, and 293 cells treated with TGF-beta1 at 4 degrees C. Microscopic examination of 293 cells coexpressing RI fused to green fluorescent protein (RI-GFP) and RII indicated that, after treatment with TGF-beta1 at 4 degrees C, RI-GFP formed aggregates at the cell surface at this temperature. RI-GFP was not detected at the surface of these cells after TGF-beta1 treatment at 37 degrees C. Our results suggest a two phase mechanism for TGF-beta1 receptor down-regulation involving receptor modulation (aggregation) at the cell surface and internalization.     

Down-regulation of insulin receptors in the heart: studies on primary cultured adult cardiac myocytes

Primary cultured cardiac myocytes from adult rats have been used to study insulin receptor regulation. Culturing of cells in the presence of insulin induced a dose-dependent down-regulation of insulin binding with a maximal effect of 35% at an insulin concentration of 1.7 X 10(-7) mol/l. The number of high-affinity sites decreased from 110 000 to 70 000 sites per cell in control and down-regulated cells, respectively, with no change in the apparent affinity constant. Down-regulation was found to be rapid (t 1/2 = 3 h) and fully reversible. Culturing of cells in the presence of cycloheximide (0.1 mmol/l) or Tris (35 mmol/l) resulted in a further time-dependent increase in insulin-induced receptor loss with no effect on insulin binding to control cells. The action of both agents was found to be additive reaching a down-regulation of 51% after a culture period of 16 h. Recovery of insulin binding activity after removal of insulin remained unaffected in the presence of cycloheximide, whereas Tris inhibited this process by 74%. In conclusion our results show that the concept of insulin-induced receptor down regulation can be extended to the adult heart muscle. Moreover the data suggest involvement of protein synthesis and receptor recycling in this process.     

Down-regulation and recycling of insulin receptors. Effect of monensin on IM-9 lymphocytes and U-937 monocyte-like cells

Receptor down-regulation is the result of various cellular processes including receptor internalization, new synthesis, and recycling. Monensin, a monocarboxylic acid ionophore, has been used to characterize the role of recycling in the metabolism of insulin receptors on two cultured human cell lines, U-937 and IM-9, which have different rates of internalization. The U-937 monocyte-like cell internalizes insulin receptors readily. Incubation with monensin at low doses (10(-6) to 10(-7) M) for 2 h did not affect subsequent surface insulin binding. However, the drug markedly enhanced insulin-induced down-regulation. Monensin had little effect on ligand internalization in this cell line as demonstrated by quantitative morphometric analysis. The IM-9 lymphocyte, a slow internalizer, was less sensitive to monensin exposure. Prolonged exposure (12 h) to this compound of either cell line resulted in apparent inhibition of insertion into the surface membrane of both newly synthesized and recycled receptors. When solubilization was used to quantitate total cell receptors, there was essentially no difference in intact cell binding (i.e. surface receptors) and total cell binding in IM-9 cells when insulin-induced down regulation alone was compared to insulin and monensin. By contrast for the U-937 cells there was only a small further decrease in binding when monensin was added to insulin in the solubilized cells compared to the marked augmentation of down-regulation when monensin was added to insulin in intact cells. These data demonstrate that cells with a rapid internalization rate have an associated active recycling process. By contrast cells with a slow internalization rate have a similarly slow recycling rate. This is consistent with relatively equal rates of receptor biosynthesis and plasma membrane insertion in both cell types.     

The de novo phospholipid effect of insulin is associated with increases in diacylglycerol, but not inositol phosphates or cytosolic Ca2+.

We have previously reported that insulin increases the synthesis de novo of phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2) and diacylglycerol (DAG) in BC3H-1 myocytes and/or rat adipose tissue. Here we have further characterized these effects of insulin and examined whether there are concomitant changes in inositol phosphate generation and Ca2+ mobilization. We found that insulin provoked very rapid increases in PI content (20% within 15 s in myocytes) and, after a slight lag, PIP and PIP2 content in both BC3H-1 myocytes and rat fat pads (measured by increases in 32P or 3H content after prelabelling phospholipids to constant specific radioactivity by prior incubation with 32Pi or [3H]inositol). Insulin also increased 32Pi incorporation into these phospholipids when 32Pi was added either simultaneously with insulin or 1 h after insulin. Thus, the insulin-induced increase in phospholipid content appeared to be due to an increase in phospholipid synthesis, which was maintained for at least 2 h. Insulin increased DAG content in BC3H-1 myocytes and adipose tissue, but failed to increase the levels of inositol monophosphate (IP), inositol bisphosphate (IP2) or inositol trisphosphate (IP3). The failure to observe an increase in IP3 (a postulated 'second messenger' which mobilizes intracellular Ca2+) was paralleled by a failure to observe an insulin-induced increase in the cytosolic concentration of Ca2+ in BC3H-1 myocytes as measured by Quin 2 fluorescence. Like insulin, the phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA) increased the transport of 2-deoxyglucose and aminoisobutyric acid in BC3H-1 myocytes. These effects of insulin and TPA appeared to be independent of extracellular Ca2+. We conclude that the phospholipid synthesis de novo effect of insulin is provoked very rapidly, and is attended by increases in DAG but not IP3 or Ca2+ mobilization. The insulin-induced increase in DAG does not appear to be a consequence of phospholipase C acting upon the expanded PI + PIP + PIP2 pool, but may be derived directly from PA. Our findings suggest the possibility that DAG (through protein kinase C activation) may function as an important intracellular 'messenger' for controlling metabolic processes during insulin action.     

Down-regulation of Transforming Growth Factor-β Receptors: Cooperativity between the Types I, II, and III Receptors and Modulation at the Cell Surface

The types I, II, and III receptors (RI, RII, RIII) for transforming growth factor-β (TGF-β) become down-regulated in response to ligand, presumably via their internalization from the cell surface. This report examines the down-regulation of full-length RI, RII, and RIII in cells endogenously or transiently expressing these receptors. Down-regulation occurred rapidly (within 2 h after TGF-β1 treatment at 37°C) and showed a dose response, between 10 and 200 pM TGF-β1, in cells expressing RI, RII, and RIII (Mv1lu and A549 cells). A comparison between Mv1Lu and mutant cell derivatives R-1B (lacking RI) or DR-26 (lacking RII) indicated that all three receptors were necessary for efficient down-regulation. Down-regulation experiments, utilizing TGF-β-treated 293 cells transiently expressing different combinations of these receptors indicated that neither RII or RIII were down-regulated when expressed alone and that RI was required for maximal down-regulation of RII. RII and RIII were partially down-regulated when these receptors were coexpressed in the absence of RI (in R-1B and 293 cells). Surprisingly, TGF-β receptors were partially down-regulated in Mv1Lu, A549, and 293 cells treated with TGF-β1 at 4°C. Microscopic examination of 293 cells coexpressing RI fused to green fluorescent protein (RI–GFP) and RII indicated that, after treatment with TGF-β1 at 4°C, RI–GFP formed aggregates at the cell surface at this temperature. RI–GFP was not detected at the surface of these cells after TGF-β1 treatment at 37°C. Our results suggest a two phase mechanism for TGF-β1 receptor down-regulation involving receptor modulation (aggregation) at the cell surface and internalization.     

The role of tyrosine kinase activity in endocytosis, compartmentation, and down-regulation of the epidermal growth factor receptor.

Occupancy-induced down-regulation of cell surface epidermal growth factor (EGF) receptors attenuates signal transduction. To define mechanisms through which down-regulation of this class of growth factor receptors occurs, we have investigated the relative roles of ligand-induced internalization and recycling in this process. Occupied, kinase-active EGF receptors were internalized through a high affinity, saturable endocytic system at rates up to 10-fold faster than empty receptors. In contrast, full length EGF receptors lacking tyrosine kinase activity underwent internalization at a rate independent of occupancy. This "kinase-independent" internalization rate appeared to reflect constitutive receptor internalization since it was similar to the internalization rate of both receptors lacking a cytoplasmic domain and of antibodies bound to empty receptors. EGF internalized by either kinase-active or kinase-inactive receptors was efficiently recycled and was found within endosomes containing recycling transferrin receptors. However, targeting of internalized receptors to lysosomes did not require receptor kinase activity. All receptors that displayed ligand-induced internalization also underwent down-regulation, indicating that the proximal cause of down-regulation is occupancy-induced endocytosis. Tyrosine kinase activity greatly enhances this process by stabilizing receptor association with the endocytic apparatus.     

Calcium dependent aggregation of marine sponge cells is provoked by leukotriene B4 and inhibited by inhibitors of arachidonic acid oxidation

The mechanism of agonist-induced desensitization of the beta adrenergic receptor coupled adenylate cyclase has been studied in a smooth muscle cell line, BC3H-1, which expresses both alpha and beta adrenergic receptors and nicotinic receptors. beta receptors have been investigated in intact cells using as radioligand 3HCGP-12177, an hydrophilic compound which labels only surface receptors. The treatment of BC3H-1 cells with the agonist Isoproterenol, at 37 degrees but not at 4 degrees, induced a dose dependent internalization of the beta adrenergic receptor. Agonist-induced internalization was very rapid, in the order of few minutes. beta adrenergic receptor internalization was very specific: the alpha adrenergic agonist Phenylefrine had almost no effect on beta receptor levels, while Isoproterenol treatment had no effect on the number of alpha adrenergic or nicotinic receptors expressed at the cell surface of these cells. beta adrenergic receptor internalization is probably the major mechanism responsible for catecholamine desensitization in smooth muscle cells.     

Vanadate down-regulates cell surface insulin and growth hormone receptors and inhibits insulin receptor degradation in cultured human lymphocytes

Insulin is able to down-regulate its specific cell surface receptor in cultured human lymphocytes. The effect of vanadate, a known insulinomimetic agent, was examined to determine whether it could mimic insulin to down-regulate the insulin receptor. Exposure of cultured human lymphocytes (IM-9) to vanadate (0-200 microM) resulted in a time- and dose-dependent decrease in cell surface insulin receptors to 60% of control, while insulin (100 nM) down-regulated to 40%. The vanadate effect, in contrast to the rapid effect of insulin, was slow to develop (4-6 h). Surface receptor recovery after 18 h exposure was rapid after vanadate removal (20 min), but it required hours after insulin suggesting the presence of an intracellular (cryptic) pool of receptors after vanadate treatment. Insulin binding to Triton X-100-solubilized whole cells after 18 h treatment revealed that total cell receptors had decreased to 50% of control after insulin but increased to 120 and 189% of control after 100 and 200 microM vanadate, respectively. Furthermore, vanadate inhibited the insulin-mediated loss of total cell receptors from 50 to 28%. Removal of cell surface receptors by trypsin before cell solubilization revealed that 100 microM vanadate increased insulin binding to 321% of control indicating an accumulation of intracellular receptors. Labeling of cell surface proteins with Na125I and lactoperoxidase followed by immunoprecipitation of solubilized receptors with anti-receptor antibody after incubation for various times up to 20 h and quantitation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, while insulin shortened t1/2 from 7.3 to 5.3 h, vanadate prolonged receptor t1/2 to 14 h. No effect of vanadate was detected on insulin receptor tyrosine kinase activity with up to 4 h incubation at the vanadate concentrations used in this study. Furthermore, human growth hormone surface receptors were similarly down-regulated by vanadate. We conclude that 1) vanadate has an apparent insulin-like effect to down-regulate cell surface insulin receptors in cultured human lymphocytes; 2) in contrast to insulin-induced down-regulation which is associated with receptor degradation vanadate causes an accumulation of intracellular (cryptic) receptors and inhibits insulin receptor degradation; and 3) these effects of vanadate may be exerted on other cell surface receptors.     

Stoichiometric translocation of adipocyte insulin receptors from the cell-surface to the cell-interior. Studies using a novel method to rapidly remove detergent and concentrate soluble receptors

A rapid one-step method was developed for harvesting and concentrating insulin receptors from solubilized adipocytes, which entails precipitating soluble receptors with polyethylene glycol and resuspending the receptor-containing pellet in a reduced volume of binding buffer. With this procedure 90-100% of receptors were recovered, while 80% of cellular protein was removed, thus resulting in a marked reduction of both ligand and receptor proteases and about a 5-fold purification of the receptor. More importantly, greater than 98% of the Triton X-100 detergent was removed during this procedure so that the reduced receptor affinity observed in solubilized extracts (due to detergent) was restored to normal. Reconstituted receptors exhibited normal binding characteristics similar to those observed for plasma membrane receptors. The general utility of our receptor precipitation-reconstitution method is highlighted by studies on insulin-induced translocation of receptors from the cell-surface to the cell-interior of adipocytes and studies on the assessment of the binding affinity of nascent intracellular receptors. The results of these studies are consistent with the following. 1) Insulin initiates endocytotic uptake of insulin receptors, which then recycle back to the cell-surface. 2) Chloroquine impairs the recycling of internalized receptors while preventing receptor degradation, resulting in the progressive trapping and accumulation of receptors within cells during insulin treatment. 3) Receptor translocation during acute insulin-induced down-regulation is stoichiometric in that receptors lost from the cell-surface can be quantitatively recovered within the cell-interior. 4) In the absence of ligand, these receptors within adipocytes are mainly newly synthesized receptors enroute to the cell-surface, and they possess an affinity similar, if not identical, to mature receptors on the plasma membrane.     

Protein kinase C activation patterns are determined by methodological variations. Studies of insulin action in BC3H-1 myocytes and rat adipose tissue

In BC3H-1 myocytes, insulin has been reported to (a) increase diacyglycerol (DAG) production and provoke increases in protein kinase C enzyme activity of crude or DEAE-Sephacel-purified cytosol and membrane fractions in BC3H-1 myocytes (Cooper et al. (1987) J. Biol. Chem. 262, 3633-3739), but (b) decrease cytosolic, and transiently increase membrane, immunoreactive protein kinase C (Acevedo-Duncan et al. (1989) FEBS Lett. 244, 174-176). Presently, we used a Mono-Q column to purify protein kinase C and found that, similar to immunoblot findings, enzyme activity decreased in the cytosol, and increased in the membrane during insulin treatment. Similar differences in protein kinase C activation patterns were observed in rat adipose tissue: insulin stimulated cytosolic protein kinase C enzyme activity as measured after DEAE-Sephacel chromatography, but decreased cytosolic enzyme activity when measured after Mono-Q chromatography or by immunoblotting. We presently evaluated the possibility that insulin-induced increases in endogenous DAG may influence protein kinase C during assay in vitro. Crude cytosol from BC3H-1 myocytes contained 25-35% of total and [3H]glycerol-labelled DAG and insulin increased this DAG. Considerable amounts of [3H]glycerol-labelled DAG were present in insulin-stimulated protein kinase C-containing column fractions following DEAE-Sephacel chromatography of cytosol fractions, whereas lesser amounts were recovered after Mono-Q column chromatography. This difference in recovery of DAG and activation of the enzyme by this endogenous DAG may explain why we were able to discern insulin-induced (presumably translocation 'provoked') decreases in cytosolic protein kinase C in the present Mono-Q column preparations of both BC3H-1 myocytes and rat adipose tissue.     

Retention of specific protein kinase C isozymes following chronic phorbol ester treatment in BC3H-1 myocytes

Since insulin effects on glucose transport persist in phorbol ester "desensitized" or "down-regulated" BC3H-1 myocytes, we reexamined the evidence for protein kinase C (PKC) depletion. After 24 hrs of 5 microM 12-0-tetradecanoyl phorbol-13-acetate (TPA) treatment, PKC-directed histone phosphorylation and acute TPA effects on glucose transport were lost, but PKC-dependent vinculin phosphorylation was still evident. Hydroxylapatite (HAP) chromatography revealed loss of a type III, but not a type II, PKC-dependent vinculin phosphorylation. Immunoblots of cytosolic preparations of PKC-"depleted" myocytes confirmed the retention of PKC. Our findings indicate that TPA "down-regulated" BC3H-1 myocytes contain immunoreactive and functionally active PKC. The latter may explain the continued effectiveness of both insulin and diacylglycerol (DiC8) for stimulating glucose transport in "down-regulated" cells.     

Replacement of the human insulin receptor transmembrane and cytoplasmic domains by corresponding domains of the oncogene product v-ros leads to accelerated internalization, degradation, and down-regulation

Internalization, degradation, and insulin-induced down-regulation of insulin receptors were studied comparatively in transformed Chinese hamster ovary (CHO) cell lines, CHO.T and CHO.IR.ros, respectively expressing either the wild-type human insulin receptor (hIR) or a mutated hybrid receptor in which the transmembrane and cytoplasmic domains of hIR were replaced by corresponding domains of the transforming protein p68gag-ros (v-ros) of avian sarcoma virus UR2. At 37 degrees C, degradation of insulin receptors photoaffinity labeled on the cell surface (440 kDa) was most rapid for the hybrid hIR.ros (t1/2 1.0 +/- 0.1 h), intermediate for the wild-type hIR (t1/2 2.7 +/- 0.5 h), and slowest for the endogenous CHO insulin receptors (t1/2 3.7 +/- 0.7 h). Initial intracellular accumulation of the hIR.ros hybrid was also most rapid, reaching maximal amounts in 20 min following which the receptors disappeared rapidly from the intracellular compartment. In contrast, intracellular accumulation of the receptors in the CHO.T and CHO cells was slower, reaching maximal amounts in 60 min, and rapid disappearance of the receptors from the intracellular compartment did not occur. Chloroquine, a lysosomotropic agent, inhibited degradation of both the wild-type hIR and the chimeric hIR.ros and increased their intracellular accumulation. However, the chloroquine effect was much more marked for the hIR.ros receptors whose intracellular accumulation was increased by greater than 300% (in comparison with approximately 60% increase for the wild-type hIR), demonstrating marked intracellular degradation of the hybrid hIR.ros at chloroquine-sensitive sites. Insulin-induced down-regulation of the cell surface hIR.ros (52% loss in 3 h) was also more marked than the wild-type hIR (approximately 30% loss in 3 h). Thus, in the hybrid hIR.ros receptor, which was shown previously to exhibit insulin-stimulated autophosphorylation and kinase activity but not insulin-stimulated metabolic function, the capacity for internalization and down-regulation is not only preserved but is also markedly accelerated. These findings suggest that 1) the postreceptor coupling mechanisms mediating insulin-induced receptor internalization, degradation, and down-regulation are different from those mediating metabolic functions; and 2) v-ros may contain the structural information directing accelerated receptor catabolism.     

Insulin increases the intracellular concentration of total oxalyl thiolesters in BC3H-1 myocytes: potential anti-insulin mediators.

Oxalyl thiolesters (RS-CO-COOH) may represent negative intracellular messengers for insulin action. Using a reverse-phase, ion-pair high pressure liquid chromatographic technique, total intracellular oxalyl thiolesters were measured in insulin-sensitive BC3H-1 myocytes after the addition of insulin. The total oxalyl thiolester concentration increased to a maximum of 2.9 times the basal concentration by 30 min after the addition of 100 microU/ml insulin and decreased to 1.8 times by 180 min. Insulin's stimulation of pyruvate dehydrogenase as measured by lactate oxidation ([1-14C]-lactate --> 14CO2) in intact BC3H-1 myocytes reached a maximum at 15-30 min and returned to basal activity during the 60-90 min measurement interval. These results suggest that oxalyl thiolesters are increased in concentration following insulin-induced signal transduction to reverse insulin-stimulated metabolic events.     

Mutation of tyrosine residues 1162 and 1163 of the insulin receptor affects hormone and receptor internalization

Insulin internalization and degradation, insulin receptor internalization and recycling, as well as long term receptor down-regulation were comparatively studied in Chinese hamster ovary (CHO) cell lines, either parental or expressing the wild-type human insulin receptor (CHO.R) or a mutated receptor in which the tyrosine residues in positions 1162 and 1163 were replaced by phenylalanines (CHO.Y2). The two transfected cell lines presented very similar binding characteristics, and their pulse labeling with [35S]methionine revealed that the receptors were processed normally. As expected, the mutation of these twin tyrosines resulted in a defective insulin stimulation of both receptor kinase activity and glycogen synthesis. We now present evidence that compared to CHO.R cells, which efficiently internalized and degraded insulin, CHO.Y2 cells exhibited a marked defect in hormone internalization, leading to impaired insulin degradation. Moreover, the mutated receptors were found to be less effective than the wild-type receptors in transducing the hormone signal for receptor internalization, whereas the process of receptor recycling after internalization seemed not to be altered. In parental CHO cells, insulin induced long term receptor down-regulation, but was totally ineffective in both transfected cell lines. These results reveal that the tyrosines 1162 and 1163 in the kinase regulatory domain of the receptor beta-subunit play a pivotal role in insulin and receptor internalization.     

Tyrosine kinase-defective insulin receptors undergo insulin-induced microaggregation but do not concentrate in coated pits

Biologically active colloid-gold complexes were used to compare ligand-induced microaggregation, redistribution, and internalization of insulin receptors on Rat 1 fibroblasts expressing wild type (HIRc) or tyrosine kinase-defective (HIR A/K1018) human insulin receptors. Insulin-like growth factor I (IGF I) and alpha 2-macroglobulin receptors also were compared. On both cell types, all four unoccupied receptor types occurred predominantly as single receptors. Ligand binding caused receptor microaggregation. Microaggregation of wild type or kinase-defective insulin receptors or IGF I receptors was not different. alpha 2-Macroglobulin receptors formed larger microaggregates. Compared to wild type insulin or IGF I receptors, accumulation of kinase-defective insulin receptor microaggregates in endocytic structures was decreased, and the size of microaggregates in coated pits was significantly smaller. As a result, receptor-mediated internalization of gold-insulin by HIR A/K1018 cells was less than 6% of the cell-associated particles compared to approximately 60% of the particles in HIRc cells. On HIR A/K1018 cells, alpha 2-macroglobulin and IGF I were internalized via coated pits demonstrating that those structures were functional. These results suggest that: 1) ATP binding, receptor autophosphorylation, and activation of receptor kinase activity are not required for receptor microaggregation; 2) receptor microaggregation per se is not sufficient to cause ligand-induced receptor-mediated internalization or the biological effects of insulin; and 3) autophosphorylation of the beta-subunit or activation of the receptor kinase activity is required for the insulin-induced concentration of occupied receptors in coated pits.     

Coupling of agonist-induced AMPA receptor internalization with receptor recycling

Excitatory post-synaptic currents in the CNS are primarily mediated by alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors in response to glutamate. Internalization of cell-surface receptors has been shown to be one mechanism by which to control receptor function. To test for agonist control of AMPA receptor plasma membrane expression we used biochemical assays to study AMPA receptor internalization and insertion processes. In heterologous cells, we observed a slow constitutive internalization and a rapid agonist-induced internalization of AMPA receptors. To our surprise, however, agonist treatment had no effect on the steady-state levels of AMPA receptors on the cell surface. To examine whether this could be explained by an agonist-induced increase in the insertion rate of AMPA receptors into the plasma membrane we developed an assay to independently measure receptor insertion. Remarkably, agonist treatment of cells also dramatically increased AMPA receptor plasma membrane insertion rates. In addition, using an assay to measure recycling of internalized pools we found that internalized receptors are rapidly recycled to the cell surface. These results suggest that agonist-induced receptor internalization is coupled to increases in receptor recycling. This increase in receptor flux through intracellular pools may allow for rapid changes in receptor surface expression by independent regulatory control of internalization and insertion.     

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.