The release of lipoprotein lipase from 3T3-L1 adipocytes is regulated by microvessel endothelial cells in an insulin-dependent manner.

Lipoprotein lipase (LPL) is the rate-limiting enzyme in the hydrolysis of serum triglycerides associated with the lipoprotein particles very low density lipoprotein and chylomicrons. The cell biology of LPL is complex. It functions while tethered to the extracellular matrix of the capillary endothelium. LPL is synthesized, however, in the parenchymal cells (for example, adipocytes or muscle cells) subtending the endothelium. Thus, after synthesis in and release by the parenchymal cell, LPL must move to the endothelial cell and across this cell monolayer before expression at its physiologically relevant location. LPL expression on the endothelium is regulated by insulin. The intent of this study was to ascertain the role of microvessel endothelial cells in the release of LPL from 3T3-L1 adipocytes and to ascertain whether insulin regulates the function of the endothelial cells. Endothelial cells were treated with insulin, and the resultant culture medium conditioned by the endothelial cells was placed on 3T3-L1 adipocytes. The release of LPL from the adipocytes induced by the endothelial cell-conditioned medium was then quantitated. Insulin concentrations as low as 100 pM stimulated the release of a factor from the endothelial cells. This factor, when added to adipocytes, caused the quantitative release of LPL from the plasma membrane of the adipocytes. The effect of insulin on the endothelial cells was maximal within 15 min of insulin addition to the endothelial cells. Repeated challenges of the endothelial cells with insulin resulted in the repeated release of the LPL release factor from the endothelial cells if the challenges were separated by periods of 2-3 h. However, if the endothelial cells were chronically stimulated with insulin for 18 h, a subsequent acute stimulation with insulin did not generate any LPL release factor. Thus, microvessel endothelial cells regulate the mobilization of LPL from adipocytes in an insulin-dependent manner.

Multisite phosphorylation of ornithine decarboxylase in transformed macrophages results in increased intracellular enzyme stability and catalytic efficiency.

Ornithine decarboxylase (ODC) is the initial inducible enzyme in the polyamine biosynthetic pathway. In the transformed macrophage-derived RAW264 cell line, ODC was overproduced and existed in both unphosphorylated and phosphorylated forms. To date, the only protein kinase known to phosphorylate mammalian ODC is casein kinase II (CKII). ODC was phosphorylated in vitro by CKII and subjected to exhaustive sequential proteolysis with trypsin and V8 protease. Two-dimensional peptide mapping showed only a single phosphopeptide; two-dimensional phosphoamino acid analysis of the phosphopeptide revealed only 32P-labeled serine. ODC was metabolically radiolabeled with 32Pi in RAW264 cells and also subjected to proteolysis, two-dimensional peptide mapping, and phosphoamino acid analysis. Two phosphopeptides were generated from the metabolically radiolabeled ODC, including one that migrated similarly to the peptide phosphorylated by CKII in vitro. Each of the in situ radiolabeled ODC peptides contained both 32P-labeled serine and threonine residues. Thus, in RAW264 cells, ODC is phosphorylated on at least one serine residue in addition to that phosphorylated by CKII and on at least two threonine residues. Phosphorylated ODC had an increased stability to intracellular proteolysis compared with unphosphorylated ODC, their half-lives being 49.2 +/- 3.78 and 23.9 +/- 2.6 min (p = 0.001), respectively. The phosphorylated and unphosphorylated forms of ODC were independently purified to homogeneity. Kinetic analysis revealed that the catalytic efficiency of the phosphorylated form of ODC was 50% greater than that of the unphosphorylated form; the unphosphorylated ODC had a Vmax of 20.54 +/- 1.65 micromol/min/mg, whereas the phosphorylated form had a Vmax of 30.61 +/- 2.6 micromol/min/mg (p = 0.005). Phosphorylation of ODC by CKII has no effect on enzyme activity. Taken together, these findings demonstrate that regulation of ODC activity is governed by as yet unidentified protein kinases.

Insulin-induced in situ phosphorylation of the insulin receptor located in the plasma membrane versus endosomes.

The binding of insulin to the plasma membrane insulin receptor initiates two dynamic processes: (i) autophosphorylation of the receptor on tyrosine residues, activating the intrinsic tyrosine kinase activity required for insulin signaling, and (ii) endocytosis of the receptor. Recent evidence (Kublaoui et al., J. Biol. Chem. 270, 59-65, 1995) demonstrates that in stimulated adipocytes, the internalized insulin receptor is not only more highly phosphorylated than the receptor remaining on the plasma membrane of the cell, but that IRS-1 binding and phosphorylation also occur in the endosomes. These data suggest that the intracellular insulin receptor mediates insulin action. Utilizing 3T3-L1 adipocytes, we substantiate and extend these findings to document the distribution of receptor between the plasma membrane and the endosomal compartment of insulin-stimulated cells and map the extent and location of the tyrosine phosphorylation sites on the receptor residing in these two cellular locations. We find that following insulin stimulation (i) 90% of the receptor-associated phosphate is located in the endosomal compartment, (ii) the endosomal receptor is most highly phosphorylated in the tyrosine kinase domain, and (iii) significant levels of juxtamembrane domain phosphorylation are detected in the endosomal receptor. These data support the role of the endosomal insulin receptor as the major transducer of insulin action.

Insulin resistance is mediated by a proteolytic fragment of the insulin receptor.

Insulin resistance is a common clinical feature of obesity and non-insulin-dependent diabetes mellitus, and is characterized by elevated serum levels of glucose, insulin, and lipids. The mechanism by which insulin resistance is acquired is unknown. We have previously demonstrated that upon chronic treatment of fibroblasts with insulin, conditions that mimic the hyperinsulinemia associated with insulin resistance, the membrane-associated insulin receptor beta subunit is proteolytically cleaved, resulting in the generation of a cytosolic fragment of the beta subunit, beta', and that the generation of beta' is inhibited by the thiol protease inhibitor E64 (Knutson, V. P. (1991) J. Biol. Chem. 266, 15656-15662). In this report, we demonstrate that in 3T3-L1 adipocytes: 1) cytosolic beta' is generated by chronic insulin administration to the cells, and that E64 inhibits the production of beta'; 2) chronic administration of insulin to the adipocytes leads to an insulin-resistant state, as measured by lipogenesis and glycogen synthesis, and E64 totally prevents the generation of this insulin-induced cellular insulin resistance; 3) E64 has no effect on the insulin-induced down-regulation of insulin receptor substrate-1, and therefore insulin resistance is not mediated by the down-regulation of insulin receptor substrate-1; 4) under in vitro conditions, partially purified beta' stoichiometrically inhibits the insulin-induced autophosphorylation of the insulin receptor beta subunit; and 5) administration of E64 to obese Zucker fatty rats improves the insulin resistance of the rats compared to saline-treated animals. These data indicate that beta' is a mediator of insulin resistance, and the mechanism of action of beta' is the inhibition of the insulin-induced autophosphorylation of the beta subunit of the insulin receptor.

High-resolution slab gel isoelectric focusing: methods for quantitative electrophoretic transfer and immunodetection of proteins as applied to the study of the multiple isoelectric forms of ornithine decarboxylase.

A high-resolution isoelectric focusing vertical slab gel method which can resolve proteins which differ by a single charge was developed and this method was applied to the study of the multiple isoelectric forms of ornithine decarboxylase. Separation of proteins at this high level of resolution was achieved by increasing the ampholyte concentration in the gels to 6%. Various lots of ampholytes, from the same or different commercial sources, differed significantly in their protein binding capacity. Ampholytes bound to proteins interfered both with the electrophoretic transfer of proteins from the gel to immunoblotting membranes and with the ability of antibodies to interact with proteins on the immunoblotting membranes. Increasing the amount of protein loaded into a gel lane also decreased the efficiency of the electrophoretic transfer and immunodetection. To overcome these problems, both gel washing and gel electrophoretic transfer protocols for disrupting the ampholyte-protein binding and enabling a quantitative electrophoretic transfer of proteins were developed. Two gel washing procedures, with either thiocyanate or borate buffers, and a two-step electrophoretic transfer method are described. The choice of which method to use to optimally disrupt the ampholyte-protein binding was found to vary with each lot of ampholytes employed.

Insulin receptor characterization and function in bovine aorta endothelial cells: insulin degradation by a plasma membrane, protease-resistant insulin receptor.

The functional significance of the insulin receptor on bovine aorta endothelial (BAE) cells is not well defined. The insulin receptor expressed on BAE cells does not mediate insulin hormonal effects and does not mediate the transcytosis of insulin from the apical to the basolateral domain of the cell monolayer. To assess the role of the insulin receptor on BAE cells, the physical characteristics of the BAE cell receptor were investigated, and the time-dependent interaction of insulin and insulin degradation products with BAE cell monolayers was quantitated. The BAE cell insulin receptor was found to be highly resistant to the proteolytic action of trypsin, pronase, and proteinase K at either 4 degrees C or 37 degrees C. This resistance may permit the receptor to maintain insulin binding capabilities in spite of the high concentrations of proteases which are normally present in blood. Scatchard analysis of cell-surface and total cellular insulin receptor demonstrated dissociation constants similar to values obtained with other cells and tissues. However, whereas other cells and tissues contain an intracellular pool of receptor that ranges from 20-40% of the total cellular receptor content, no intracellular population of insulin receptors was detected in BAE cells. Upon incubation of intact BAE cell monolayers with insulin, no endocytosis of cell-surface insulin receptor could be demonstrated. However, insulin degradation by the BAE cells was readily quantitated, at a rate of 16.3 fmol/10(6) cells/h at an insulin concentration of 2 nM. This rate of degradation was not inhibited by chloroquine, which inhibits insulin degradation in fibroblasts, hepatocytes, and adipocytes, nor by phenylarsine oxide, which inhibits endocytosis. Bacitracin inhibited insulin binding to the cell monolayers and inhibited insulin degradation with identical IC50 values (80 microM). These data suggest that in BAE cells, insulin degradation occurs in the absence of receptor-mediated endocytosis and is mediated by binding of insulin to its receptor. Therefore, it is concluded that the functional role of the insulin receptor expressed in BAE cells is to bind blood-borne insulin at the plasma membrane of the cell and thereby facilitate the degradation of insulin at the BAE cell plasma membrane.

Ligand-independent internalization and recycling of the insulin receptor. Effects of chronic treatment of 3T3-C2 fibroblasts with insulin and dexamethasone.

Upon binding insulin at the plasma membrane, the insulin receptor internalizes into the endosomal compartment of the cell with a half-time of approximately 10 min. Our earlier work demonstrated that receptor inactivation (loss of insulin binding capacity) is a regulated process. Long term treatment of cultured cells with insulin or the glucocorticoid dexamethasone increases or decreases, respectively, the rate constant for insulin receptor inactivation (Knutson, V. P., Ronnett, G. V., and Lane, M. D. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2822-2826). In these studies, monolayer cultures of 3T3-C2 fibroblasts were chronically treated with insulin or dexamethasone. Subsequently, the surface receptors were labeled with the photoactivatable cross-linking agent 125I-labeled 2-(p-azidosalicylamido)ethyl-1,3'- dithiopropionate -insulin. Following equilibration of the radiolabeled receptor between the plasma membrane and internal pools, the steady-state rate constant for receptor recycling was determined by quantitating the rate at which internal radiolabeled receptor was inserted into the plasma membrane. The steady-state rate constant for this recycling process was the same in control, insulin-treated, or steroid-treated cells (t1/2 = 2h). In contrast, the rate constant for receptor internalization was regulated; the half-times were 10 h for control cells, 5 h for insulin-treated cells, and 19 h for dexamethasone-treated cells. These changes in rate constants for internalization and inactivation lead to changes in the relative numbers of receptor molecules undergoing recycling versus inactivation. Therefore, whereas the recycling of the insulin receptor is not a regulated process, the internalization of surface receptor in the absence of bound ligand is a metabolically controlled step in receptor processing.

Proteolytic processing of the insulin receptor beta subunit is associated with insulin-induced receptor down-regulation.

This report describes the use of an antibody directed against the carboxyl terminus of the insulin receptor beta subunit to assess the fate of the insulin receptor protein over the time course of insulin-induced receptor down-regulation. The insulin receptor beta subunit is lost from the cellular membranes of insulin-treated 3T3-C2 fibroblasts with a time course superimposable with the insulin-induced loss of cellular insulin binding activity. Concomitant with the time-dependent loss of the intact beta subunit from the membranes, a 61,000-Da fragment of the insulin receptor beta subunit accumulates in the cytosol of the cells in a time-dependent manner. The insulin-induced loss of the intact beta subunit from the cellular membranes is inhibited by cycloheximide. Chloroquine and the thiol protease inhibitors leupeptin and E-64 inhibit the insulin-induced loss of the intact beta subunit from the membranes and induce an accumulation of the intact subunit in the membranes. However, in the presence of leupeptin, E-64, or chloroquine, the insulin-induced loss of insulin binding activity occurs normally. These data indicate that down-regulation results in the loss of the intact beta subunit from the cellular membranes with the production of a fragment of the beta subunit in the cytosol. The protease responsible for the generation of the fragment is a thiol protease which requires acidic conditions. Since the insulin-induced proteolysis of the beta subunit can be totally inhibited under conditions where the insulin-induced loss of insulin binding activity proceeds normally, the proteolysis of the beta subunit is a process which is separate and distinguishable from the insulin-induced loss of insulin binding activity.

Cellular trafficking and processing of the insulin receptor.

The insulin receptor is a dynamic cellular macromolecule that moves through various compartments of the cell throughout its lifetime. This review addresses the processes involved in the synthetic assembly of the insulin receptor; the interaction of insulin with the receptor protein; the receptor-mediated endocytosis of insulin; and the role of receptor tyrosine and serine phosphorylation in both endocytosis and recycling. This discussion is concluded by examining the data available on the intracellular inactivation and degradation of the receptor protein. Emphasis is given to the cellular regulation imposed at each of these steps in receptor processing, and how the use of pharmacologic and physiologic perturbants has afforded experimental insights into the mechanisms the cell utilizes in modulating the expression and functioning of the insulin receptor.

Comparison of insulin receptor tyrosine phosphorylation under in vitro and in situ conditions: assessment of specific protein tyrosine phosphorylation without the use of 32P-phosphate-labeled substrates.

An assay which permitted a comparison of insulin receptor tyrosine phosphorylation induced under in vivo and in situ conditions was developed. It was demonstrated that the level of tyrosine phosphorylation of the receptor induced by insulin under in situ conditions exceeded by 2.3-fold the level of tyrosine phosphorylation induced under in vitro conditions. In addition, chronically treated, down-regulated cells demonstrated a significant decrease in the level of insulin receptor tyrosine phosphorylation compared to cells acutely treated with insulin. The procedure utilized an antibody specific for the insulin receptor which allowed separation of the receptor from other cellular proteins which are potential substrates for tyrosine phosphorylation. A second iodinated antibody specifically directed against phosphotyrosine residues allowed quantitation of the phosphorylated residues. Since the use of 32P-labeled substrates for phosphorylation was not required, this procedure allowed a comparison of protein phosphorylation induced under in vitro and in situ conditions. This procedure should permit investigations into the roles of other cellular factors, such as phosphatases and serine kinases, in modulating protein tyrosine phosphorylation.

Purification of a murine monoclonal antibody of the IgM class.

A simple method is described for the purification of murine monoclonal antibodies of the IgM class. Ascites fluid was subjected to ammonium sulfate precipitation. The precipitate was redissolved and dialyzed and subsequently subjected to gel filtration chromatography on Ultrogel AcA22 and finally ion exchange chromatography on DEAE-Sepharose. The purity of the antibody was assessed by reducing SDS gel electrophoresis, and estimated to be greater than 85% pure after gel filtration, and greater than 95% pure after the ion exchange chromatography. The immuno-activity of the antibody was assessed throughout the purification scheme by the ability of the antibody to bind to immunogen immobilized to nitrocellulose. Approximately 50% antibody activity was recovered from this purification scheme after gel filtration, but only 10% of the initial activity could be recovered after ion exchange chromatography. This result with ion exchange chromatography underscores the lability of IgM antibodies to immobilization. Therefore, if the recovery of immunoreactive IgM antibody is a goal, purification schemes of IgM antibodies should avoid ion exchange or affinity chromatography.

Comparison of the function of the tight junctions of endothelial cells and epithelial cells in regulating the movement of electrolytes and macromolecules across the cell monolayer.

In cell culture, both endothelial and epithelial cell monolayers have been found to generate structurally similar tight junctional complexes, as assessed by thin complexes of the two cell types are, at least in part, responsible for the very different permeability characteristics of native endothelial and epithelial cell monolayers. The purpose of this work was to compare cultured endothelial and epithelial cells with respect to the function of their tight junctional complexes in regulating the movement of macromolecules and ions across the cell monolayers, and define functional parameters to characterize the tight junctional complexes. Bovine aorta endothelial cells and T84 colonic carcinoma epithelial cells were cultured on a microporous membrane support. The permeability coefficients of inulin, albumin, and insulin were determined with the cell monolayers and compared with the permeability coefficients obtained with 3T3-C2 fibroblasts, a cell line that does not generate tight junctions. Electrical resistance measurements across the monolayer-filter systems were also compared. The permeability coefficient of albumin across the endothelial cell monolayer compared favorably with other reported values. Likewise, the electrical resistance across the T84 cell monolayer was in good agreement with published values. Utilizing permeability coefficients for macromolecules as an index of tight junction function, we found that a distinction between a lack of tight junctions (fibroblasts), the presence of endothelial tight junctions, and the presence of epithelial tight junctions was readily made. However, when utilizing electrical resistance as an index of tight junction function, identical measurements were obtained with fibroblasts and endothelial cells. This indicates that more than one index of tight junction function is necessary to characterize the junctional complexes. Although structurally similar, epithelial cell and endothelial cell tight junctions perform very different functions, and, from our data, we conclude that the demonstration of tight junctional structures by electron microscopy is not relevant to the functional nature of the junction: structure does not imply function. A minimal assessment of tight junction function should rely on both the determination of the electrical resistance across the cell monolayer, and the determination of the permeability coefficients of selected macromolecules.

Insulin-binding peptide. Design and characterization.

The design and characterization of a six-amino acid-containing peptide that binds insulin is described. The amino acid sequence of the insulin-binding peptide (IBP) was determined from the strand of DNA complementary to the strand of DNA coding for the insulin molecule in the domain of the insulin monomer believed to interact with the insulin receptor. The IBP (Cys-Val-Glu-Glu-Ala-Ser) binds specifically to insulin in a saturable manner with a Kd of 3 nM. This binding process is time dependent and slightly temperature dependent, and the peptide appears to interact with insulin near the carboxyl terminus of the B-chain of insulin. Incubation of insulin with the peptide decreases insulin binding to the insulin receptor by 50%, with no effect on the affinity of insulin for the receptor and no effect on cellular insulin-stimulated deoxyglucose uptake. A polyclonal antibody produced against the IBP will inhibit specific insulin binding to intact cells by approximately 50%, with no effects on insulin-stimulated glucose uptake. From this data, we suggest that there are at least two domains of the insulin molecule through which it interacts with its receptor, the "binding region" of insulin, which is the domain blocked by the IBP, and the "message region" of insulin, through which insulin not only binds to the receptor, but also generates the cellular signal.

The covalent tagging of the cell surface insulin receptor in intact cells with the generation of an insulin-free, functional receptor. A new approach to the study of receptor dynamics.

A new method is described in which the cell surface insulin receptor can be radioactively tagged in a specific manner with a small insulin-free probe. After protecting the amino groups of insulin essential for binding and bio-activity, insulin is coupled to the heterobifunctional, cleavable cross-linking reagent SASD (sulfosuccinimidyl 2-(p-azidosalicylamido)-1,3'-dithiopropionate), via displacement of the N-hydroxysuccinimide moiety of SASD. Removal of the protecting groups results in the formation of 2-(p-azidosalicylamido)-1,3'-dithiopropionate (ASD)-insulin with insulin receptor binding activity equivalent to unmodified insulin. Iodination of ASD-insulin results in the incorporation of 125I into both the azidohydroxybenzoyl moiety of SASD and a tyrosine residue of insulin. Following binding of 125I-ASD-insulin to intact monolayers of 3T3-C2 cells, radiolabel is incorporated exclusively into a 135-kDa protein in a manner dependent upon the length of exposure of the cells to short wavelength ultraviolet light. This protein corresponds in molecular weight to the alpha subunit of the insulin receptor. Labeling of this protein can be inhibited by excess unlabeled insulin. Reduction of the disulfide bond of ASD with 10 mM glutathione causes the release of the 125I-insulin portion of the reagent from the receptor complex, with the iodinated photoactivated end of ASD covalently attached to the receptor. Insulin receptor labeled in this manner retains its ability to bind insulin. General metabolic processes of the intact cells do not appear to be perturbed by this labeling procedure, and the cellular processing of the insulin receptor does not appear to be modified by the covalent labeling of the receptor protein. This procedure therefore provides a way to specifically label the cell surface insulin receptor in a manner which does not perturb the normal functioning of the labeled cell and equally importantly, does not perturb the normal cellular processing of the insulin receptor itself.

The acute and chronic effects of glucocorticoids on insulin receptor and insulin responsiveness. Transient fluctuations in intracellular receptor level parallel transient fluctuations in responsiveness.

The treatment of confluent embryonic Swiss mouse fibroblasts (3T3-C2 cells) with glucocorticoids has been shown to result in a time- and dose-dependent increase in the number of cellular insulin receptors (Knutson, V. P., Ronnett, G. V., and Lane, M. D. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2822-2826). Cellular events relating to the insulin receptor and insulin responsiveness which occur over the time course of the transition to the "up-regulated" steady state are described. Over the 48-h transition from the basal to the up-regulated steady state, a transient increase in the level of intracellular receptor was detected with a 3-5-fold increase in intracellular receptor found 12 h after steroid administration. Through the use of the heavy isotope density shift, this increase in the intracellular receptor population was preceded by a decrease in the rate of receptor inactivation, with no change in receptor synthesis. Insulin-induced receptor down-regulation was abolished after 12 h of dexamethasone treatment, when the intracellular receptor level was elevated. Nevertheless, the cells maintained the ability to internalize receptor in response to insulin binding. The hormonal responsiveness of the cells over the glucocorticoid-induced transition was assessed by the ability of insulin to stimulate the cellular uptake of 2-deoxyglucose and aminoisobutyric acid. Glucose transport was transiently increased at a time when the intracellular population of receptor was transiently elevated. Glucocorticoid treatment ultimately led to a loss of insulin-sensitive glucose transport. Insulin-stimulated amino acid transport was transiently abolished when the intracellular population of receptor was high. With chronic steroid treatment, the sensitivity of the amino acid transporter was increased above control levels. These data would indicate that glucocorticoids have no short- or long-term effect on insulin receptor synthesis; the insulin-induced internalization of insulin receptor alone is not sufficient to induce a cellular response to insulin; and the cellular events leading to the transient accumulation of intracellular receptor are coupled to the cellular responsiveness of the cells to insulin.

The effects of cycloheximide and chloroquine on insulin receptor metabolism. Differential effects on receptor recycling and inactivation and insulin degradation.

The effects of protein synthesis inhibitors and the lysosomotropic agent chloroquine on the metabolism of the insulin receptor were examined. Through the use of the heavy-isotope density shift technique, cycloheximide was found to inhibit both the synthesis of new insulin receptor and the inactivation of old cellular insulin receptor. Upon investigation of the locus of this effect of protein synthesis inhibition, it was found that cycloheximide did not inhibit 1) the translocation of receptor from the cell surface to an intracellular site, 2) the recycling of receptor from the internal site back to the plasma membrane, nor 3) the degradation of insulin. Cycloheximide did, however, rapidly and completely inhibit the inactivation of the insulin receptor. In the presence of extracellular insulin, this effect of cycloheximide resulted in the long-term (6 h) accumulation of receptor in a trypsin-resistant intracellular compartment. Puromycin and pactamycin, protein synthesis inhibitors with mechanisms of action which differ from cycloheximide, produced the same effects on insulin receptor metabolism as cycloheximide, indicating that this effect on receptor metabolism is due to the inhibition of protein synthesis and not a secondary effect of cycloheximide. Actinomycin D also inhibited the inactivation of receptor. Chloroquine inhibited the receptor-mediated degradation of insulin, but had no effect on either the internalization or inactivation of the insulin receptor. The insulin-induced recycling of the internalized receptor was inhibited by chloroquine, possibly through the inhibition of the discharge of insulin from the insulin-receptor complex. From these observations, we suggest that 1) a protein factor is required to inactivate the insulin receptor, 2) this protein and the messenger RNA coding for the protein have short cellular half-lives, and 3) insulin degradation and insulin receptor inactivation are distinct, separable processes which not only occur at different rates, but possibly occur in distinct subcellular locations.

Role of glycosylation in the processing of newly translated insulin proreceptor in 3T3-L1 adipocytes.

A procedure was developed for the immunoprecipitation of glycosylated and nonglycosylated forms of the insulin receptor and its precursors without prior purification using lectins. 3T3-L1 adipocytes were labeled with [35S]methionine after which 35S-labeled receptor polypeptides were specifically immunoprecipitated and characterized by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The first 35S-polypeptide detected was a 190-kDa glycosylated proreceptor which was rapidly (t1/2 approximately equal to 15 min) processed to a 210-kDa intermediate. The latter precursor was more slowly (t1/2 approximately equal to 2 h) proteolytically processed to 125-kDa (alpha') and 83-kDa (beta') precursors of the mature alpha- and beta-receptor subunits. Immediately prior to insertion into the plasma membrane, i.e. about 3 h after translation, the alpha'- and beta'-precursor polypeptides were converted to the mature 135-kDa alpha- and 95-kDa beta-receptor subunits. The characteristics of the oligosaccharide moieties of the receptor precursors and products were investigated. The 210-kDa precursor and its two products, the 125-kDa alpha'- and 83-kDa beta'-species, and the mature alpha- and beta-receptor subunits bind tightly to wheat germ lectin, whereas the 190-kDa proreceptor species is not bound. Upon incubation with endoglycosidase H, both the 210- and 190-kDa species are converted to a 180-kDa species. The 125-kDa alpha'- and 83-kDa beta'-species are also cleaved by endoglycosidase H, being reduced in size to 97 and 79 kDa, respectively. Based on their sensitivity to endoglycosidase H and insensitivity to neuraminidase, the oligosaccharide chains of the receptor precursors (190, 210, 125, and 83 kDa) do not contain terminal sialic acid (or other capping sugars). However, near the time of insertion into the plasma membrane, capping of the alpha'- and beta'-species by sialic acid occurs, giving rise to the mature 135-kDa alpha- and 95-kDa beta-receptor subunits, which are partially endoglycosidase H-resistant and neuraminidase-sensitive. When 3T3-L1 adipocytes are treated with tunicamycin, a 180-kDa proreceptor aglycopolypeptide is synthesized which is incapable of undergoing further processing and proteolytic cleavage to the alpha- and beta (or alpha'- and beta'-)-subunits. The 180-kDa species, which appears to be the aglyco-form of hte 190-kDa proreceptor generated by endoglycosidase H, is resistant to trypsin in the intact cell and apparently has not reached the cell surface. Thus, the oligosaccharide moieties of the insulin receptor precursor are crucial for proper processing, intracellular translocation, and formation of functionally competent insulin re

Rapid, reversible internalization of cell surface insulin receptors. Correlation with insulin-induced down-regulation.

Chronic treatment of 3T3-C2 fibroblasts with insulin causes the slow (t1/2 = 3-4 h) down-regulation of cellular insulin receptor to a new steady state level by accelerating receptor decay (Knutson, V.P., Ronnett, G.V., and Lane, M.D. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2822-2826). In the present investigation, the synthesis and turnover of the receptor during the transition to the down-regulated state was examined by the heavy isotope density-shift method. It was observed that within two h after insulin addition, receptor decay increased abruptly for several hours then gradually declined until the "down-regulated" rate was achieved. The abrupt increase in receptor decay induced by insulin was preceded by a more rapid (t1/2 less than or equal to 10 min) translocation of cell surface receptor to an "intracellular" trypsin-resistant compartment. Thus, upon exposure to ligand, insulin receptor rapidly redistributes from the cell surface to an intracellular compartment, without an initial net loss of cellular receptors. The translocation process was rapidly reversed (t1/2 less than or equal to 20 min) upon removal of insulin. With prolonged exposure to insulin, the initial rapid translocation of receptor was followed by a slower inactivation of receptor apparently in the intracellular compartment. Cycloheximide, which lengthens receptor half-life by blocking a step in receptor inactivation, had no effect on receptor internalization. Internalization of insulin receptor and its bound ligand were, however, rapidly (less than 10 min) blocked by phenylarsine oxide. These results support the following sequence of events. Upon exposure to ligand, insulin receptors are translocated from the cell surface to an intracellular site which results in accelerated receptor decay and ultimately to a lower steady state cellular receptor level.

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.