Stimulation of receptor-mediated low density lipoprotein endocytosis in neuraminidase-treated cultured bovine aortic endothelial cells

Sialic acids, occupying a terminal position in cell surface glycoconjugates, are major contributors to the net negative charge of the vascular endothelial cell surface. As integral membrane glycoproteins, LDL receptors also bear terminal sialic acid residues. Pretreatment of near-confluent, cultured bovine aortic endothelial cells (BAEC) with neuraminidase (50 mU/ml, 30 min, 37 degrees C) stimulated a significant increase in receptor-mediated 125I-LDL internalization and degradation relative to PBS-treated control cells. Binding studies at 4 degrees C revealed an increased affinity of LDL receptor sites on neuraminidase-treated cells compared to control BAEC (6.9 vs. 16.2 nM/10(6) BAEC) without a change in receptor site number. This enhanced LDL endocytosis in neuraminidase-treated cells was dependent upon the enzymatic activity of the neuraminidase and the removal of sialic acid from the cell surface. Furthermore, enhanced endocytosis due to enzymatic alteration of the 125I-LDL molecules was excluded. In contrast to BAEC, neuraminidase pretreatment of LDL receptor-upregulated cultured normal human fibroblasts resulted in an inhibition of 125I-LDL binding, internalization, and degradation. Specifically, a significant inhibition in 125I-LDL internalization was observed at 1 hr after neuraminidase treatment, which was associated with a decrease in the number of cell surface LDL receptor sites. Like BAEC, neuraminidase pretreatment of human umbilical vein endothelial cells resulted in enhanced receptor-mediated 125I-LDL endocytosis. These results indicate that sialic acid associated with either adjacent endothelial cell surface molecules or the endothelial LDL receptor itself may modulate LDL receptor-mediated endocytosis and suggest that this regulatory mechanism may be of particular importance to endothelial cells.     

Insulin receptor recycling in vascular endothelial cells. Regulation by insulin and phorbol ester

Endothelial cell insulin receptors mediate the transcytosis of insulin from luminal to abluminal cell surface. We have investigated the kinetics of insulin receptor translocation by immunoprecipitation of radiolabeled receptors at various times before and after trypsin treatment of intact endothelial cells. Insulin receptors were constitutively internalized with t1/2 = 18 +/- 2 min and were recycled to the cell surface. Insulin stimulated receptor internalization and externalization rates 2.6- and 2.4-fold, respectively. Changes in cell-surface binding of 125I-insulin were consistent with the receptor translocation rates observed in surface-labeling experiments. Phorbol myristate acetate (PMA) treatment increased the rate of insulin-stimulated receptor externalization 1.7-fold. PMA treatment increased the constitutive externalization rate 3.5-fold without affecting the constitutive internalization rate, suggesting that recycling might occur via a mobilization of receptors from intracellular sites in a manner independent of internalization rate. Analysis of the intracellular distribution of receptors by 125I-insulin binding and immunogold electron microscopy revealed that less than one-third of the total insulin receptor pool resided on the cell surface. In summary, endothelial cell insulin receptors are constitutively recycled, and internalization and externalization rates are increased by receptor occupancy and PMA treatment.     

The two isotypes of the human insulin receptor (HIR-A and HIR-B) follow different internalization kinetics.

Human insulin receptor (HIR) is expressed in two isoforms which differ in the C-terminal end of the alpha-subunit (HIR-A = -12 aa, HIR-B = +12 aa). We studied internalization kinetics of HIR-A and HIR-B in Rat1 fibroblasts. Internalized receptors were quantified by 125I-insulin binding after cell trypsinisation and solubilization, surface receptors were determined by 125I-insulin binding to intact cells and by chemical crosslinking with B26-125I-insulin. HIR-A and HIR-B show different kinetics of receptor internalization. While in HIR-A cells the maximum of internalization (approx. 65% of total) is reached after 10 min followed by a high recycling rate (approx. 80% of internalized receptors after 20 min), the internalization in HIR-B cells reaches a maximum (approx. 60% of total) after 15 min without detectable recycling within 30 min. The data show that the different alpha-subunits of both receptor types determine different velocities of internalization and determine whether a fast recycling occurs.     

Calcium ions are required for the intracellular routing of insulin and its receptor.

We have studied the role of the cytosolic-free calcium concentration ([Ca2+]i) on the early and later internalization steps of insulin and its receptor. As before, we find that the rate of 125I-insulin internalization in HL60 cells remains normal when [Ca2+]i is lowered 10 times below normal resting level by the use of an intracellular Ca2+ chelator. By contrast, the subsequent intracellular steps, i.e. insulin receptor recycling and insulin degradation, are inhibited in calcium-depleted cells. Under low [Ca2+]i conditions, the association of 125I-insulin with late endosomes and lysosomes is also reduced. This suggests that calcium ions are required for fusion processes occurring at the endosomal or postendosomal stage of internalization. Thus, by regulating insulin receptor recycling and by controlling insulin degradation, Ca2+ ions play a key role in the regulation of insulin action.     

Structural and Functional Characteristics of Insulin Receptors in Rat Neuroblastoma Cells

Insulin receptors were detected in a variety of rat neuroblastoma and glioma cell lines. The binding of 125I-insulin to B103 neuroblastoma cells had characteristics typical of insulin receptors in other tissues, including high affinity for insulin, low affinity for insulin-like growth factor I (IGF-I), and curvilinear Scatchard plots. Using photoaffinity labeling procedures and sodium dodecyl sulfate (SDS) gel electrophoresis to analyze the subunit structure of insulin receptors in B103 cells, the predominantly labeled protein had an apparent molecular weight of 125K and the mobility of this protein was shifted after removal of sialic acid residues. On the basis of size and susceptibility to neuraminidase, the insulin binding subunit in neuroblastoma cells was identical to the alpha-subunit of insulin receptors in adipocytes and different from the 115K subunit found in brain. The presence of an "adipocyte" form of the insulin receptor in clonal cells derived from brain is probably a consequence of transformation and results from more extensive oligosaccharide processing of the 115K receptor expressed in normal brain cells. The fully glycosylated receptors in neuroblastoma cells were capable of exerting functions typical of insulin receptors in adipocytes such as internalization of insulin and stimulation of glucose transport.     

Structural differences between liver- and muscle-derived insulin receptors in rats

The structure of insulin receptors, solubilized from rat skeletal muscle and liver, was studied. The alpha subunit was identified by specific cross-linking to A14 125I-insulin with disuccinimidyl suberate. Muscle- and liver-derived alpha subunits migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a Mr of 131,000 and 135,000, respectively. There was no significant difference in insulin binding affinity. Treatment of cross-linked, immunoprecipitated receptors with either neuraminidase or endoglycosidase H decreased the Mr of muscle- and liver-derived alpha subunits but did not affect the difference in Mr. Autophosphorylated beta subunits migrated with a Mr of 98,000 for muscle and 101,000 for liver. After partial V8 digestion of autophosphorylated, immunoprecipitated receptors the major phosphopeptide fragment migrated on SDS-PAGE at Mr 57,000 from muscle and 60,000 from liver. Glycosidase digestion of autophosphorylated receptors suggested that Mr heterogeneity was due in part to differences in the sialic acid content of beta subunits. Muscle and liver are the major target organs of insulin; the apparent heterogeneity of insulin receptor structure may be relevant to tissue-specific differences in insulin action.     

Kinetics of insulin receptor internalization and recycling in adipocytes. Shunting of receptors to a degradative pathway by inhibitors of recycling

The kinetics of receptor internalization and recycling was directly determined in adipocytes by measuring 125I-insulin binding to total, intracellular, and cell-surface insulin receptors. In the absence of insulin 90% of all receptors were on the cell-surface and 10% were intracellular. Insulin (100 ng/ml) rapidly altered this distribution by translocating surface receptors to the cell-interior through a temperature and energy dependent process. Surface-derived receptors were seen within cells as early as 30 s and accumulated intracellularly at the rate of approximately 20,000/min (t 1/2 = 2.7 min). After 6 min the size of the intracellular receptor pool plateaued (for up to 2 h), with 30% of surface receptors residing within the cell. This plateau was due to the attainment of an equilibrium between receptor uptake and recycling, since removal of insulin (to stop receptor uptake) was followed by both a rapid depletion of intracellular receptors and a a concomitant and stoichiometric reappearance of receptors on the cell-surface. Receptors were efficiently recycled, with little or no net loss observed even after 4 h of insulin treatment; however, recycling could be partially inhibited (approximately 10%) by several agents (e.g. chloroquine and Tris). Tris treatment of adipocytes in the presence of insulin led to 50% loss of surface and total receptors at 2 and 4 h, respectively. Since chloroquine prevented the decrease in total receptors, but not the loss of surface receptors, it appears that Tris impairs recycling by diverting a portion of incoming receptors to a chloroquine-inhibitable degradative site. From these results we conclude that: 1) insulin triggers endocytotic uptake of insulin-receptor complexes; 2) internalized receptors are then rapidly reinserted into the plasma membrane, and the receptors can traverse this recycling pathway within 6 min; 3) prolonged recycling does not normally result in measurable receptor loss, but when receptors are prevented from recycling, they become trapped intracellularly and are shunted to a chloroquine-sensitive degradative pathway; and 4) chloroquine and Tris are only partially effective inhibitors of receptor recycling.     

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.     

Effects of vesicle-adipocyte interaction on regulation of insulin receptor recycling

The effect of interacting isolated rat adipocytes with small, unilammelar vesicles on insulin receptor internalization and processing was studied. Treatment of freshly isolated cells with vesicles containing phosphatidylcholine and phosphatidylserine followed by incubation in 35 mM Tris-containing buffer considerably reduced the chloroquine-induced increase in cell-associated 125I-insulin and significantly inhibited the time and insulin dependent loss of surface insulin receptors. The internal receptor pool, as measured by insulin binding to detergent solubilized adipocytes, was relatively smaller in vesicle-treated cells. Concomitant with a slower rate of receptor internalization, insulin-sensitive hexose uptake also demonstrated significantly slower kinetics of decreased response with time. These results support the conclusion that pretreatment of fat cells with phospholipid vesicles inhibits normal insulin receptor cycling.     

Purification and characterization of the human brain insulin receptor

The insulin receptor from human brain cortex was purified by a combination monoclonal antibody affinity column and a wheat germ agglutinin column. This purified receptor preparation exhibited major protein bands of apparent Mr = 135,000 and 95,000, molecular weights comparable to those for the alpha and beta subunits of the purified human placental and rat liver receptors. A minor protein band of apparent Mr = 120,000 was also observed in the brain receptor preparation. Crosslinking of 125I-insulin to all three receptor preparations was found to preferentially label a protein of apparent Mr = 135,000. In contrast, cross-linking of 125I-labeled insulin-like growth factor I to the brain preparation preferentially labeled the protein of apparent Mr = 120,000. The purified brain insulin receptor was found to be identical with the placental insulin receptor in the amount of neuraminidase-sensitive sialic acid and reaction with three monoclonal antibodies to the beta subunit of the placental receptor. In contrast, a monoclonal antibody to the insulin binding site recognized the placental receptor approximately 300 times better than the brain receptor. These results indicate that the brain insulin receptor differs from the receptor in other tissues and suggests that this difference is not simply due to the amount of sialic acid on the receptor.     

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.     

Internalization of the human insulin receptor. The insulin-independent pathway.

Internalization of the human insulin receptor requires the activation by insulin of the intrinsic kinase of the receptor. However, even in the absence of kinase activation, insulin receptors slowly enter the cells. In the present study, we addressed the question of this insulin-independent pathway of internalization. To that end, we traced insulin receptor internalization with a monoclonal antibody (mAb 83-14) directed against the alpha-subunit of the human insulin receptor. Internalization of this antibody was followed in Chinese hamster ovary (CHO) cells transfected with either normal (CHO.HIRC2) or kinase-deficient (CHO.A1018) human insulin receptors. The internalization rate of 125I-mAb 83-14 was comparable in CHO cells expressing kinase-active or kinase-inactive receptors and was similar to that observed for 125I-insulin in CHO.A1018 cells. Moreover, in CHO.HIRC2 cells, the internalization of 125I-mAb 83-14 was identical with that of its 125I-Fab fragments. Thus, mAb 83-14 represents an appropriate tool to study the constitutive internalization of the insulin receptor. Internalization of insulin receptors tagged with 125I-mAb 83-14 was unaffected by cytochalasin B, which excluded a macropinocytotic process. By contrast, internalization was sensitive to hypertonia, which abrogates clathrin-coated pits-mediated endocytosis. The implication of clathrin-coated pits in this internalization process was directly demonstrated by quantitative electron microscopic autoradiography, which showed that 125I-mAb 83-14 present on the nonvillous domain of the cell surface preferentially associate with clathrin-coated pits at all time points.     

Tyrosine phosphorylation of the insulin receptor during insulin-stimulated internalization in rat hepatoma cells

We have studied the phosphorylation state of the insulin receptor during receptor-mediated endocytosis in the well-differentiated rat hepatoma cell line Fao. Insulin induced the rapid internalization of surface-iodinated insulin receptors into a trypsin-resistant compartment, with a 3-fold increase in the internalization rate over that seen in the absence of insulin. Within 20 min of insulin stimulation, 30-35% of surface receptors were located inside the cell. This redistribution was half-maximal by 10.5 min. Similar results were obtained when the loss of surface receptors was measured by 125I-insulin binding. Tyrosyl phosphorylation of internalized insulin receptors was measured by immunoprecipitation with antiphosphotyrosine antibody. Immediately after insulin stimulation, 70-80% of internalized receptors were tyrosine phosphorylated. Internalized receptors persisted in a phosphorylated state after the dissociation of insulin but were dephosphorylated prior to their return to the plasma membrane. After 45-60 min of insulin stimulation, the tyrosine phosphorylation of the internal receptor pool decreased by 45%, whereas the phosphorylation of surface receptors was unchanged. These data suggest that insulin induces the internalization of phosphorylated insulin receptors into the cell and that the phosphorylation state of the internal receptor pool may be regulated by insulin.     

Evidence for a subtype of insulin-like growth factor I receptor in brain

We examined the structure of receptors for insulin-like growth factor I (IGF-I), insulin, and epidermal growth factor (EGF) in human brain and human placenta using affinity cross-linking procedures and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In human brain, proteins specifically cross-linked to 125I-IGF-I, 125I-insulin, and 125I-EGF had apparent molecular weights of 120,000, 115,000 and 170,000, respectively. In human placenta, proteins cross-linked to 125I-IGF-I and 125I-insulin were 10 kDa larger than the corresponding subunits in brain. The receptor labeled by 125I-EGF in placenta was indistinguishable from the EGF receptor in brain. The size discrepancy of IGF-I receptors in brain and placenta was no longer apparent after removing the carbohydrate moieties of the proteins with endo-beta-N-acetylglucosaminidase F (EndoF). Furthermore, the brain IGF-I receptor was not cleaved by neuraminidase, whereas, the placental IGF-I receptor had increased mobility on SDS gels following neuraminidase treatment. The results indicate that receptors for IGF-I and insulin in human brain are structurally distinct from the corresponding receptors in human placenta, the structural heterogeneity of the receptors involves differences in N-linked glycosylation, particularly the terminal processing steps, and EGF receptors are present in human brain and human placenta but are structurally similar in these tissues. We conclude that there is a selective modification in the glycosylation of receptors for IGF-I and insulin in brain.     

Down-regulation of insulin receptors is related to insulin internalization

In the present study, we have tested the influence of inhibition of endocytosis by hypertonic medium on the regulation of cell surface insulin receptors. We show that active internalization of 125I-insulin is markedly inhibited by hypertonic media and that, in parallel, cell surface invaginations are significantly diminished. These two events are accompanied by a marked inhibition of cell surface insulin receptor down-regulation. These data provide further strong evidence that receptor-mediated endocytosis is the major mechanism by which insulin receptors are regulated at the surface of target cells.     

Role of Hsp70 synthesis in the fate of the insulin-receptor complex after heat shock in cultured fetal hepatocytes

The influence of a mild heat shock on the fate of the insulin-receptor complex was studied in cultured fetal rat hepatocytes whose insulin glycogenic response is sensitive to heat [Zachayus and Plas (1995): J Cell Physiol 162:330–340]. After exposure from 15 min to 2 hr at 42.5°C, the amount of ¹²⁵I-insulin associated with cells at 37°C was progressively decreased (by 35% after 1 hr), while the release of ¹²⁵I-insulin degradation products into the medium was also inhibited (by 75%), more than expected from the decrease in insulin binding. Heat shock did not affect the insulin-induced internalization of cell surface insulin receptors but progressively suppressed the recycling at 37°C of receptors previously internalized at 42.5°C in the presence of insulin. When compared to the inhibitory effects of chloroquine on insulin degradation and insulin receptor recycling, which were immediate (within 15 min), those of heat shock developed within 1 hr of heating. The protein level of insulin receptors was not modified after heat shock and during recovery at 37°C, while that of Hsp72/73 exhibited a transitory accumulation inversely correlated with variations in insulin binding, as assayed by Western immunoblotting from whole cell extracts. Coimmunoprecipitation experiments revealed a heat shock-stimulated association of Hsp72/73 with the insulin receptor. Affinity labeling showed an interaction between ¹²⁵I-insulin and Hsp72/73 in control cells, which was inhibited by heat shock. These results suggest that increased Hsp72/73 synthesis interfered with insulin degradation and prevented the recycling of the insulin receptor and its further thermal damage via a possible chaperone-like action in fetal hepatocytes submitted to heat stress. © 1996 Wiley-Liss, Inc.     

Hormone-induced conformational changes in the hepatic insulin receptor

The insulin receptor can exist in either a lower or a higher affinity state. Hormone binding alters the equilibrium between the two states of the insulin receptor, favoring the formation of that of higher affinity (Corin, R.E., and Donner, D.B. (1982), J. Biol. Chem. 257, 104-110). After brief or extended incubations with hormone, during which the fraction of higher affinity receptors increased, 125I-insulin was covalently coupled to the alpha subunits of its receptor using disuccinimidyl suberate. Some 125I-insulin remained bound to higher affinity receptors after dissociation of hormone from lower affinity sites. This hormone could also be covalently coupled to the alpha subunit of the receptor. During extended incubations between 125I-insulin and liver plasma membranes, components of the receptor were cleaved to yield degradation products of 120,000 and 23,000 Da. The significance of this process remains undetermined. Unoccupied insulin receptors were cleaved by trypsin to produce fragments of 94,000 and 37,000 Da which remained membrane-bound and could be covalently coupled to 125I-insulin. Trypsin treatment after binding yielded an additional receptor fragment of 64,000 Da. As the incubation time between 125I-insulin and membranes was lengthened, components of the receptor became progressively less sensitive to trypsin. Higher affinity binding sites isolated after release of rapid dissociating insulin were less sensitive to trypsin than were mixtures of higher and lower affinity receptors. These observations suggest that hormone binding produces two conformational changes (alterations of tryptic lability) in the hepatic insulin receptor. The first change is rapid and exposes parts of the receptor to tryptic degradation. The second, slower conformational change renders the receptor less sensitive to trypsin and occurs with the same time course as the increase of receptor affinity mediated by site occupancy.     

Internalization of the neonatal brain insulin receptor

Using 10-15 day neonatal rabbit brain cells, we studied the internalization (n = 6) and intracellular degradation (n = 8) of specifically bound 125I-insulin. In addition we investigated the association between the internalization of the specifically bound 125I-insulin and the metabolic effects of insulin such as glucose (n = 13) and amino-acid (leucine) uptake (n = 6). Phenylarsine oxide (10 microM), an agent that inhibits the internalization of the insulin receptor (n = 6) decreased the specifically bound 125I-insulin in the intact and trypsin-resistant (inside) part of the brain cells by 50% (p less than 0.05). On the other hand chloroquine (100 microM), a lysosomotropic agent that interferes with the intracellular degradation of the insulin receptor (n = 8) increased two-fold the 125I-insulin specifically bound to the intact and trypsin resistant part of the cells (p less than 0.05). Both these agents did not alter the time-dependent basal glucose uptake by the brain cells. Glucose alone regulated its own uptake (n = 4) whereas 1 X 10(-6) M insulin did not augment the glucose uptake (n = 11+13) above basal. Similarly leucine regulated the leucine uptake (n = 4) but insulin did not alter this basal uptake by the brain cells (n = 6). In summary we observed no associated glucose or leucine uptake along with the presence of internalization and intracellular degradation of specifically bound 125I-insulin in the brain cells.     

Phospholipid environment alters hormone-sensitivity of the purified insulin receptor kinase.

Insulin receptor kinase, affinity-purified by adsorption and elution from immobilized insulin, is stimulated 2-3-fold by insulin in detergent solution. Reconstitution of the receptor kinase into leaky vesicles containing phosphatidylcholine and phosphatidylethanolamine (1:1, w/w) by detergent removal on Sephadex G-50 results in the complete loss of receptor kinase sensitivity to activation by insulin. Insulin receptors in these vesicles also exhibit an increase in their apparent affinity for 125I-insulin (Kd = 0.12 nM versus 0.76 nM). Inclusion of 8.3-16.7% phosphatidylserine into the reconstituted vesicles restores 40-50% of the insulin-sensitivity to the receptor kinase. An elevated apparent affinity for 125I-insulin of insulin receptors in vesicles containing phosphatidylcholine and phosphatidylethanolamine is also restored to the value observed in detergent solution by the inclusion of phosphatidylserine in the reconstituted system. The effect of phosphatidylserine on insulin receptor kinase appears specific, because cholesterol, phosphatidylinositol and phosphatidic acid are all unable to restore insulin-sensitivity to the receptor kinase. Autophosphorylation sites on the insulin receptor as analysed by h.p.l.c. of tryptic 32P-labelled receptor phosphopeptides are not different for insulin receptors autophosphorylated in detergent solution or for the reconstituted vesicles in the presence or absence of phosphatidylserine. These data indicate that the phospholipid environment of insulin receptors can modulate its binding and kinase activity, and phosphatidylserine acts to restore insulin-sensitivity to the receptor kinase incorporated into phosphatidylcholine/phosphatidylethanolamine vesicles.     

Cell surface proteolysis and down-regulation of the hepatic insulin receptor. Evidence for selective sorting of intact and degraded receptors after internalization

Insulin binding to rat liver plasma membranes promotes proteolysis of the Mr 135,000 alpha subunit of the insulin receptor to a fragment of Mr 120,000 (Lipson, K. E., Yamada, K., Kolhatkar, A. A., and Donner, D. B. (1986) J. Biol. Chem. 261, 10833-10838). The enzyme that catalyzes this degradation copurifies with plasma membranes and cannot be identified in any other cellular organelle or in cytosol. The proteinase has optimal activity above pH 7 and is an integral protein based upon its resistance to extraction with 2 M NaCl. After affinity labeling, degraded insulin receptors were identified in plasma membranes isolated from a liver perfused with 1 nM 125I-insulin for 10 min at 37 degrees C, indicating that proteolysis occurs in the hepatocyte cell membrane under physiological conditions. Microsomes do not contain the receptor degrading activity or a detectable amount of degraded receptors under basal conditions. After perfusion of a liver with 125I-insulin, Mr 135,000 and Mr 120,000 complexes were detected in microsomes, suggesting that both intact and degraded receptors can be internalized. The initial absence of degraded receptors in plasma membranes suggests that, following internalization, such sites do not recycle. Thus, hormone-induced proteolysis of the insulin receptor begins at the surface of the rat hepatocyte and can lead to loss of receptors from the plasma membrane.