This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Eriksson, J. W. Articles by Smith, U. Search for Related Content PubMed PubMed Citation Articles by Eriksson, J. W. Articles by Smith, U.

Insulin Promotes and Cyclic Adenosine 3',5'-Monophosphate Impairs Functional Insertion of Insulin Receptors in the Plasma Membrane of Rat Adipocytes: Evidence for Opposing Effects of Tyrosine and Serine/Threonine Phosphorylation¹

Lundberg Laboratory for Diabetes Research, Department of Medicine, Sahlgrenska University Hospital (J.W.E., P.L., C.W., U.S.), Goteborg; and the Department of Medicine, Norrland University Hospital (J.W.E.), Umea, Sweden

Address all correspondence and requests for reprints to: Dr. Jan Eriksson, Department of Medicine, Norrland University Hospital, S-901 85 Umea, Sweden.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The aim of the present study was to elucidate events in the plasma membrane (PM) associated with the previously described effect of insulin to rapidly enhance the number of cell surface insulin binding sites in rat adipocytes. [¹²⁵I]insulin was cross-linked to cell surface insulin receptors of intact cells that had been preincubated with or without insulin. Subsequently prepared PM displayed a 3-fold increase in bound [¹²⁵I]insulin when cells had been pretreated with 6 nM insulin for 20 min compared to membranes from control cells, and SDS-PAGE with autoradiography showed that this occurred at the insulin receptor -subunit. The magnitude of the effect was similar to that found for insulin binding to intact cells that had been preincubated with insulin. In contrast, the insulin binding capacity in the PM was not affected by prior treatment of cells with insulin when assessed with the addition of [¹²⁵I]insulin directly to solubilized PM; this suggests an unchanged total number of PM receptors. Thus, the enhancement of cell surface insulin binding capacity produced by insulin is not due to the translocation of receptors, but instead appears to be confined to receptors already present in the PM. The addition of phospholipase C (from Clostridium perfringens), which cleaves PM phospholipids, mimicked the effect of insulin to enhance cell surface binding in adipocytes, and this suggests a pool of cryptic PM receptors. Both the nonmetabolizable cAMP analog N⁶-monobutyryl cAMP (N⁶-mbcAMP) and the serine/threonine phosphatase inhibitor okadaic acid abolished the effect of concomitant insulin treatment to increase binding capacity. In contrast, the tyrosine phosphatase inhibitor vanadate increased insulin binding even in the presence of okadaic acid or N⁶-mbcAMP. The effect of N⁶-mbcAMP to impair cell surface insulin binding was also evident in the presence of a peptide derived from the major histocompatibility complex type I that effectively impairs receptor internalization, but the amount of PM receptors assessed by immunoblot was unaltered.

Taken together, the data suggest that insulin exposure leads to the uncovering of cryptic receptors associated with the PM. It is also suggested that tyrosine phosphorylation promotes this process, whereas enhanced serine phosphorylation, e.g. produced by cAMP, impairs the functional insertion of the receptors, rendering them unable to bind insulin.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
WE PREVIOUSLY showed that insulin can rapidly enhance the insulin binding capacity at the cell surface of intact rat adipocytes (1, 2). This effect is attributed to an increased number of binding sites without any change in apparent receptor affinity (1). In insulin-resistant cells (e.g. cAMP treatment, obesity), this effect of insulin is severely attenuated (1, 2). However, in such cells the effect can be elicited by the insulin-mimicking agent vanadate, and in parallel, cellular insulin sensitivity becomes restored (3). Thus, this novel mechanism appears to be linked to the cellular regulation of insulin sensitivity. The enhanced cell surface binding occurs despite a concomitant receptor internalization (1, 2, 4, 5) and, therefore, may involve receptors within the plasma membrane (PM). Two mechanisms are conceivable: 1) exposure of additional binding sites on each receptor, or 2) uncovering of previously nonbinding receptors. The latter mechanism is compatible with previous results indicating the presence of a large pool of cryptic insulin receptors within the PM, which has been shown in adipocytes, hepatocytes, placental cells, and lymphocytes (6, 7, 8, 9). The aim of the present study was to elucidate events in the adipocyte PM that are associated with the rapid increase in cell surface binding capacity produced by insulin. We employed cross-linking of [¹²⁵I]insulin to cell surface receptors followed by PM preparation to establish whether the increase in insulin binding is recovered within the PM. Moreover, phospholipase C (PLC) was used to cleave PM phospholipids and unmask possible cryptic insulin receptors (6).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Porcine monocomponent insulin and mono-[¹²⁵I]Tyr-A₁₄-insulin (SA, 1.2–1.7 mCi/nmol) were obtained from Novo Nordisk (Copenhagen, Denmark), and human mono-[¹²⁵I]Tyr-B₂₆-insulin was obtained from Amersham (Aylesbury, UK). Adenosine deaminase was purchased from Boehringer Mannheim (Mannheim, Germany), and medium 199 was obtained from Statens Bakteriologiska Laboratorium (Stockholm, Sweden). Okadaic acid was obtained from Life Technologies (Gaithersburg, MD). Sodium orthovanadate, disuccinimidyl suberate, PLC (from Clostridium perfringens), and phosphatidylinositol (PI)-specific PLC (from Bacillus cereus) were purchased from Sigma Chemical Co. (St. Louis, MO). Rabbit antibodies directed to the insulin receptor ß-subunit were kindly provided by Dr. B. I. Posner, Montreal, Canada (antibody 960) or were obtained from Transduction Laboratories (Lexington, KY). A dimerized peptide fragment [Ala⁸⁵]D^K-(69–85) derived from the 1-domain of the major histocompatibility complex class I (MHC-I) was a generous gift from Dr. Lennart Olsson, Receptron (San Francisco, CA). All other chemicals were of the highest quality commercially available.

Cell incubation conditions and [¹²⁵I]insulin binding
Male Sprague-Dawley rats (150–200 g), fed ad libitum, were stunned and decapitated. The epididymal fat pads were immediately excised and minced, and the fat cells were isolated in medium 199 containing 5.6 mM D-glucose with 40 mg/ml BSA and 1 mg/ml collagenase. The cells were then filtered and washed three times with fresh medium. The preincubations were performed in medium 199 at a lipocrit of 5–10% for 20 min at 37 C in the presence of 1 U/ml adenosine deaminase. After the preincubations, the cells were ATP-depleted with 2 mM KCN, which was also present during the binding assays. This procedure effectively stops receptor internalization (1). After 5 min, the cells were thoroughly washed before the binding assays. The washing procedure has been shown to remove at least 95% of insulin bound during the preincubation period (1).

[¹²⁵I]Insulin binding to intact cells was performed essentially as previously described (1). Briefly, aliquots of cells and medium with 1% BSA and 2 mM KCN were, unless otherwise indicated, transferred to 16 C and 34 pM [¹²⁵I]insulin was added. After 1 h (unless otherwise indicated), when steady state was clearly established, specific [¹²⁵I]insulin binding was measured as cell-associated radioactivity after subtracting nonspecific binding (in the presence of 600 nM unlabeled insulin).

Affinity labeling of insulin receptors at the cell surface
[¹²⁵I]Insulin was cross-linked to its receptors (affinity labeling) essentially as previously described (10, 11). After preincubation and washing in the presence of KCN, the cells were transferred to 23 C and 430 pM [¹²⁵I]insulin was added. After 1 h, 0.5 mM disuccinimidyl suberate was added, and the incubation was continued at 15 C for 30 min. The cells were then homogenized in 20 mM Tris-HCl, 1 mM EDTA, and 255 mM sucrose buffer (TES), pH 7.4, at 4 C with protease inhibitors (aprotinin, leupeptin, pepstatin, and phenylmethylsulfonylfluoride) and PM were prepared through gradient centrifugation (12). Briefly, the cell homogenate (H) was centrifuged in TES at 11,000 x g at -4 C for 15 min. The resuspended pellet was layered on a 1.12-M sucrose cushion and centrifuged at 82,000 x g for at 4 C for 20 min when the PM were collected and pelleted at 103,000 x g for 10 min. The original supernatant was centrifuged at 30,000 x g for 30 min. The resulting supernatant was then centrifuged at 370,000 x g for 75 min. The pellet containing the low density microsomal membranes was homogenized. Control experiments assessing the activity of 5'-nucleotidase, a marker enzyme for the PM fraction, indicated similar relative amounts as previously reported (12) (in H, 0.78 µmol/h·mg protein; PM, 5.01 µmol/h·mg protein; low density microsomal membranes 0.03 µmol/h·mg protein), implying appropriate purity of the PM preparations. PM were solubilized through boiling in 1% SDS for 1 min with or without 50 mM dithiothreitol. Samples were applied on SDS-PAGE (5% or 7.5%) overnight, and the gels were subjected to autoradiography (Agfa Curix RP 1, Agfa, Kista, Sweden), thus assessing bound [¹²⁵I]insulin.

[¹²⁵I]Insulin binding to solubilized and isolated PM
PM were prepared essentially as described above. [¹²⁵I]Insulin binding to samples of isolated PM (100 µg membrane protein) was assessed by adding 340 pM radioligand at 16 C for 1 h. Solubilized receptors were obtained by treating PM samples with 1% Thesit (Boehringer Mannheim, Mannheim, Germany) in the presence of the protease inhibitors for 20 min at 20 C. Then, receptors were precipitated, and [¹²⁵I]insulin binding was assessed essentially as previously described (1). In some experiments solubilized PM receptors were immunoprecipitated using ß-subunit antibody 960, which per se does not interfere with insulin binding (9) (data not shown), and then [¹²⁵I]insulin was bound to receptors.

Immunoblotting of PM insulin receptors
PM were obtained and solubilized under reducing conditions essentially as described above. The solubilized proteins were separated on a 10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and immunoblotted with insulin receptor ß-subunit antibody (Transduction Laboratories) according to the manufacturer. An enhanced chemiluminiscence kit with a secondary horseradish peroxidase-labeled antibody (Amersham) was employed, and the bands were visualized on an autoradiography film.

Statistical analysis
Statistical significance of differences was tested with Student’s two-tailed t test for paired data. Results are expressed as the mean ± SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of preincubation with insulin on [¹²⁵I]insulin binding to intact cells and PM
Table 1 shows the binding of [¹²⁵I]insulin to intact cells, isolated PM, and the PM fraction after cross-linking of [¹²⁵I]insulin to intact cells. As also previously found (1, 2, 3), intact cells pretreated with insulin (6 nM), followed by washing in the presence of 2 mM KCN, displayed a 3- to 4-fold increase in cell surface binding capacity compared to untreated cells. When binding to isolated PM was performed, this effect was not recovered, and binding was similar regardless of whether the cells had been pretreated with insulin. This was also true when binding was assessed in solubilized PM from cells that had been preincubated with insulin (binding was 106 ± 14% of the control value; n = 3) as well as when receptors from solubilized PM of these cells were immunoprecipitated with the 960 antibody before the binding assay (binding was 90–110% of the control value). To ensure that possible receptor redistribution due to dephosphorylation processes did not occur during the membrane preparation, control experiments were performed with phosphatase inhibitors (1 mM vanadate, 10 mM sodium pyrophosphate, or both) present throughout the membrane preparation. However, also under those conditions, binding to immunoprecipitated receptors from solubilized PM was similar regardless of whether cells had been pretreated with insulin (data not shown).

Note that a direct stoichiometric comparison of binding between the different preparations shown in Table 1 was not possible due to different receptor recoveries and binding conditions. However, the effect of insulin could be appropriately assessed within each group.

Effects of okadaic acid and cAMP
Preincubating the cells with okadaic acid (1 µM) alone tended to decrease basal, nonstimulated binding, although this was not statistically significant (Table 2). Okadaic acid completely inhibited the effect of insulin to increase insulin binding, and, in fact, insulin and okadaic acid together exerted a decrease (30%) in binding compared to that in untreated cells. As previously shown (2), the nonmetabo-lizable (13) cAMP analog, N⁶-monobutyryl cAMP (N⁶-mbcAMP), also completely prevented the stimulatory effect of insulin (see Table 3). Vanadate alone increased insulin binding, and this effect was also demonstrated in the presence of okadaic acid (Table 2) or N⁶-mbcAMP (data not shown) (3).

View larger version (64K): [in this window] [in a new window]   Figure 1. SDS-PAGE of solubilized PM prepared after cross-linking of [125I]insulin to intact cells. Adipocytes were preincubated in the presence or absence of insulin (6 nM) and N6-mbcAMP (4 mM) for 20 min as indicated. [125I]Insulin binding and cross-linking were performed as described in Materials and Methods. Unlabeled insulin (600 nM) was absent (lanes 1, 3, and 5) or present as a control (nonspecific binding; lanes 2, 4, and 6). The cells were then homogenized and fractionated, and the PM fraction was solubilized in the presence of dithiothreitol (50 mM). Samples (20 µg membrane protein) were subjected to SDS-PAGE (7.5%) with subsequent autoradiography. The indicated molecular sizes refer to protein standards: myosin, 205 kDa; ß-galactosidase, 116 kDa; BSA, 80 kDa. Insulin receptor -subunit, 135 kDa.

View larger version (21K): [in this window] [in a new window]   Figure 2. Immunoblotting of PM insulin receptors. Adipocytes were preincubated with or without the dimerized MHC-I peptide fragment (10 nM) for 60 min at 37 C. During the final 30 min of the preincubation, N6-mbcAMP (4 mM) was added when indicated. Cell surface [125I]insulin binding to intact cells was measured as described in Table 1: control, 0.225; MHC-I peptide, 0.255; N6-mbcAMP, 0.046; and MHC-I peptide plus N6-mbcAMP, 0.052 fmol/105 cells. PM were prepared and solubilized, and the PM proteins were separated with SDS-PAGE (10%), transferred to a nitrocellulose membrane, and immunoblotted with an antibody directed to the insulin receptor ß-subunit. Enhanced chemiluminescence and autoradiography were then performed. The data shown are from one representative experiment of three performed. There were no consistent changes in PM insulin receptor abundance among the different preincubation conditions. The numbers refer to different protein standards (insulin receptor ß-subunit, 95 kDa).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
It may seem difficult to reconcile earlier observations that insulin rapidly induces internalization of the insulin receptor (4, 5) with our previous and current results demonstrating an enhanced number of binding sites for insulin at the cell surface of rat adipocytes (1, 2). However, the present data with affinity labeling of intact cells followed by cell fractionation show that the effect to enhance receptor binding is recovered in the PM with a similar magnitude as in whole cells. [¹²⁵I]Insulin binding to receptors from solubilized PM, with or without prior immunoprecipitation, should reflect the total PM receptor pool. As this was not augmented by insulin, the recruitment of cell surface binding sites must occur within a pool of receptors located in the PM. Taken together, the results with affinity labeling and the data on binding to solubilized membranes thus indicate that the receptor up-regulation in response to insulin is confined to receptors already associated with the PM. Therefore, in the nonstimulated state, only part of the total receptor pool in the PM appears to be fully and functionally inserted in the membrane and capable of binding insulin at the cell surface. This concept is also in agreement with previous studies in different cell types, suggesting cryptic insulin receptors associated with the PM but not fully exposed at the cell surface (6, 7, 8, 9). Insulin exposure may lead to uncovering of such cryptic receptors, e.g. by promoting their functional PM insertion, and this can be elicited by other neighboring receptors that are already fully inserted and accessible for ligand binding.

The failure to recover the increased binding capacity in response to insulin when [¹²⁵I]insulin was added directly to isolated intact PM shows that this effect is not preserved during the fractionation procedure. Probably, binding to this PM preparation only represents a part of the total pool of PM receptors, as previous results indicate a large proportion of cryptic receptors in such preparations (6). Thus, the loss of the insulin effect during the fractionation procedure suggests that the receptors that have been exposed at the cell surface in response to insulin return to the cryptic state when cellular integrity is destroyed. Moreover, previous data have shown that insulin pretreatment of isolated PM does not alter their insulin-binding capacity (1), indicating that the effect of insulin on cell surface binding capacity requires an intact cell. The effect of insulin was demonstrated in cells that after the insulin pretreatment were ATP depleted to prevent receptor recycling during the binding assay. However, our previous data with the insulin mimicker vanadate have shown that this mechanism is also demonstrable under non-ATP-depleted conditions (1, 3). In fact, the expression of this effect of insulin requires ATP, as KCN added before insulin treatment prevents the up-regulation of receptors (1).

The possibility of a conformational change in the receptors in response to insulin leading to the exposure of several binding sites per receptor appears unlikely, as previous work indicates that maximally two insulin molecules can bind to each receptor (17). Furthermore, the apparent molecular size of the cross-linked ligand-receptor complex was not altered by insulin pretreatment, and this supports, but does not prove, that the number of binding sites per receptor was unchanged.

A more probable mechanism is that initially nonbinding receptors become accessible to ligand binding. This possibility is also attractive, because PLC, which cleaves PM phospholipids, in the present as well as previous studies (6, 7, 8) was shown to uncover PM receptors, indicating a pool of cryptic receptors associated with the PM. Moreover, insulin has been reported to activate some isoforms of PLC (15, 16). Interestingly, the addition of exogenous crude PLC dose dependently increased cell surface binding to at least the same extent as insulin. The time course for the enzyme action was rapid (<5 min). PI-specific PLC hydrolyses phosphatidylinositols, including a glycosylphosphatidylinositol (GPI) that is hydrolyzed upon insulin stimulation and tentatively may be involved in insulin signaling (16). The PI-specific PLC did, however, not exert a consistent effect on binding. This would argue against a mechanism by which insulin, through activation of PLC isoforms, leads to uncovering of cryptic insulin receptors. This possibility, however, is not completely excluded, as endogenous insulin-sensitive (G)PI-specific PLC in the cytoplasm (or in the PM) could exert more pronounced local effects on insulin receptor binding than the extracellular addition of enzyme targeted to the outer PM leaflet. One tentative mechanism is that GPI may anchor the insulin receptor in the PM, restraining its full exposure. Such a role of GPI has previously been shown for some PM enzymes that become fully active upon hydrolysis of the GPI anchor (18, 19).

The site of the pool of cryptic insulin receptors is not yet defined. Possibly, these receptors can reside in membrane vesicles associated with the cytoplasmic side of the PM, e.g. clathrin-coated pits or, less likely, caveolae (20). Alternatively, the cryptic state could be purely functional, involving alterations in receptor conformation and/or phosphorylation.

Both N⁶-mbcAMP and okadaic acid, an inhibitor of the serine/threonine phosphatases PP1 and PP2A (21), completely abolished the effect of insulin on receptor recruitment. These findings are compatible with an inhibitory effect of serine phosphorylation on this process. In accordance with this, cAMP has been shown to promote serine phosphorylation of isolated receptors in vitro (22), and phosphorylation on specific serine sites of the insulin receptor may lead to both impairment of receptor tyrosine kinase activity as well as altered conformation and insertion of the receptor in the PM (23). Another possibility is that these agents influence the insulin signaling cascade through serine phosphorylation of insulin receptor substrate-1 (IRS-1) and impaired PI-3-kinase activation (24). Other peptides involved in the insulin signal transduction pathways may also be involved. Moreover, key steps for docking and fusion of receptors with the PM may be involved, including IRS-1 itself, which could, beside its action in insulin signaling, act as a coupling protein, forming complexes with insulin receptors and promoting their full PM insertion. This process might be impaired by serine phosphorylation of IRS-1, whereas tyrosine phosphorylation could promote the formation of such complexes.

The effect of cAMP to impair cell surface insulin binding was also shown when insulin receptors were "locked" in the PM by pretreatment with the peptide derived from the MHC class I complex, which inhibits internalization of several insulin-regulated membrane proteins, e.g. insulin and insulin-like growth factor II (IGF-II) receptors and the glucose transporter GLUT4 (14, 25). As this occurred without any corresponding change in insulin receptor abundance in the PM, these data suggest that an important effect of cAMP is exerted at the level of the functional insertion of insulin receptors within the PM. The ability of elevated cAMP levels to impair receptor recruitment is associated with cellular insensitivity to insulin with respect to glucose transport activation, and IGF-II receptor translocation (2, 26, 27). In analogy, okadaic acid has previously been shown to impair insulin action in adipocytes on both glucose transport (28) and IGF-II receptors (29).

The present findings, thus, suggest that the number of receptor sites available to insulin is, to a large extent, regulated by mechanisms occurring within the PM. Interestingly, similar events also appear to influence the accessibility and functional state of GLUT 4. Recent data suggest that docking and/or fusion of the glucose transporters with the PM are critical steps for their inherent activity, and this process also seems to be regulated by insulin, cAMP, and catecholamines in a manner similar to the presently proposed mechanisms (30, 31). Another insulin-regulated PM protein, the IGF-II receptor, seems to be regulated in an analogous manner by cAMP and insulin (27). Functionally, cryptic IGF-II receptors seem to be associated with the PM, and we have recently reported that the number of such cryptic receptors may be increased after cAMP exposure (32). The present and pre-vious (3) results suggest that vanadate-induced tyrosine phosphorylation overcomes the inhibitory action of serine/threonine phosphorylation, as vanadate produced a clear enhancement of cell surface insulin binding in the presence of okadaic acid as well as cAMP. This supports the concept that tyrosine phosphorylation of insulin-regulated proteins plays a role not only in insulin signaling and associated translocation processes, but also in the functional insertion of GLUT4 and insulin receptors in the PM.

In conclusion, the present study shows that the effect of insulin to amplify its cell surface binding in intact cells is attributable to recruitment of receptors recovered in the PM fraction. The data suggest that this effect occurs through the uncovering of functionally cryptic PM receptors and that tyrosine phosphorylation may be an important mechanism. This mechanism is impaired in insulin-resistant states (1, 2, 3), and we suggest that this can involve serine/threonine phosphorylation of the receptors or other adjacent proteins. Thus, tyrosine and serine/threonine phosphorylation may play a pivotal role. Further studies are in progress to clarify whether these regulatory mechanisms involve changes in the fusion and insertion of receptors at the PM or the conformation of individual receptors.

	Acknowledgments

 
We are grateful to Birgitta Karlsson-Svalstedt, Aino Johansson, and the late Barbro Carlander for expert technical assistance, and to Anna-Lena Dahlgren for skillful secretarial help.

	Footnotes

 
¹ This work was supported by grants from the Swedish Medical Research Council (B-3506), the Swedish Diabetes Association, the Gothenburg Diabetes Association, the Swedish Society of Medicine, the Gothenburg Medical Society, and the IngaBritt and Arne Lundberg, Novo-Nordisk, Tore Nilson, Magnus Bergvall, and King Gustaf V and Queen Victoria Foundations.

Received June 14, 1996.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Eriksson J, Lönnroth P, Smith U 1992 Insulin can rapidly increase cell surface insulin binding capacity in rat adipocytes. Diabetes 41:707–714[Abstract]
2. Eriksson JW, Lönnroth P, Smith U 1992 Cyclic AMP impairs the rapid effect of insulin to enhance cell-surface insulin-binding capacity in rat adipocytes. Biochem J 288:625–629
3. Eriksson JW, Lönnroth P, Smith U 1992 Vanadate increases cell surface insulin binding and improves insulin sensitivity both in normal and insulin-resistant rat adipocytes. Diabetologia 35:510–516[CrossRef][Medline]
4. Marshall S, Olefsky JM 1981 Characterization of insulin-induced receptor loss and evidence for internalization of the insulin receptor. Diabetes 30:746–753[Medline]
5. Simpson IA, Hedo JA, Cushman SW 1984 Insulin-induced internalization of the insulin receptor in the isolated rat adipose cell. Diabetes 33:13–18[Abstract]
6. Cuatrecasas P 1971 Unmasking of insulin receptors in fat cells and fat cell membranes. J Biol Chem 246:6532–6542[Abstract/Free Full Text]
7. McCaleb ML, Donner DB 1981 Affinity of the hepatic insulin receptor is influenced by membrane phospholipids. J Biol Chem 256:11051–11057[Free Full Text]
8. Clark S, DeLuise M, Larkins RG, Melick RA, Harrison LC 1978 The effects of digestive enzymes on characteristics of placental insulin receptor. Biochem J 174:37–43[Medline]
9. Goodman DW, Isakson PC 1993 Mitogen activation of resting lymphocytes exposes cryptic insulin receptors. J Biol Chem 268:4207–4215[Abstract/Free Full Text]
10. Pilch PF, Czech MP 1980 The subunit structure of the high affinity insulin receptor. J Biol Chem 255:1722–1731[Abstract/Free Full Text]
11. Massagué J, Czech MP 1982 The subunit structures of two distinct receptors for insulin-like growth factors I and II and their relationship to the insulin receptor. J Biol Chem 257:5038–5045[Free Full Text]
12. Weber TM, Joost H-G, Simpson IA, Cushman SW 1988 Methods for assessment of glucose transporter activity and the number of glucose transporters in isolated rat adipose cells and membrane fractions. In: Kahn CR, Harrison LC (eds) Insulin Receptors, part B. Liss, New York, pp 171–187
13. Beebe SJ, Holloway R, Rannels SR, Corbin JD 1984 Two classes of cAMP analogs which are selective for the two different cAMP-binding sites of type II protein kinase demonstrate synergism when added togehter to intact adipocytes. J Biol Chem 259:3539–3547[Abstract/Free Full Text]
14. Stagsted J, Olsson L, Holman GD, Cushman SW, Satoh S 1993 Inhibition of internalization of glucose transporters and IGF-II receptors. J Biol Chem 268:22809–22813[Abstract/Free Full Text]
15. Saltiel AR 1990 Second messengers of insulin action. Diabetes Care 13:244–256[Abstract]
16. Fox JA, Soliz NM, Saltiel AR 1987 Purification of a phosphatidylinositol-glycan-specific phospholipase C from liver plasma membranes: a possible target of insulin action. Proc Natl Acad Sci USA 84:2663–2667[Abstract/Free Full Text]
17. Sweet LJ, Morrison BD, Pessin JE 1987 Isolation of functional ß heterodimers from the purified human placental ₂ß₂ heterotetrameric insulin receptor complex. J Biol Chem 262:6939–6942[Abstract/Free Full Text]
18. Low MG, Saltiel AR 1988 Structural and functional roles of glycosyl-phosphatidylinositol in membranes. Science 239:268–275[Abstract/Free Full Text]
19. Romero G, Luttrell L, Rogol A, Zeller K, Hewlett E, Larner J 1988 Phosphatidylinositol-glycan anchors of insulin mediators. Science 240:509–511[Abstract/Free Full Text]
20. Carpentier JL, McClain D 1995 Insulin receptor kinase activation releases a constraint maintaining the receptor on microvilli. J Biol Chem 270:5001–5006[Abstract/Free Full Text]
21. Bialojan C, Takai A 1988 Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Biochem J 256:283–290[Medline]
22. Roth RA, Beaudoin J 1987 Phosphorylation of purified insulin receptor by cAMP kinase. Diabetes 36:123–126[Abstract]
23. Stadtmauer L, Rosen OM 1986 Increasing the cAMP content of IM-9 cells alters the phosphorylation state and protein kinase activity of the insulin receptor. J Biol Chem 261:3402–3407[Abstract/Free Full Text]
24. Jullien D, Tanti J-F, Heydrick SJ, Gautier N, Grémeaux T, van Obberghen E, Le Marchand-Brustel Y 1993 Differential effects of okadaic acid on insulin-stimulated glucose and amino acid uptake and phosphatidylinositol 3-kinase activity. J Biol Chem 268:15246–15251[Abstract/Free Full Text]
25. Stagsted J, Reaven GM, Hansen T, Goldstein A, Olsson L 1990 Regulation of insulin receptor functions by a peptide derived from a major histocompatibility complex class I antigen. Cell 62:297–307[CrossRef][Medline]
26. Kirsch D, Kemmler W, Häring HU 1983 Cyclic AMP modulates insulin binding and induces post-receptor insulin resistance of glucose transport in isolated rat adipocytes. Biochem Biophys Res Commun 115:398–405[CrossRef][Medline]
27. Lönnroth P, Appell KC, Wesslau C, Cushman SW, Simpson IA, Smith U 1988 Insulin-induced subcellular redistribution of insulin-like growth factor II receptors in the rat adipose cell. J Biol Chem 263:15386–15391[Abstract/Free Full Text]
28. Corvera S, Jaspers S, Pasceri M 1991 Acute inhibition of insulin-stimulated glucose transport by the phosphatase inhibitor, okadaic acid. J Biol Chem 266:9271–9275[Abstract/Free Full Text]
29. Tanti J-F, Grémeaux T, Cormont M, van Obberghen E, Le Marchand-Brustel Y 1993 Okadaic acid stimulates IGF-II receptor translocation and inhibits insulin action in adipocytes. Am J Physiol 264:E868–E873
30. Vannucci SJ, Nishimura H, Satoh S, Cushman SW, Holman GD, Simpson IA 1992 Cell surface accessibility of GLUT4 glucose transporters in insulin-stimulated rat adipose cells. Biochem J 288:325–330
31. Timmers KI, Clark AE, Omatsu-Kanbe M, Whiteheart SW, Bennet MK, Holman GD, Cushman SW 1995 Identification of SNAP receptors in rat adipose cell membrane fractions and membrane fusion complexes co-immunoprecipitated with NSF. Diabetes [Suppl 1] 44:32A (Abstract)
32. Yu Z-W, Wickman A, Eriksson JW 1996 Cryptic receptors for insulin-like growth factor II in the plasma membrane of rat adipocytes–a possible link to cellular insulin resistance. Biochim Biophys Acta 1282:57–62[Medline]

This article has been cited by other articles:

				L. Braiman, A. Alt, T. Kuroki, M. Ohba, A. Bak, T. Tennenbaum, and S. R. Sampson Insulin Induces Specific Interaction between Insulin Receptor and Protein Kinase C{{delta}} in Primary Cultured Skeletal Muscle Mol. Endocrinol., April 1, 2001; 15(4): 565 - 574. [Abstract] [Full Text]

				T. Amit, O. Bar-Am, F. Dastot, M. B. H. Youdim, S. Amselem, and Z.'e. Hochberg The Human Growth Hormone (GH) Receptor and Its Truncated Isoform: Sulfhydryl Group Inactivation in the Study of Receptor Internalization and GH-Binding Protein Generation Endocrinology, January 1, 1999; 140(1): 266 - 272. [Abstract] [Full Text]

				S. Yu, A. Castle, M. Chen, R. Lee, K. Takeda, and L. S. Weinstein Increased Insulin Sensitivity in Gsalpha Knockout Mice J. Biol. Chem., June 1, 2001; 276(23): 19994 - 19998. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Eriksson, J. W. Articles by Smith, U. Search for Related Content PubMed PubMed Citation Articles by Eriksson, J. W. Articles by Smith, U.