Photoaffinity labeling of insulin receptor with an insulin analog selectively modified at the amino terminal of the B chain

Yeung, Clement W.T.; Moule, Margaret L.; Yip, Cecil C.

doi:10.1021/bi00551a031

Cited by 49 publications

(19 citation statements)

References 43 publications

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“…The most intensely labeled polypeptide that significantly penetrated the gel had a Mr of 130,000 (Fig. 1, lane 2), a value similar to that reported for the insulin-binding component of the receptor in other cells (4,6,11,13,14). The Mr 130,000 polypeptide migrated with the insulin-binding subunit identified when affinity crosslinking with "2I-insulin was restricted to proteins accessible on the surface of intact 3T3-L1 adipocytes (Fig.…”

Section: Resultssupporting

confidence: 73%

“…1). This size contrasts markedly to the Mr of 130,000 established for the insulin-binding subunit on the surface of a number of target tissues and cells (4,6,11,13,14). Because the Mr 200,000 polypeptide (i) has a short halflife, (ii) is the only insulin-binding protein resistant to functional inactivation by externally added proteolytic enzymes, and (iii) contains a hormone-binding site inaccessible at the cell surface ( Fig.…”

Section: Resultsmentioning

confidence: 91%

See 1 more Smart Citation

Latent insulin receptors and possible receptor precursors in 3T3-L1 adipocytes.

Deutsch¹,

Wan²,

Rosen³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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Cell surface and cryptic insulin receptors were solubilized from the particulate fraction of murine 3T3-L1 adipocytes with buffer containing 1% Triton X-100. Solubilized receptors were affinity crosslinked with 125I-labeled insulin and disuccinimidyl suberate and characterized by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography after specific immunoprecipitation. Two insulin-binding polypeptides were identified: the more abundant protein had a Mr of 130,000, corresponding to the size-ofthe hormone-binding subunit of insulin receptors on the surface of target cells; the second polypeptide exhibited a Mr of 200,000 and appears to be a component of the latent pool because it was unaffected when 3T3-L1 adipocytes were exposed to trypsin under conditions that result in a 95% reduction in cell surface insulin-binding activity and the loss ofthe Mr 130,000 polypeptide in. crosslinking experiments. Unexpectedly, the population of Me 200,000 molecules in intact cells was accessible for limited cleavage by chymotrypsin, yielding a Mr 195,000 insulin-binding polypeptide. When 3T3-LI adipocytes received a 15-min pulse of P5S]methionine, the predominant immunoprecipitated polypeptide had a Mr of 180,000. During a 1.5-hr chase, radioactivity in the Mr 180,000 species rapidly declined while the latent Mr 200,000 polypeptide became intensely labeled.After a 5-hr chase period, broad protein bands-with M1s of 130,000 and 90,000 were visualized as the major immunoprecipitated radioactive polypeptides. Thus, theMr 180,000species maybe a very early biosynthetic precursor that may be subsequently processed to a Mr 200,000 form and one or both of the smaller receptor subunits at the cell surface.After treatment with dexamethasone and isobutylmethylxanthine, murine 3T3-L1 fibroblasts uniformly differentiate into cells that exhibit the morphological and biochemical properties of adipocytes (1). The developing adipocytes become physiologically responsive to low concentrations ofinsulin and increase their cell surface complement of high-affinity insulin.receptors 15-to 35-fold over a period of 3-4 days (1, 2). Therefore, 3T3-Li adipocytes provide a highly suitable system.for detailed investigations on the biogenesis, posttranslational modification, internalization/recycling; and turnover of the insulin receptor. Moreover, studies on these characteristics in cells exposed to various concentrations ofinsulin and other effectors may reveal some of the mechanisms that.modulate target cell sensitivity to insulin. .In recent studies (3) we demonstrated that 3T3-L1 adipocytes contain a latent pool of insulin receptors that cannot bind extracellular insulin and-are resistant to inactivation.by exogenous proteolytic enzymes. The cryptic binding sites were revealed when particulate material from cell homogenates was solubilized in buffers containing the nonionic detergent Triton X-100 (3). The latent receptors closely resemble cell surface insulin receptors in their kinetics of association with hormone, equilib...

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Section: Resultssupporting

confidence: 73%

Section: Resultsmentioning

confidence: 91%

Latent insulin receptors and possible receptor precursors in 3T3-L1 adipocytes.

Deutsch¹,

Wan²,

Rosen³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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“…The insulin receptor has been well established to exist minimally as a heterotetrameric disulphide-linked complex consisting of two Mr-130000 (a) and two Mr-95000 (,O) subunits (Hedo et al, 1981;Massague et al, 1981;Van Obberghen et al, 1981;Fujita-Yamaguchi, 1984;Boyle et al, 1985). It is thought that the a subunit encompasses the high-affinity insulin-binding site (Yip et al, 1978(Yip et al, , 1980Jacobs et al, 1979;Pilch & Czech, 1980;Yeung et al, 1980), whereas the , subunit contains a single hydrophobic transmembrane domain (Ebina et al, 1985;Ullrich et al, 1985), an intracellular ATPbinding site (Roth & Cassell, 1983;Van Obberghen et al, 1983), as well as tyrosine-autophosphorylation receptor sites (Avruch et al, 1982;Kasuga et al, 1982aKasuga et al, ,b,c, 1983aPetruzzelli et al, 1982Petruzzelli et al, , 1984Tamura et al, 1983;Zick et al, 1983).…”

Section: Introductionmentioning

confidence: 99%

Differential sensitivity of the insulin-receptor kinase to thiol and oxidizing agents in the absence and presence of insulin

Wilden

Pessin

1987

Biochemical Journal

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The purified human placental insulin-receptor beta-subunit autophosphorylating activity was found to be inhibited, in a time- and concentration-dependent manner, by the specific thiol-alkylating agents N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid). The insulin-receptor kinase was observed to be more sensitive to inhibition by N-ethylmaleimide in the presence [IC50 (concn, giving 50% inhibition) = 25 +/- 3 microM] than in the absence (IC50 = 73 +/- 6 microM) of insulin. Similarly, inhibition by 5,5'-dithiobis-(2-nitrobenzoic acid) occurred with IC50 = 30 +/- 6 microM in the presence and 155 +/- 35 microM in the absence of insulin. Examination of the exogenous-substrate protein kinase activity demonstrated that the differential sensitivity to N-ethylmaleimide was due to direct inhibition of protein kinase activity, as opposed to blockade of the phospho-acceptor properties of the insulin receptor. In contrast, iodoacetamide had essentially no effect on the insulin-receptor beta-subunit autophosphorylating activity and was able to protect partially against the N-ethylmaleimide inhibition in both the presence and the absence of insulin. Consistent with these findings, none of the thiol-specific agents were able to alter significantly insulin binding at concentrations which maximally inhibited the beta-subunit autophosphorylation. Further, in the presence of insulin, the insulin-receptor kinase activity was also observed to be more sensitive to oxidation by H2O2 and FeCl3/ascorbate compared with insulin receptors in the absence of insulin. These results indicate that there is a critical thiol group(s) necessary for the beta-subunit autophosphorylating activity of the insulin-receptor kinase and that in the presence of insulin is more susceptible to exogenously added thiol and oxidizing agents.

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“…Specific functional properties are associated with the individual subunits: the a subunit contains the hormone binding site (Massague et al, 1980;Siegel et al, 1981;Jacobs et al, 1979;Wisher et al, 1980;Yeung et al, 1980;Kasuga et al, 198 1 b) while serine and tyrosine residues in the /3 subunit serve as phosphate acceptors in insulin-stimulated phosphorylation reactions (Kasuga et al, 1982a,b;Petruzzelli et al, 1982;Avruch et al, 1982). Recent reports suggest that the j3 subunit exhibits an intrinsic tyrosine protein kinase activity and catalyzes the autophosphorylation of the receptor (Roth & Cassell, 1983;Van Obberghen et al, 1983;Shia et al 1983;Roth et al 1983).…”

mentioning

confidence: 99%

Biogenesis, transit, and functional properties of the insulin proreceptor and modified insulin receptors in 3T3-L1 adipocytes

Salzman¹,

Wan²,

Rubin³

1984

Biochemistry

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The biogenesis, intracellular transport, and functional properties of the insulin proreceptor and modified insulin receptors were studied in hormone-responsive 3T3-L1 adipocytes. After control cells were labeled with [35S]Met for 7 min, the principal polypeptide that was precipitated by anti-insulin receptor antibodies had a molecular weight (Mr) of 180 000. This initial precursor was rapidly converted (t1/2 = 35 min) to a 200-kilodalton (kDa) polypeptide, designated the insulin proreceptor, by the apparent posttranslational addition of N-linked, high mannose core oligosaccharide units. Mature alpha (Mr 130 000) and beta (Mr 90 000) subunits were derived from sequences within the proreceptor by proteolytic cleavage and late processing steps, and these subunits appeared on the cell surface 2-3 h after synthesis of the 180-kDa precursor. The cation ionophore monensin was used in combination with metabolic labeling, affinity cross-linking, and external proteolysis to probe aspects of proreceptor function, transit, and the development of insulin sensitivity at the target cell surface. At 5 micrograms/mL, monensin potently inhibited the proteolytic cleavage step, and the 200-kDa polypeptide accumulated. Lower concentrations of the ionophore selectively blocked late processing steps in 3T3-L1 adipocytes so that apparently smaller alpha' (Mr 120 000) and beta' (Mr 85 000) subunits were produced. Proreceptor and alpha' and beta' subunits were translocated to the cell surface, indicating that the signal for intracellular transit occurs in the 200-kDa polypeptide and is independent of the posttranslational proteolysis and late processing steps. The alpha' subunit bound insulin both at the surface of intact cells and after solubilization with Triton X-100; the beta' subunit was phosphorylated in an insulin-stimulated manner. The detergent-solubilized 200-kDa proreceptor also exhibited both functional properties. However, the proreceptor that was transported to and exposed on the cell surface was incapable of binding insulin in intact adipocytes. Thus, late processing is not essential for the expression of functions associated with mature alpha and beta subunits. In contrast, it appears that the proteolytic generation of subunits is required for the correct orientation of the hormone binding site in the plasma membrane bilayer and the development of insulin responsiveness in 3T3-L1 adipocytes.

show abstract

Photoaffinity labeling of insulin receptor with an insulin analog selectively modified at the amino terminal of the B chain

Cited by 49 publications

References 43 publications

Latent insulin receptors and possible receptor precursors in 3T3-L1 adipocytes.

Latent insulin receptors and possible receptor precursors in 3T3-L1 adipocytes.

Differential sensitivity of the insulin-receptor kinase to thiol and oxidizing agents in the absence and presence of insulin

Biogenesis, transit, and functional properties of the insulin proreceptor and modified insulin receptors in 3T3-L1 adipocytes

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