Characterization of insulin receptor subunits in brain and other tissues by photoaffinity labeling

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

doi:10.1016/0006-291x(80)91366-2

Cited by 111 publications

(34 citation statements)

References 15 publications

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“…The a-subunit of the glioblastoma receptor moved with an M -~140,000, while that of the normal brain had an Mr -18,000 (Fig. 4), similar to that previously reported with the whole rat brain as well as several regions from the rat brain (9)(10)(11)(12). In addition, the electrophoretic mobility of the rat brain insulin receptor subunits was not changed by neuraminidase treatment (10,11).…”

Section: Insulin-stimulated Glucose Uptakesupporting

confidence: 85%

“…The finding of identical insulin binding activity per unit of protein of the normal and malignant brain tissue despite the marked difference in both the structure of the binding (a) subunit ofthe insulin receptor deserves a comment. It has been assumed that the lower apparent molecular weight ofthe brain receptor's a-subunit is due to its decreased or absent neuraminidase-sensitive sialic acid content (10)(11)(12). In rat adipocytes removal of sialic acid preserves insulin binding but impairs insulin action (32,33).…”

Section: Insulin-stimulated Glucose Uptakementioning

confidence: 99%

“…130,000-135,000 for the peripheral receptor) (9)(10)(11)(12). In addition, neuraminidase treatment, removing sialic acid residues, failed to alter the electrophoretic properties of the a-subunit of the brain receptor while significantly decreasing the apparent molecular weight of the a-subunit of the peripheral receptors (10, 1 1), suggesting a difference of glycosylation of the two types of insulin receptors.…”

Section: Introductionmentioning

confidence: 99%

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Insulin receptor of human cerebral gliomas. Structure and function.

et al. 1986

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The insulin receptor from human brain tumors of glial origin was examined for the first time using intact cells (from an established cultured human glioblastoma cell line) and partially purified solubilized membranes (from cultured cells and freshly isolated human brain tumors). The structure of the glial insulin receptor subunits was assessed by affinity cross-linking of 125I-insulin with the a-subunit of the receptor, neuraminidase treatment of the cross-linked receptor, behavior of the receptor on lectin columns, and electrophoretic mobility of the phosphorylated ,8-subunit. The functions of the insulin receptor were examined by measuring specific "SI-insulin binding (receptor concentration, affinity, specificity, pH-, time-, and temperature dependence), insulin-induced down-regulation of the receptor, insulin-stimulated autophosphorylation of the 13-subunit, and phosphorylation of exogenous substrates as well as insulin-stimulated glucose uptake in glioblastoma cells. All of these properties were typical for the insulin receptor from target tissues for insulin action. The insulin receptor of the normal human brain showed the altered electrophoretic mobility and lack of neuraminidase sensitivity of its a-subunit previously reported for the rat brain receptor. There was no difference, however, in the functions of the receptor subunits (binding, phosphorylation) from the normal brain tissue and the eight human gliomal tumors. Since the glial elements compose a majority of the brain cells, the "normal" structure and function of their insulin receptor might provide a key to understanding the role of insulin in the carbohydrate metabolism of the human central nervous system.

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Section: Insulin-stimulated Glucose Uptakesupporting

confidence: 85%

Section: Insulin-stimulated Glucose Uptakementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Insulin receptor of human cerebral gliomas. Structure and function.

et al. 1986

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“…Sa) whereas that of the fl-subunit (Mr -94000) is similar (Fig. Sb) (Yip et al, 1980;Heidenreich et al, 1983;Gammeltoft et al, 1984a;Rees-Jones et al, 1984). The binding properties show striking differences in comparison with other cell types, because negatively co-operative interactions are absent (Gammeltoft et al, 1984b;Van Schravendijk et al, 1984) and the insulin specificity is different (Gammeltoft et al, 1984b(Gammeltoft et al, , 1985b.…”

Section: Heterogeneity Of Insulin Receptorsmentioning

confidence: 72%

Protein kinase activity of the insulin receptor

Gammeltoft¹,

Obberghen²

1986

Biochemical Journal

139

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The insulin receptor is an integral membrane glycoprotein (Mr approximately 300,000) composed of two alpha-subunits (Mr approximately 130,000) and two beta-subunits (Mr approximately 95,000) linked by disulphide bonds. This oligomeric structure divides the receptor into two functional domains such that alpha-subunits bind insulin and beta-subunits possess tyrosine kinase activity. The amino acid sequence deduced from cDNA of the single polypeptide chain precursor of human placental insulin receptor revealed that alpha- and beta-subunits consist of 735 and 620 residues, respectively. The alpha-subunit is hydrophilic, disulphide-bonded, glycosylated and probably extracellular. The beta-subunit consists of a short extracellular region which links the alpha-subunit through disulphide bridges, a hydrophobic transmembrane region and a longer cytoplasmic region which is structurally homologous with other tyrosine kinases like the src oncogene product and EGF receptor kinases. The cellular function of insulin receptors is dual: transmembrane signalling and endocytosis of hormone. The binding of insulin to its receptor on the cell membrane induces transfer of signal from extracellular to cytoplasmic receptor domains leading to activation of cell metabolism and growth. In addition, hormone-receptor complexes are internalized leading to intracellular proteolysis of insulin, whereas receptors are recycled to the membrane. These phenomena are kinetically well-characterized, but their molecular mechanisms remain obscure. Insulin receptor in different tissues and animal species are homologous in their structure and function, but show also significant differences regarding size of alpha-subunits, binding kinetics, insulin specificity and receptor-mediated degradation. We suggest that this heterogeneity of receptors may be linked to the diversity in insulin effects on metabolism and growth in various cell types. The purified insulin receptor phosphorylates its own beta-subunit and exogenous protein and peptide substrates on tyrosine residues, a reaction which is insulin-sensitive, Mn2+-dependent and specific for ATP. Tyrosine phosphorylation of the beta-subunit activates receptor kinase activity, and dephosphorylation with alkaline phosphatase deactivates the kinase. In intact cells or impure receptor preparations, a serine kinase is also activated by insulin. The cellular role of two kinase activities associated with the insulin receptor is not known, but we propose that the tyrosine- and serine-specific kinases mediate insulin actions on metabolism and growth either through dual-signalling or sequential pathways.(ABSTRACT TRUNCATED AT 400 WORDS)

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“…The main reason for this is the fact that the small amounts of adipose tissue that can be obtairred from patients for experimental studies are usually insufficient for detailed experimental characterizations using routine biochemical procedures. The technique of photoaffinity labeling of insulin receptors (16,18,(24)(25)(26) Photoreactive insulin derivative. The preparation and characterization of the photosensitive insulin analogue used in this study, B2(2-nitro-4-azidophenylacetyl)des-PheB-insulin (NAPA-DP-insulin), have been described previously (27,28).…”

mentioning

confidence: 99%

Insulin receptors in isolated human adipocytes. Characterization by photoaffinity labeling and evidence for internalization and cellular processing.

Berhanu¹,

Kolterman²,

Baron³

et al. 1983

J. Clin. Invest.

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A B S T R A C T We photolabeled and characterized insulin receptors in isolated adipocytes from normal human subjects and then studied the cellular fate of the labeled insulin-receptor complexes at physiologic temperatures. The biologically active photosensitive insulin derivative, B2(2-nitro-4-azidophenylacetyl)des-PheBl_ insulin (NAPA-DP-insulin) was used to photoaffinity label the insulin receptors, and the specifically labeled cellular proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography.At saturating concentrations, the binding of 12511 NAPA-DP-insulin to the isolated adipocytes at 16'C was rapid (half-maximal in -1 min and maximal in '10 min) and -25% of the specifically bound ligand was covalently linked to the cells by a 3-min exposure to long-wave (366 nm) ultraviolet light. Analysis disulfide bonds with dithiothreitol, all three unreduced forms of the insulin receptor were converted into a major labeled Mr-125,000 band and a less intensely labeled M,-90,000 band. The labeling of the Mr-125,000 receptor subunit was saturable and native porcine insulin effectively inhibited (half-maximal inhibition at 12 ng/ml) the photolabeling of this binding subunit by NAPA-DP insulin.When intact adipocytes photolabeled at 16°C (a temperature that inhibits endocytosis) were immediately trypsinized, all of the labeled receptor bands were converted into small molecular weight tryptic fragments, indicating that at 16°C all of the labeled insulinreceptor complexes remained on the cell surface. However, when the photolabeled cells were further incubated at 370C and then trypsinized, a proportion of the labeled receptors became trypsin insensitive, indicating that this fraction has been translocated to the cell interior and thus was inaccessible to the trypsin in the incubation medium. The intracellular translocation of the labeled receptors was observed within 2 min, became half-maximal by 10 min, and maximal by '-30 min of incubation at 370C.

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Characterization of insulin receptor subunits in brain and other tissues by photoaffinity labeling

Cited by 111 publications

References 15 publications

Insulin receptor of human cerebral gliomas. Structure and function.

Insulin receptor of human cerebral gliomas. Structure and function.

Protein kinase activity of the insulin receptor

Insulin receptors in isolated human adipocytes. Characterization by photoaffinity labeling and evidence for internalization and cellular processing.

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