The Effect of Solubilization on the Properties of the Insulin Receptor of Human Placental Membranes*

Harrison, Len C.; Billington, Timothy; East, I.J.; Nichols, Robyn J.; Clark, Stella

doi:10.1210/endo-102-5-1485

Cited by 104 publications

(35 citation statements)

References 27 publications

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“…The insulin receptor characteristics of the human erythrocytes were shown to be similar to those of other tissue ceUs (l, 2,8,10,12,13,16,20,24). Parallel alterations of insulin receptors of cells of various body tissues and monocytes on one hand (40) and of monocytes and erythrocytes on the other hand (27,31,33,38,41,42) have led to the conclusion that erythrocytes thus can be considered äs representätive of insulin receptors on the cells of other body tissues.…”

Section: Introductionmentioning

confidence: 76%

“…The magnitude of this response depends on the hormone concentration, the receptor concentration and the affinity of the receptor, whereby alterations in any one of these can alter the biological response. Insulin receptors have been defined in human mononuclear cells (4)(5)(6)(7)(8) adipocytes (2,5), placental cells (9,10), cultured lymphocytes (l 1), hepatocytes (12), cultured fibroblasts (11), myocytes (13), gjanulocytes (14), reticulocytes (15) and erythrocytes (11,.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Insulin Binding to Erythrocytes from Pregnant, Postpartum, Follicular and Luteal States

Dwenger¹,

Mitzkat²,

Holle³

et al. 1982

CCLM

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By using a modified Scatchard analysis, statistically significant differences were observed between the receptor affinities of the groups A aiid D, B and D, A and C.The receptor affinities and concentrations were not significantly different between the follicular and the luteal phases. From the data, no inverse correlation between the plasma insulin concentration and receptor binding was seen, i.e. the phenömenon of downiegulation pf insulin receptor concentration with hyperinsulinaemia seemed not to apply to erythrocytes.The present results suggest that insulin binding to erythrocytes is mpdulated preferably or even exclusively by an alteration of receptor affinity and that short-term chariges in insulin binding to erythroeytes are not caused by an alterätion of receptor concentration. Vergleich der Bindung von Insulin an Erythrocyten während und nach Schwangerschaft und in der Proliferationsund der Sekretionsphase des Menstruations-Cyclus

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Section: Introductionmentioning

confidence: 76%

Section: Introductionmentioning

confidence: 99%

Insulin Binding to Erythrocytes from Pregnant, Postpartum, Follicular and Luteal States

Dwenger¹,

Mitzkat²,

Holle³

et al. 1982

CCLM

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“…In addition, formation of a high-affinity state or positive co-operativity was observed at low receptor occupancies (Gammeltoft et al, 1978;Donner & Corin, 1980;Marsh et al, 1984). The molecular basis of these affinity changes is unknown, but the fact that they are also present in solubilized receptor preparations (Harrison et al, 1978) may suggest that they result from conformational changes within a bivalent insulin receptor structure (Pang & Schafer, 1984;Czech, 1984).…”

Section: Functional Properties Of Insulin Receptormentioning

confidence: 99%

Protein kinase activity of the insulin receptor

Gammeltoft¹,

Obberghen²

1986

Biochemical Journal

138

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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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“…These results indicate that the insulin receptor of both liver and placenta has a subunit of molecular weight 135,000 that binds insulin and that the receptor may be composed of at least two different subunits that are linked together or greatly stabilized by disulfide bonds. The molecular size of insulin receptors solubilized from several tissues, including membranes from rat liver (1), rat adipocytes (1), human placenta (2,3), and turkey erythrocytes (4), has been determined by gel filtration. In each tissue the molecular weight has been estimated as being approximately 300,000.…”

mentioning

confidence: 99%

Insulin receptor: covalent labeling and identification of subunits.

Jacobs¹,

Hazum²,

Shechter³

et al. 1979

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

238

100

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Two methods were used to label insulin receptors covalently with 125I. In the first, an aryl azide derivative of insulin, 125I-labeled 4-azido-2-nitrophenyl-insulin, was synthesized and used to photolabel the binding region of the insulin receptor in rat liver membranes and human placenta membranes. In the second, insulin receptors were purified from rat liver membranes and labeled with 125I by use of chloramine-T; this method presumably has no specificity for the binding region of the receptor. The proteins labeled by both methods were anal zed by sodium dodecyl sulfate/poyacrylamide gel electrophoresis after or without reduction by dithiothreitol. The photoaffinity label specifically labeled a single band in both liver and placenta that had an apparent molecular weight of 135,000 after reduction. A band with similar mobility was present in the chloramine-T-labeled preparation, which also contained a second major band with an apparent molecular weight of 45,000. Without reduction, both methods resulted in a single labeled band with an apparent molecular weight of about 310,000. These results indicate that the insulin receptor of both liver and placenta has a subunit of molecular weight 135,000 that binds insulin and that the receptor may be composed of at least two different subunits that are linked together or greatly stabilized by disulfide bonds. The molecular size of insulin receptors solubilized from several tissues, including membranes from rat liver (1), rat adipocytes (1), human placenta (2, 3), and turkey erythrocytes (4), has been determined by gel filtration. In each tissue the molecular weight has been estimated as being approximately 300,000. This high molecular weight species of the receptor has been reported to dissociate in the presence of insulin into forms of lower molecular weight (3-7). Consistent with this, the major Coomassie blue-staining band seen in sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gels of a highly purified preparation of insulin receptors from liver membranes has an apparent molecular weight of 135,000 (8). A band of similar molecular weight has been specifically labeled in fat cells by using a photoaffinity label (9) or by covalently crosslinking 125I-labeled insulin to its receptor (10).In the present studies the photoaffinity label, 4-azido-2-nitrophenyl-insulin (ANP-insulin), was synthesized and used to label insulin receptors in membranes from rat liver and human placenta. The photoaffinity-labeled bands were compared with the bands present in the purified insulin receptor preparation by NaDodSO4/polyacrylamide gel electrophoresis. METHODSPurification of Insulin Receptor from Rat Liver. Liver membranes were prepared by differential centrifugation as described (11). Insulin receptors were solubilized with Triton X-100 and purified by sequential chromatography on DEAEcellulose, insulin-agarose, and concanavalin A-agarose by methods described previously (8).Iodination of Purified Insulin Receptor. Fifty microliters of 100 mM sodium phosphate buffer (pH 7.4), c...

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The Effect of Solubilization on the Properties of the Insulin Receptor of Human Placental Membranes*

Cited by 104 publications

References 27 publications

Insulin Binding to Erythrocytes from Pregnant, Postpartum, Follicular and Luteal States

Insulin Binding to Erythrocytes from Pregnant, Postpartum, Follicular and Luteal States

Protein kinase activity of the insulin receptor

Insulin receptor: covalent labeling and identification of subunits.

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