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

Salzman, Alan; Wan, Catherine F.; Rubin, Charles S.

doi:10.1021/bi00321a043

Cited by 25 publications

(5 citation statements)

References 60 publications

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“…Hedo & J. M. Podskalny, unpublished work). Other laboratories have reported similar-sized precursors of the insulin receptor in 3T3-L1 adipocytes (Deutsch et al, 1983;Salzman et al, 1984;Ronnet et al, 1984) and IM-9 lymphocytes (Jacobs et al, 1983). However, the present report demonstrates the existence of the .…”

Section: Discussioncontrasting

confidence: 43%

Biosynthesis of the insulin receptor in rat adipose cells. Intracellular processing of the Mr-190 000 pro-receptor

Hedo

Simpson²

1985

Biochemical Journal

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We investigated the biosynthesis of the insulin receptor in primary cultures of isolated rat adipose cells. Cells were pulse-chase-labelled with [3H]mannose, and at intervals samples were homogenized. Three subcellular membrane fractions were prepared by differential centrifugation: high-density microsomal (endoplasmic-reticulum-enriched), low-density microsomal (Golgi-enriched), and plasma membranes. After detergent solubilization, the insulin receptors were immunoprecipitated with anti-receptor antibodies and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and autoradiography. After a 30 min pulse-label [3H]mannose first appeared in a band of Mr 190 000. More than 80% of the Mr-190 000 component was recovered in the microsomal fractions. Its intensity reached a maximum at 1 h in the high-density microsomal fraction and at 2 h in the low-density microsomal fraction, and thereafter declined rapidly (t 1/2 approx. 3 h) in both fractions. In the plasma-membrane fraction, the radioactivity in the major receptor subunits, of Mr 135 000 (alpha) and 95 000 (beta), rose steadily during the chase and reached a maximum at 6 h. The Mr-190 000 precursor could also be detected in the high-density microsomal fraction by affinity cross-linking to 125I-insulin. In the presence of monensin, a cationic ionophore that interferes with intracellular transport within the Golgi complex, the processing of the Mr-190 000 precursor into the alpha and beta subunits was completely inhibited. Our results suggest that the Mr-190 000 pro-receptor originates in the endoplasmic reticulum and is subsequently transferred to the Golgi complex. Maturation of the pro-receptor does not seem to be necessary for the expression of the insulin-binding site. Processing of the precursor into the mature receptor subunits appears to occur during the transfer of the pro-receptor from the Golgi complex to the plasma membrane.

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Section: Discussioncontrasting

confidence: 43%

Biosynthesis of the insulin receptor in rat adipose cells. Intracellular processing of the Mr-190 000 pro-receptor

Hedo

Simpson²

1985

Biochemical Journal

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“…This suggests that it may be an incompletely processed proreceptor molecule that does not exhibit the proper conformation for ligand binding or AbP3 recognition. Certainly, incompletely or improperly processed mammalian insulin proreceptors have been reported not to bind insulin (23). The difference, however, is that in D. melanogaster the 170-kDa protein appears to be largely or completely incorporated into the receptor oligomer and to be disposed with the rest of the complex on the cell surface.…”

Section: Resultsmentioning

confidence: 99%

Structure and ligand specificity of the Drosophila melanogaster insulin receptor.

Fernandez-Almonacid¹,

Rosen²

1987

Mol. Cell. Biol.

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The insulin-binding and protein tyrosine kinase subunits of the Drosophila melanogaster insulin receptor homolog have been identified and characterized by using antipeptide antibodies elicited to the deduced amino acid sequence of the a and ( subunits of the human insulin receptor. In D. melanogaster embryos and cell lines, the insulin receptor contains insulin-binding a subunits of 110 or 120 kilodaltons (kDa), a 95-kDa 18 subunit that is phosphorylated on tyrosine in response to insulin in intact cells and in vitro, and a 170-kDa protein that may be an incompletely processed receptor. All of the components are synthesized from a proreceptor, joined by disulfide bonds, and exposed on the cell surface. The Di subunit is recognized by an antipeptide antibody elicited to amino acids 1142 to 1162 of the human insulin proreceptor, and the a subunit is recognized by an antipeptide antibody elicited to amino acids 702 to 723 of the human proreceptor. Of the polypeptide ligands tested, only insulin reacts with the D. melanogaster receptor. Insulinlike growth factors type I and II, epidermal growth factor, and the silkworm insulinlike prothoracicotropic hormone are unable to stimulate autophosphorylation. Thus despite the evolutionary divergence of vertebrates and invertebrates, the essential features of the structure and intrinsic functions of the insulin receptor have been remarkably conserved.The subunit structure of the prototypic mammalian insulin receptor, the human placental insulin receptor, has been well characterized, and its primary amino acid sequence has been deduced from cDNA cloning (5, 27). The processed receptor is a tetramer composed of two 135-kilodalton (kDa) a subunits that bind insulin and two 95-kDa 1 subunits that traverse the plasma membrane and, in their cytoplasmic domain, possess protein tyrosine kinase activity (5, 27). All four subunits are glycosylated and are linked to each other by disulfide bonds (12). In an effort to find a biological system susceptible to genetic analysis of the insulin receptor, we turned to Drosophila melanogaster. Insects are the only nonvertebrate organisms from which an insulinlike molecule with biological activity has been fully purified (25). It seemed likely, therefore, that D. melanogaster would possess a homolog of the mammalian insulin receptor, the Drosophila insulin receptor homolog (DIRH). Previous work in our laboratory has demonstrated the existence of a D. melanogaster membrane-associated glycoprotein, Mr 300,000 to 400,000, that binds bovine and porcine insulin with a kD of approximately 10 nM (17). An insulin-dependent protein tyrosine kinase activity which peaks during embryogenesis was detected, as were 100-and 120-kDa proteins that could be specifically cross-linked to 1251-insulin (19). Finally a genomic sequence encoding a protein homologous to the kinase domain of the human insulin receptor, as well as the human insulinlike growth factor type I (IGF-I) receptor (28), was cloned and shown to represent a single-copy gene in the D. melanogaster g...

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“…These intermediates are sensitive to endoglycosidase H and neuraminidase resistant. Salzman et al (44) and Herzberg et al (45) reported that the mature a-and /-subunits from 3T3-L1 adipocytes and IM-9 lymphocytes contain both endoglycosidase H-sensitive and -insensitive sugar side chains. The latter report also showed that endoglycosidase F could not remove all the sugar side chains from the /3-subunit but treatment with a chemical deglycosylating agent resulted in an additional decrease in molecular weight suggesting the presence of 0-linked side chains.…”

Section: Resultsmentioning

confidence: 99%

Diabetes-induced functional and structural changes in insulin receptors from rat skeletal muscle.

Burant¹,

Treutelaar²,

Buse³

1986

J. Clin. Invest.

114

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The effect of diabetes on the structure and function of insulin receptors was studied in rats 7 d after streptozotocin injection, using solubilized, partially purified receptors from rat hindlimb muscles. Diabetes increased the number of insulin receptors per gram of muscle 60-70% without apparent change in insulin binding affinity. Incubation of receptors at 40C with 1y-32PIATP and insulin resulted in dose-dependent autophosphorylation of the (i-subunit on tyrosine residues; receptors from diabetic rats showed decreased base-line phosphorylation, as well as a decrease in autophosphorylation at maximally stimulating insulin concentrations. These receptors also showed diminished exogenous substrate kinase activity using histone H2b and angiotensin II as phosphoacceptors. The electrophoretic mobility (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of a subpopulation of (i-subunits derived from diabetics was slightly decreased; differences in electrophoretic mobility between controland diabetic-derived (i-subunits were enhanced by generating fragments by partial Staphylococcus aureus V8 protease digestion. Endoglycosidase-H or neuraminidase treatment increased the electrophoretic mobility of (-subunits in both groups, but only neuraminidase appeared to decrease or abolish differences in electrophoretic mobility between controls and diabetics, suggesting that excess sialidation may account, in part, for the altered mobility of diabetic derived (i-subunits. All structural and functional alterations in insulin receptors were prevented by treating diabetic rats with insulin for 60 h. Peripheral insulin resistance associated with insulinopenic diabetes may be related to modifications in insulin receptor structure, resulting in impaired signal transmission.

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

Cited by 25 publications

References 60 publications

Biosynthesis of the insulin receptor in rat adipose cells. Intracellular processing of the Mr-190 000 pro-receptor

Biosynthesis of the insulin receptor in rat adipose cells. Intracellular processing of the Mr-190 000 pro-receptor

Structure and ligand specificity of the Drosophila melanogaster insulin receptor.

Diabetes-induced functional and structural changes in insulin receptors from rat skeletal muscle.

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