An antipeptide antibody that specifically inhibits insulin receptor autophosphorylation and protein kinase activity.

Herrera, Román; Petruzzelli, Lilli; Thomas, Nancy E.; Bramson, H. Neal; Kaiser, E. T.; Rosen, Ora M.

doi:10.1073/pnas.82.23.7899

Cited by 70 publications

(29 citation statements)

References 44 publications

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“…[3]). [y-32P]ATP (3000 Ci/mmol) was from New England Nuclear Corp. Antipeptide antibodies are detailed in [4] and the monoclonal antibody in [5]. Sources of other materials are given in [I].…”

Section: Introduction 2 Materials and Methodsmentioning

confidence: 99%

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Polylysine activates and alters the divalent cation requirements of the insulin receptor protein tyrosine kinase

Rosen

Lebwohl

1988

FEBS Letters

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Protamine and poly(Lys) activate the protein tyrosine kinase of both the human placental insulin receptor and its purified recombinant cytoplasmic domain. Spermidine, poly(Arg) (average molecular mass 15 kDa), poly(Glu), Arg or Lys are not effective. Activation is stable, reversible, and optimal when the enzyme is preincubated with activator, divalent cation and ATP prior to the addition of exogenous protein substrates. The most striking feature of the activation is that it results in 2&30-fold stimulation of the kinase in the presence of 0.2-0.4 mM MnZ+ and induces equivalent activity in the presence of MgZ + alone (0.44.0 mM). The activated protein tyrosine kinase has a specific activity (0.25-0.5 pmol/mg protein) that approaches that of well characterized protein serine kinases.

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Section: Introduction 2 Materials and Methodsmentioning

confidence: 99%

“…Quantitation was achieved by cutting out phosphoproteins after PAGE and counting them directly. Immunoprecipitation was performed as in [4]. Alkali treatment of polyacrylamide gels is outlined in [7] and phosphoamino acid analysis in (61.…”

Section: Protein Kinase Assaysmentioning

confidence: 99%

Polylysine activates and alters the divalent cation requirements of the insulin receptor protein tyrosine kinase

Rosen

Lebwohl

1988

FEBS Letters

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“…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 genome (18) and to hybridize to a single chromosomal band by in situ analysis (unpublished results). The amino acid sequence (1142 to 1152) of the human proreceptor was completely conserved in the cloned D. melanogaster genomic fragment, predicting that an antipeptide antibody (AbP2), elicited to that peptide in the human sequence (9), would be able to immunoprecipitate the ,B subunit of the DIRH. In agreement with that prediction, a * Corresponding author.…”

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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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“…To test whether the oocytes expressed the human insulin receptor in a functionally active state, we evaluated the protein kinase activity of the human receptor by studying insulin-dependent autophosphorylation after immunoprecipitation with human insulin receptor-specific anti-peptide antibody AbP5 (16). As expected, insulin stimulated the phosphorylation of the subunit of the wild-type human receptor expressed in X. raevis (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The peptide was coupled to Affi-gel 10 (Bio-Rad Laboratories), emulsified with complete Freund adjuvant, and injected into rabbits; this procedure was followed by two boosts in incomplete adjuvant. The (16,40).…”

mentioning

confidence: 99%

Reconstitution of an insulin signaling pathway in Xenopus laevis oocytes: coexpression of a mammalian insulin receptor and three different mammalian hexose transporters.

Vera

Rosen

1990

Mol. Cell. Biol.

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We report the functional expression of the mammalian muscle-adipocyte insulin-sensitive hexose transporter in Xenopus laevis oocytes. Oocytes microinjected with RNA synthesized in vitro showed enhanced hexose transport activity compared with uninjected controls. However, like the endogenous oocyte hexose transporter, activity was stimulated only twofold by 1 ,uM insulin. X. laevis oocytes injected with in vitro-synthesized RNA encoding the human insulin proreceptor expressed a functionally active insulin receptor that enhanced the insulin sensitivity of injected oocytes. This increase was not observed in oocytes expressing a mutant insulin receptor that lacked protein tyrosine kinase activity. In the presence of the coexpressed human insulin receptor, insulin induced a two-to threefold increase in hexose transport. The muscle-, brain-, and liver-type hexose carriers normally expressed in tissues with different responses to insulin exhibited the same insulin sensitivity when expressed in oocytes. This was observed whether or not the insulin signal was transduced through a coexpressed human insulin receptor or the endogenous oocyte insulin-like growth factor I receptor. We conclude that the expressed human insulin receptor is able to couple efficiently with preexisting postreceptor regulatory pathways in oocytes and that the regulation of hexose transport in these cells can be mediated through the combined actions of the expressed human insulin receptor and the endogenous oocyte insulin-like growth factor I receptor.Transport of glucose in most mammalian cell types occurs by carrier-mediated facilitated diffusion down a concentration gradient (41,48). Recent studies indicate that this facilitated hexose uptake is mediated by a family of structurally related integral membrane proteins. Thus, a number of putative hexose transport proteins that have marked differences in their patterns of tissue-specific expression have been cloned. These include a cDNA clone encoding a protein expressed largely, but not exclusively, in HepG2 hepatoma cells and the brain (3,29); one expressed in the liver, the pancreatic islets, and the kidneys (13, 44); one expressed in fetal skeletal muscle (24); and another expressed almost exclusively in fat cells and in adult skeletal and cardiac muscles (2,5,12,18,21). Functional studies indicate that at least three of these proteins (HepG2 brain, liver, and adult skeletal muscle) are, indeed, hexose carriers (2,37,44,45).Comparison of the deduced amino acid sequences of the cloned transporters indicates homology in primary structure (2,3,5,12,13,18,21,24,29,44). In addition, the hydropathy plots of the different transporters are virtually superimposable and compatible with the proposed model for the orientation of the HepG2 glucose transporter in the cell membrane (29, 44). Sequence similarity is higher in the putative hydrophobic membrane-spanning segments than in the extramembranous more hydrophilic domains. Thus, each transporter differs in the length and/or the amino acid sequence of the a...

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An antipeptide antibody that specifically inhibits insulin receptor autophosphorylation and protein kinase activity.

Cited by 70 publications

References 44 publications

Polylysine activates and alters the divalent cation requirements of the insulin receptor protein tyrosine kinase

Polylysine activates and alters the divalent cation requirements of the insulin receptor protein tyrosine kinase

Structure and ligand specificity of the Drosophila melanogaster insulin receptor.

Reconstitution of an insulin signaling pathway in Xenopus laevis oocytes: coexpression of a mammalian insulin receptor and three different mammalian hexose transporters.

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