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Protein kinase activity of the insulin receptor.

ABSTRACT: 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)

SUBMITTER: Gammeltoft S

PROVIDER: S-EPMC1146640 | biostudies-other | 1986 Apr

REPOSITORIES: biostudies-other

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Project description:Two systems in vitro are described that show insulin-stimulated phosphorylation of the insulin receptor on serine residues. In the first system, insulin receptor was purified partially from Fao rat hepatoma cells by direct solubilization of the cells in Triton X-100 and chromatography on wheat-germ-agglutinin-agarose. Phosphorylation of these preparations with [gamma-32P]ATP in the presence or absence of insulin resulted in 32P incorporation exclusively into phosphotyrosine residues. Serine kinase activity towards the insulin receptor was reconstituted by adding extracts of Fao cells. Prior exposure of the cells to insulin stimulated serine kinase activity towards the insulin receptor in extracts 7.2-fold. A receptor serine kinase activity enhanced by treatment of cells with cyclic AMP analogues was also retained in the reconstituted system. In the second system, insulin receptor and insulin-sensitive serine kinase activity towards the insulin receptor were co-purified from human placenta. The protocol involved preparation of membranes, before solubilization and chromatography on wheat-germ-agglutinin-agarose, by using gentle procedures designed not to disrupt a potentially labile association between the insulin receptor and the serine kinase. Serine kinase activity in these preparations towards the insulin receptor was stimulated up to 10-fold by insulin, and the stoicheiometry of serine phosphorylation was estimated to be approx 0.8 mol/mol of insulin receptor for phosphorylations performed in the presence of insulin. Thus a preparation of insulin receptor is described for the first time that is phosphorylated to high stoicheiometry on serine in an insulin-dependent manner. Conditions that facilitate recovery and assay of serine kinase activity are defined and discussed. These systems provide a basis for characterizing the nature of the insulin-sensitive serine kinase that phosphorylates the insulin receptor, and defining its role in insulin action and control of receptor function.

Project description:Hyperaldosteronism alters cardiac function, inducing adverse left ventricle (LV) remodeling either via increased fibrosis deposition, mitochondrial dysfunction, or both. These harmful effects are due, at least in part, to the activation of the G protein-coupled receptor kinase 2 (GRK2). In this context, we have previously reported that this kinase dysregulates both β-adrenergic receptor (βAR) and insulin (Ins) signaling. Yet, whether aldosterone modulates cardiac Ins sensitivity and βAR function remains untested. Nor is it clear whether GRK2 has a role in this modulation, downstream of aldosterone. Here, we show in vitro, in 3T3 cells, that aldosterone impaired insulin signaling, increasing the negative phosphorylation of insulin receptor substrate 1 (ser307pIRS1) and reducing the activity of Akt. Similarly, aldosterone prevented the activation of extracellular signal-regulated kinase (ERK) and the production of cyclic adenosine 3',5'-monophosphate (cAMP) in response to the β1/β2AR agonist, isoproterenol. Of note, all of these effects were sizably reduced in the presence of GRK2-inhibitor CMPD101. Next, in wild-type (WT) mice undergoing chronic infusion of aldosterone, we observed a marked GRK2 upregulation that was paralleled by a substantial β1AR downregulation and augmented ser307pIRS1 levels. Importantly, in keeping with the current in vitro data, we found that aldosterone effects were wholly abolished in cardiac-specific GRK2-knockout mice. Finally, in WT mice that underwent 4-week myocardial infarction (MI), we observed a substantial deterioration of cardiac function and increased LV dilation and fibrosis deposition. At the molecular level, these effects were associated with a significant upregulation of cardiac GRK2 protein expression, along with a marked β1AR downregulation and increased ser307pIRS1 levels. Treating MI mice with spironolactone prevented adverse aldosterone effects, blocking GRK2 upregulation, and thus leading to a marked reduction in cardiac ser307pIRS1 levels while rescuing β1AR expression. Our study reveals that GRK2 activity is a critical player downstream of the aldosterone signaling pathway; therefore, inhibiting this kinase is an attractive strategy to prevent the cardiac structural disarray and dysfunction that accompany any clinical condition accompanied by hyperaldosteronism.

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Protein kinase activity of the insulin receptor.

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