Richard Roth - US grants

The response of endocrine-regulated cells depends on both the binding and degradation of regulatory hormones. although the degradation of insulin has been extensively studied with whole animals, perfused organs, suspensions of cells and purified enzymes, it is still not clear how insulin is degraded. One enzyme present in the microsomes of many cells, glutathione-insulin transhydrogenase (GIT), splits insulin into its A and B chains. Another enzyme present in the cytosol of many cells, insulin degrading enzyme (IDE), cleaves several peptide bonds in the insulin backbone. However, before insulin can be degraded by either of these enzymes it must first be bound by another molecule, the insulin receptor, and taken up by the cell. In the proposed research, the functional, structural and genetic interactions of these three molecules will be studied. First, the insulin degrading enzyme will be purified from a cell line of human lymphocytes. This purified enzyme will be characterized and both monoclonal and polyclonal antibodies will be produced against it. Second, mixtures of the purified IDE, GIT and insulin receptor will be tested for their ability to degrade insulin. In addition, the possibility that the structure of the insulin receptor is effected by these enzymes will be examined. Third, the structural linkage of plasma membrane IDE and the insulin receptor will be studied by capping each molecule on the surface of the cell and determining whether the other molecule is present in the same cap. And finally, a collection of hybrid cells will be constructed containing one or several human chronosomes on the background of a mouse fibroblast cell line. The presence of human IDE, GIT and the insulin receptor in these cells will be correlated with the presence of various human chromosomes to determine which chromosomes contain the genes encoding for these molecules.

The first event in the response of target cells to insulin is the binding of the hormone to its specific receptor on the surface of cells. This binding activates an intrinsic enzymatic activity of the receptor, it catalyzes the transfer of the terminal phosphate of ATP to tyrosine residues of specific proteins. This enzymatic activity appears to be required for insulin to stimulate many of its biological responses and recently several endogenous substrates have been identified. Questions still remain however on how the tyrosine phosphorylation of these specific proteins results in subsequent biological responses. The goal of the present studies is to further elucidate this process. A better understanding of the physiologically important substrates of the insulin receptor kinase may allow one to design drugs which directly activate these proteins in cells whose receptors are impaired, a condition which may exist in non-insulin-dependent diabetics. To this end, several approaches are being pursued. First, one of the proteins that rapidly associates with the substrate called IRS-1 is itself an enzyme, a phosphatidylinositol kinase. This enzyme binds to the tyrosine phosphorylated IRS-1, its activity is stimulated and it generates two novel phospholipids, phosphatidylinositol 3,4-P and 3,4,5-P. The role of these lipids in the cell is presently not known. We have identified a protein which binds to these lipids and we propose to purify this protein, obtain a cDNA clone encoding the protein and generate antibodies to this protein to study the role of this protein in insulin signaling. In addition, we are studying another substrate of the insulin receptor kinase. By utilizing a monoclonal antibody to a 60 kDa protein we have shown that it becomes rapidly tyrosine phosphorylated in response to insulin and that it can be directly phosphorylated in vitro with the insulin receptor. This protein appears to become associated with a GTPase activating protein of ras (called GAP) but it differs from a previously observed 62 kDa tyrosine phosphorylated GAP-associated protein in SRC transformed cells. We propose to further study this protein as well to attempt to elucidate its role in mediating specific biological responses. In addition to studies of these substrates, we are attempting to develop systems for evaluating the roles of particular substrates in mediating specific responses. To this end, we have begun studies of the Drosophila insulin receptor. This receptor diverges from the human insulin receptor to a greater degree than any of the other known members of the insulin receptor family. A chimeric receptor containing the cytoplasmic domain of the Drosophila receptor with the extracellular domain of the human receptor exhibits an insulin-activated tyrosine kinase activity. Cells overexpressing this receptor will be compared to cells overexpressing the human receptor to see if one can correlate the phosphorylation of specific substrates with particular biological responses. We also plan to express this chimeric receptor and the human receptor in a line of Drosophila cells to see if any substrates are missing in this system. If so, then cDNAs encoding particular substrates can be expressed in this system to attempt to complement the receptors expressed in these cells.

The insulin receptor serves to focus the hormone on particular target tissues as well as to initiate the responses of these cells to the hormone. Extensive studies of the insulin receptor by many investigators have told us a great deal about this molecule, including its complete amino acid sequence as well as the role of specific residues in receptor function. In addition to binding insulin, the receptor has been shown to have an intrinsic tyrosine specific kinase activity. This activity is increased after the receptor binds insulin and appears to be critical for insulin to stimulate various biological responses. A reversible decrease in the receptor's intrinsic tyrosine kinase activity has been observed in cells from patients with non-insulin dependent diabetes, possibly contributing to the insulin resistance observed in these patients. In addition, a similar phenomena can be induced in cells in culture by stimulating the serine phosphorylation of the receptor with phorbol esters. In vitro, the receptor can be directly phosphorylated by a specific serine/threonine protein kinase, called protein kinase C. This phosphorylation also appears to decrease the intrinsic tyrosine kinase activity of the receptor without affecting the ability of the receptor to bind insulin. To further study the mechanisms of this regulation, we have identified one of the isozymes of protein kinase C for its ability to phosphorylate the receptor in vitro and have isolated stably transfected cell lines which overexpress the insulin receptor and this protein kinase C. We have found that the ability of insulin to activate the receptor kinase in these cells is inhibited ~ 70%. We therefore propose to further study this process by: 1) Selecting stable transfectants of this protein kinase C and various mutated insulin receptors to identify the specific sites regulated by this enzyme; 2) Test the affect of overexpression of kinase C on the ability of insulin and its receptor to be internalized and on insulin to induce various biological responses; 3) Test the specificity of this affect by isolating cell lines overexpressing the receptor and other protein kinase C isozymes and examining the same processes in these cells; and 4) Test whether insulin activates any of the protein kinase C isozymes. After responding to insulin, a cell must terminate its response. One mechanisms for accomplishing this is to degrade the hormone. One enzyme, called insulin-degrading enzyme, has been implicated in this process. We have produced monoclonal antibodies to this enzyme, and isolated a cDNA clone which codes for this molecule. We now propose to: 1) Identify residues in the active site of this protease by site-directed mutagenesis; 2) Increase and/or decrease the levels of this enzyme in cells to further test the role of this enzyme in terminating the response to insulin; and 3) Identify the regions of the protease involved in recognizing insulin by generating anti-peptide antibodies to specific sequences of the molecule. A further understanding of this enzyme may allow the design of specific inhibitors of insulin degradation.

In 1989, Shier and Watt identified a gene whose sequence predicted that it encodes a new receptor that is in the family of insulin and insulin-like growth factor I receptors. Hence, it was given the name insulin receptor-related receptor (or IRR). However, the tissues that express this receptor, the ligand which binds to it and the physiological function of this receptor are unknown. The goal of the present grant is to learn more about this novel receptor. In preliminary studies we have identified the mRNA for this receptor in rat and human kidney (both adult and fetal) and in dog gastric parietal cells. One of the objectives of the proposed studies will be to further define the specific cells expressing this receptor. To this end, specific monoclonal and polyclonal antibodies will be generated. These antibodies and cDNA probes will be used to test for the presence of the receptor protein and mRNA, respectively, in both isolated primary kidney cells and cell lines. In addition, if some of the antibodies are found to be agonists, they will be used to explore the function of this receptor. A chimera composed of portions of the extracellular domain of the insulin receptor-related receptor on the backbone of the insulin receptor has also been constructed. This chimera did not bind insulin, insulin-like growth factor I or II, or relaxin, suggesting that IRR is not a receptor for these ligands. This and other chimeras will therefore be used to screen for the presence of a ligand for the insulin receptor-related receptor by looking for samples that stimulate the intrinsic kinase activity of the chimera. In addition, we will overproduce large amounts of the extracellular domain of IRR for use in purifying the ligand for IRR. Identification of the ligand for IRR as well as the tissue distribution of this receptor should aid in the determination of the physiological function of this molecule. From its location in the kidney and stomach parietal cells, this receptor could have a role in the regulation of blood pressure, acid production, etc. In addition, since this receptor has an intrinsic tyrosine kinase activity, it is possible that it is involved in growth control and may be involved in cancerous lesions of stomach and kidney.

The insulin receptor serves to focus the hormone on particular target tissues as well as to initiate the responses of these cells to the hormone. Extensive studies of the insulin receptor by many investigators have told us a great deal about this molecule, including its complete amino acid sequence as well as the role of specific residues in receptor function. In addition to binding insulin, the receptor has been shown to have an intrinsic tyrosine specific kinase activity. This activity is increased after the receptor binds insulin and appears to be critical for insulin to stimulate various biological responses. A reversible decrease in the receptor's intrinsic tyrosine kinase activity has been observed in cells from patients with non-insulin dependent diabetes, possibly contributing to the insulin resistance observed in these patients. In addition, a similar phenomena can be induced in cells in culture by stimulating the serine phosphorylation of the receptor with phorbol esters. In vitro, the receptor can be directly phosphorylated by a specific serine/threonine protein kinase, called protein kinase C. This phosphorylation also appears to decrease the intrinsic tyrosine kinase activity of the receptor without affecting the ability of the receptor to bind insulin. To further study the mechanisms of this regulation, we have identified one of the isozymes of protein kinase C for its ability to phosphorylate the receptor in vitro and have isolated stably transfected cell lines which overexpress the insulin receptor and this protein kinase C. We have found that the ability of insulin to activate the receptor kinase in these cells is inhibited ~ 70%. We therefore propose to further study this process by: 1) Selecting stable transfectants of this protein kinase C and various mutated insulin receptors to identify the specific sites regulated by this enzyme; 2) Test the affect of overexpression of kinase C on the ability of insulin and its receptor to be internalized and on insulin to induce various biological responses; 3) Test the specificity of this affect by isolating cell lines overexpressing the receptor and other protein kinase C isozymes and examining the same processes in these cells; and 4) Test whether insulin activates any of the protein kinase C isozymes. After responding to insulin, a cell must terminate its response. One mechanisms for accomplishing this is to degrade the hormone. One enzyme, called insulin-degrading enzyme, has been implicated in this process. We have produced monoclonal antibodies to this enzyme, and isolated a cDNA clone which codes for this molecule. We now propose to: 1) Identify residues in the active site of this protease by site-directed mutagenesis; 2) Increase and/or decrease the levels of this enzyme in cells to further test the role of this enzyme in terminating the response to insulin; and 3) Identify the regions of the protease involved in recognizing insulin by generating anti-peptide antibodies to specific sequences of the molecule. A further understanding of this enzyme may allow the design of specific inhibitors of insulin degradation.

The insulin receptor (IR) serves to focus the hormone on particular target tissues as well as to initiate the response of these cells to the hormone via its intrinsic tyrosine kinase activity which is critical for various biological responses. A major question that still needs to be answered is how this IR kinase and subsequent signaling is regulated since patients with non-insulin dependent diabetes exhibit a reversible decrease in IR signalling, possibly contributing to their insulin resistance. One potential mechanism would be an increase in the ser/thr phosphorylation of the IR and/or subsequent molecules in the signaling cascade. During the prior grant period, extensive studies were focused on the regulation of IR signaling by the protein kinase C pathway. Activation of this pathway was shown to modulate insulin-stimulated signaling via an increase in the serine phosphorylation of a specific serine residue in one of the substrates (called IRS-1) of the insulin receptor kinase. In the next grant period we plan to further test the role of this residue in the ability of other agents to modulate IR signaling including via various counter regulatory hormones such as tumor necrosis factor and PDGF. In addition, we will attempt to identify various kinases capable of phosphorylating this residue including an insulin-stimulated kinase which appears to be able to readily phosphorylate this regulatory serine. Such serine phosphorylation of IRS-1 and other substrates of the IR tyrosine kinase may contribute to the insulin resistance observed in various models of NIDDM. After tyrosine phosphorylation, insulin-receptor substrates like IRS-1, 2 and 3 are bound and activate a lipid kinase called PI 3- kinase. This kinase produces phosphatidylinositol 3-phosphates which can activate various ser/thr kinases including one called Akt/PKB as well as certain isoforms of the protein kinase C family of kinases. During the last grant period we have shown that the enzymatic activity of Akt/PKB is greatly activated in insulin treated cells. In addition, we have shown that constitutively active forms of the enzyme can mediate a number of insulin-like biological responses including the stimulation of glucose uptake, GLUT4 translocation, activation of the 70 kDa S6 kinase and stimulation of mTOR enzymatic activity. In the present proposal we plan to further explore the mechanism whereby Akt can induce various biological responses. In particular, we plan to identify potential substrates of the Akt kinase as well as to investigate further the mechanism whereby Akt is capable of eliciting the above biological responses as well as several additional biological responses. Finally, we plan to explore the role of Akt in mediating the negative regulation of the IR signaling pathway.

DESCRIPTION (provided by applicant): Protein kinase C (PKC) isozymes play a key role in ischemic heart disease and in hypertrophic heart failure. Conflicting data for the role of individual PKC isozymes in modulating these functions have been reported, mainly because these studies relied on non-selective pharmacological tools. Our research focuses on identifying PKC isozyme-selective inhibitors and activators and we applied them to determine the role of individual isozymes in normal and diseased heart. We showed that epsilonPKC activation or deltaPKC inhibition provides cardioprotection from ischemia in vitro, ex vivo and recently in vivo. We find that the peptides can be effectively delivered into the heart to produce these effects and that cardioprotection by a deltaPKC inhibitor delivered only during reperfusion can be achieved when using as little as 250ng of peptide per kg. Here, we plan to examine the mechanism by which inhibition of deltaPKC protects from reperfusion injury. Several PKC isozymes (notably, beta, delta, and epsilonPKCs) have been suggested to modulate cardiac hypertrophy and transition to heart failure. We recently found that our isozyme-specific peptide regulators of PKC remain effective when delivered in vivo daily, for 10 days. Therefore, for the first time, we can determine whether these PKC-regulating peptides can prevent or reduce these pathological cardiac changes in two animal models. Together, these studies will identify the PKC isozymes and the molecular pathways that should be targeted for the development of new therapeutics for human cardiac ischemia and heart failure.