Neil Nathanson - US grants

The goal is to elucidate at the molecular level the factors involved in the development, function, and regulation of muscarinic acetylcholine receptors (mAChR) in the heart. We will study chick cardiac mAChR during embryonic development in vivo and in cardiac muscle cell culture. We will determine the molecular basis of the diminished physiological responsiveness of newly synthesized mAChR to confirm our hypothesis that the mAChR is synthesized in an immature form and is subsequently converted to a more physiologically active form. We have also obtained preliminary evidence by quantitative immunoblotting that at least 2 of the subunits of the GTP regulatory proteins (N Alpha and Beta) are increased greatly in amount in embryonic atria but not ventricles between days 10 and 15. It has been reported that an atrial-specific increase in mAChR number which is dependent on innervation also occurs at this time. This proposal will test the hypothesis that the mAChR and its GTP-binding effector proteins are coordinately regulated during embryonic development by innervation. We have obtained preliminary evidence that treatment of cultured cardiac cells with phorbol esters (which activate protein kinase C) prevents the mAChR-mediated increase in K+ permeability. We will test the hypothesis that short-term desensitization of the mAChR-induced negative chronotropic response is mediated by protein kinase C. We will use two different methods to isolate antibodies specific for the mAChR: by immunizations with purified mAChR preparations, and by immunization with several monoclonal antibodies specific for mAChR ligands to isolate anti-idiotype antibodies which recognize the ligand binding site on the mAChR. These antibodies will be used in our subsequent studies on mAChR regulation. This research should identify the factors involved in cardiac mAChR regulation, development, and function, and may aid in the understanding of a number of cardiac abnormalities.

The long-term goal of this proposal is to determine the molecular mechanisms involved in the regulation of expression of GTP-binding regulatory proteins (G-protein), the signal tranduction proteins necessary for neurotransmitters and hormone receptor action, in neural cells. This proposal uses a combination of biochemical, immunological, and molecular biological approaches to study the molecular mechanisms regulating G-protein expression. We have identified a novel form of Gs alpha mRNA in astrocytoma cells which is also found in rat and mouse brain, but not other tissues. This proposal will isolate cDNA and genomic clones to determine its origin and relationship to previously identified forms of Gs alpha. This proposal will also use expression vectors to determine the effects of overexpression of specific G-protein subunits on the synthesis, assembly, and function of the G-protein proteins in situ in intact cells. In addition, we have obtained preliminary evidence that treatment of rat PC12 pheochromocytoma cells with nerve growth factor increases the levels of G-protein subunits. This proposal will use antibody and cDNA probes to determine the mechanisms involved in the regulation of G-protein expression by nerve growth factor. The polypeptide Go alpha is expressed in particularly high levels in brain and neuronal cells but expressed at low levels or unexpressed in astrocytoma, glioma, and other non- neuronal neural cell types. This proposal will localize the regulatory elements responsible for the high level of expression of Go alpha in neuronal cells. This proposal should provide new insights into the regulation of the number and function of G- proteins in neural cells.

The obligatory role of guanine nucleotide regulatory proteins (G- proteins) in many transmembrane signalling events is now clearly established. While this has been elucidated most clearly for retinal phototransduction and for hormonal regulation of adenylate cyclase, similar mechanisms apparently are important for regulation of phospholipase C-catalyzed phosphoinositide hydrolysis, for neurotransmitter receptor-mediated modification of ion channel activity, for various secretory events, and for regulation of cell growth and differentiation. Although evidence for the involvement of a G-protein in most of these phenomena is strong, unambiguous identity of this protein has been established in only a few cases. The application of molecular cloning technology to studies of G-proteins in the last two years has resulted in major advances in knowledge of the structure of G- protein subunits and has resulted in the identification of new G- proteins, the functions of which are not yet established. This symposium proposes to bring together leaders in the field of G- protein research. A major goal is to provide a forum for interaction between individuals who are actively studying G- protein function using model systems and investigators who are actively pursuing the identity and structure of G-proteins with technologies evolved from molecular cloning. The broad areas to be covered in the symposium include: (1) regulation of ion channels by G-proteins; (2) G-protein regulation of second messenger production; (3) receptors that interact with G- proteins; and (4) structure, function and expression of G- proteins. There will be five sessions, one each on topics 1-3, and two sessions on topic 4. The meeting will also include a major keynote address and an afternoon poster session related to the general themes of the symposium. The published proceedings from the symposium are expected to make a timely and important contribution to this rapidly expanding field. This symposium on guanine nucleotide regulatory proteins (G- proteins) will provide a unique opportunity to bring together biochemists, physiologists, pharmacologists, cell biologists, and molecular biologists who are leaders in the multidisciplinary approaches being taken in this field. The critical comparisons possible in such an interaction should provide an important boost to our understanding of G-protein structure and function, and more generally to understanding of cellular signaling mechanisms.

DESCRIPTION (provided by applicant): Muscarinic acetyicholine receptors (mAChR) play a key role in the central nervous system, where they are involved in such functions as memory, learning, control of movement, and the regulation of circadian rhythms. The long-term goal of this research is to elucidate the mechanisms which regulate the expression and function of mAChR in the nervous system. Work from a number of laboratories has suggested that different subtypes of mAChR are differentially localized in cells, and this laboratory has demonstrated that different subtypes of mAChR are differentially localized in polarized cells and begun to identify sorting signals responsible for this differential localization. This proposal will determine the molecular basis and mechanisms responsible for generation of this asymmetry, and determine the relevance of these sorting pathways to the localization and function of the mAChR in the nervous system in vivo. Different mAChR subtypes have been implicated in different functions in the central nervous system, but it is difficult to unambiguously assign functions to specific receptor subtypes in vivo. This laboratory has created knockout mice lacking the M1 receptor and demonstrated that the M1 receptor plays a crucial role in the activation of phospholipase C and mitogen-activated protein kinase pathways in the forebrain and in generation of seizures in the pilocarpine model of epilepsy. This proposal will use these mice to determine the role the M1 receptor in the regulation of processing of the Alzheimer's precursor protein, to determine the pathways specifically involved in pilocarpine-induced seizures, and to determine the role of the M1 receptor in both signaling pathways and behavioral responses of the striatum. The identification of the factors responsible for the regulation of expression, localization, and function of the macromolecules involved in synaptic transmission is a problem of fundamental importance in cell and neurobiology. In addition, this research is of clinical importance because of the role of the mAChR in learning, memory, the cognitive defects in Alzheimer's disease, and the movement disorders in Parkinson' s disease. The elucidation of the factors involved in the regulation and function of mAChR may aid in understanding the etiology and possible treatment of a variety of neurological and mental abnormalities.

The overall goal of this research is to delineate the mechanisms which regulate signal transduction by the neurally active cytokines, leukemia inhibitor factor (LIF) and ciliary neurotrophic factor (CNTF). LIF and CNTF use an overlapping set of receptor polypeptides: LIF action is mediated by a heterodimeric receptor consisting of the low affinity LIF receptor (LIFR) and gp130, and CNTF uses a receptor consisting of LIFR, gp130, and the (nonsignaling) low affinity CNTF receptor. LIF and CNTF are members of a family of pluripotent cytokines that can regulate both neuronal survival and phenotypic expression of neuropeptides and neurotransmitters. The specific aims of this proposal are: (l) To determine the mechanisms responsible for the regulation of LIF receptor signaling by mltogen-activated protein kinase (MAPK). We have found that the LIFR is phosphorylated by MAPK after stimulation of cells with LIF or other growth factors. Removal of the MAPK phosphorylation site eliminates the ability of heterologous receptor activation to regulate LIFR-mediated induction of gene expression. This proposal will determine the molecular and cellular mechanisms responsible for the regulation of neurokine signal transduction by MAPK-mediated phosphorylation of the LIFR. (2) To determine the functional consequences of LIF-stimulated serine phosphorylation of gp130, and to identify the kinase responsible for this phosphorylation. We have demonstrated that gp130 is rapidly serine phosphorylated by an unknown kinase after stimulation with LIF. The site of phosphorylation is adjacent to a dileucine sequence previously shown to be involved in receptor internalization. We will determine the role of this phosphorylation in LIF-mediated signal transduction and receptor internalization, and identify the protein kinase(s) responsible for this phosphorylation, (3) To determine the role of src family kinases in the action of neurokine receptor signaling in the nervous system. Previous work has demonstrated that a number of src family kinases are associated with the activated LIF receptor, and we have found that that src kinases are activated following LIF stimulation of both neuronal cell lines and cultured neurons. This proposal will determine the role of src family kinases in neurokine action in neuronal cells. The research described here should provide new information on the function and regulation of the neurokine receptors and on the molecular basis for their diverse actions in the nervous system.

DESCRIPTION (Adapted from Investigator's Abstract): The long-term goal of this research is to elucidate the mechanisms involved in the regulation, development, and function of muscarinic acetylcholine receptors in the embryonic chick heart. The chick embryo represents an attractive system for the study of cardiac mAChR both in vivo and in cell culture. Recent work from Dr. Nathanson's laboratory has demonstrated that treatment of chick heart cells with muscarinic agonists and transforming growth factor-beta regulates the expression of mRNAs encoding the two main mAChR subtypes expressed in chick heart, cm2 and cm4. The promoter for the cm2 receptor has also been isolated and shown to drive expression when transfected into cardiac cells. This proposal will determine the cellular and molecular mechanisms responsible for the regulation of mAChR expression by muscarinic agonists and TGF-beta. Muscarinic receptors activate a G protein regulated inwardly rectifying potassium (GIRK) channel. Nathanson's laboratory has found that treatment of chick embryos in ovo with muscarinic agonists causes a large decrease in the levels of mRNA encoding the inward-rectifying potassium channel subunits GIRK1 and GIRK4 in atria, while the level of GIRK1 mRNA in ventricle is not changed. This proposal will determine the mechanism responsible for the regulation of potassium channel expression by mAChR activation and the basis for the tissue-specific differences in regulation of GIRK expression by agonist. Transcription factors of the GATA-family have been shown to be involved in the regulation of expression of a number of cardiac genes. This laboratory has found that coexpression of GATA-6 in cells which do not normally express the m2 receptor with a luciferase reporter gene under the transcriptional control of the cm2 promoter causes a 100- to 600- fold increase in transcriptional activity. This proposal will determine factors in the regulation of m2 receptor expression in cardiac cells. This research will provide valuable new information on the basic mechanisms regulating the expression and function of mAChR in the heart. In addition, this research may aid in understanding the etiology of a variety of cardiac abnormalities.

DESCRIPTION (Adapted from Applicant's Abstract): The overall goal of this research is to elucidate the mechanisms involved in the regulation and function of muscarinic acetylcholine receptors (mAChR) in the mammalian heart. Activation of the mAChR causes a decrease in the rate and force of contraction, and regulates the activities of adenylyl cyclase, phospholipase C, and potassium and calcium channels. While most cardiac mAChR are of the m2 subtype, a wide variety of pharmacological, physiological, immunological, biochemical, and molecular biological evidence indicates that the m1 receptor is also present at very low levels in the heart. The function and number of mAChR can be decreased by continued exposure of agonist. The PI has recently demonstrated that the beta-adrenergic receptor kinase and beta-arrestin synergistically regulate signal transduction by the m2 receptor in transfected cells. This proposal will test the hypothesis that the m2 and m4 receptors exhibit different selectivities for regulation by subtypes of G-protein receptor kinases and arrestins, and determine the functional consequences of elimination of the phosphorylation sites on mAChR function and regulation. Short-term agonist exposure also causes sequestration of receptors from the cell surface. Previous results indicate that the m1 and m2 receptors use distinct cellular mechanisms for agonist-induced internalization. This proposal will test the hypothesis that a portion of the m2 receptor in the sixth transmembrane domain and the third cytoplasmic domain (i3)form an contiguous sequestration domain. This proposal will identify specific residues and determine the effects of lack of sequestration on the regulation of muscarinic responsiveness. This proposal will also alter expression and introduce mutations into mAChR in the heart in vivo to provide unique information on the regulation of mAChR signaling in the heart. This proposal will use m1 knockout mice generated by this laboratory to determine the role of the m1 receptor in the physiological actions of acetylcholine and the regulation of mAChR expression in the heart. This project will determine the effect of disruption of the m2 receptor on cardiac function, responses to ACh, and expression of other mAChR subtypes, and will introduce mutations into the m2 receptor in mice to test the role of phosphorylation and sequestration on receptor function and signaling in the heart in vivo. This research will provide valuable new information on the basic mechanisms regulating the expression and function of mAChR in the heart. In addition, this research may aid in understanding the etiology of a variety of cardiac abnormalities.

DESCRIPTION (provided by applicant): The long-term goal of this research is to determine the molecular and cellular mechanisms responsible for the regulation of muscarinic acetylcholine receptor (mAChR) gene expression in the retina during development. Muscarinic receptors have an important role in the processing of visual information in the retina, and have also been implicated in the development of the embryonic retina. Our laboratory has found that there is receptor subtype- and cell-specific regulation of mAChR in the chick retina during embryonic development. The developmental regulation of the M2 receptor-in vivo can be mimicked in retina cell culture by an apparently novel developmentally-regulated secreted factor. This application will identify and determine the mechanism of action of the M2-inducing factor. The specific aims are: (1) To purify the M2 receptor-inducing factor. We have developed a serum-free culture system that supports secretion of this factor, and have used a rapid reporter gene assay to obtain highly enriched preparations of this factor. We will refine this purification scheme to purify the factor to sufficient homogeneity to allow biochemical and molecular biological analyses. (2) To isolate cDNA clones encoding the M2 receptor-inducing factor. We will use both a protein sequence determination approach and expression cloning to isolate cDNA clones encoding the factor. (3) To test the hypothesis that the M2 receptor-inducing factor regulates M2 receptor expression in vivo. We will determine if the factor identified on the basis of its ability to induce M2 receptor expression in retinal cells in culture is indeed responsible for the developmental induction of retinal M2 receptor expression in vivo. (4) To determine the molecular and cellular mechanisms responsible for the regulation of the M2 receptor by the M2 receptor-inducing factor. These experiments should provide valuable new information on the mechanisms responsible for the developmental regulation of neurotransmitter receptor gene expression in the retina.

[unreadable] DESCRIPTION (provided by applicant): Organophosphate (OP) acetylcholinesterase inhibitors are used widely in agriculture as pesticides and also have been used in terrorist attacks against civilian populations. OPs are irreversible acetylcholinesterase inhibitors which can cause death due to overstimulation of both central and peripheral cholinergic systems. Overstimulation of muscarinic acetylcholine receptors (mAChR) in the central nervous system (CNS) can cause respiratory depression and convulsions. This research will use genetic, pharmacological, and molecular approaches to define underlying mechanisms of OP action in the CNS and to identify new pharmacological targets to prevent or ameliorate OP-induced actions in the CNS. OP-induced seizures progress through three neurochemical stages: an initial phase which can be blocked by muscarinic antagonists, followed by a transitional period where both cholinergic and noncholinergic pathways contribute to the seizures, and followed by mainly noncholinergic-mediated seizure activity. This proposal will use mouse strains deleted of specific mAChR genes to determine the subtype of receptor which mediates OP-induced convulsions. This proposal will also use pharmacological and genetic approaches to determine the roles of the noradrenergic and dopaminergic systems and their receptors in OP-induced seizures. Prevention and/or termination of OP- induced seizure activity is critical both for reduction of rapid lethality and pathological neurological changes. The cell damage after OP-induced seizures is followed by proliferation of neuronal precursor cells. This increased neurogenesis has been suggested to both contribute to and to limit the long-term damage caused by OP exposure. This proposal will also determine the mechanisms and contributions of convulsive and non- convulsive pathways to OP-induced cell death and neurogenesis. Environmental exposure to OP pesticides causes thousands of death world-wide. The dangers of the use of OP nerve agents in attacks on civilian populations was demonstrated by the sarin attacks in Japan in 1994 and 1995 which resulted not only in immediate deaths but in changes in long-lasting neurological functions. This research will provide valuable new insights into the mechanisms of OP action in the CNS and identify new potential targets for specific pharmacological interventions to ameliorate the effects of OP on the CNS. PUBLIC HEALTH RELEVANCE Organophosphates are inhibitors of the nervous system enzyme acetylcholinesterase inhibitors and are used widely in agriculture as pesticides and also have been used in terrorist attacks against civilian populations. Exposure to organophosphates can cause convulsions and long-term damage to the central nervous system. This research will determine the molecular mechanism for these actions and may identify targets for improved treatment of organophosphate poisoning. [unreadable] [unreadable] [unreadable]