Robert J. Lefkowitz - US grants

This grant is requested to support a program in basic research directed at elucidating, at a molecular level, the nature and mode of functioning of the adrenergic receptors for catecholamines. Catecholamies such as norepinephrine and epinephrine are of vital importance in the hormonal control of the entire circulation. These compounds appear to act via stimulation of adenyl cyclase in cell membranes which is in turn mediated by binding to specific beta-adrenergic receptors. The specific aims of these studies include: 1) the isolation, purification and characterization of adrenergic receptors from heart muscle and other tissues, and 2) delineation of the way in which receptors interact with adenyl cyclase, and 3) study of isolated receptors in disease states. These aims will be achieved by studying the binding of radioactively labeled (negative) 3H alprenolol, a competitive beta-adrenergic antagonist, to cell membrane fragments isolated from heart muscle, and to specific proteins isolated and purified from these membranes. Detailed characterization of these receptor structures will ultimately permit evaluation of possible alterations in their structure and function in disease states such as hypertension, congestive heart failure, or in physiological processes such as aging.

Catecholamines such as norepinephrine and epinephrine are of vital importance in the hormonal control of the entire circulation. These compounds act via alpha- and beta-adrenergic receptors which are coupled through G proteins to enzymes such as adenylyl cyclase and phospholipase C as well as ion channels. Amongst a number of mechanisms which regulate receptor function, one of the most important is phosphorylation of the receptors by a novel family of G protein-coupled receptor kinases (GRKs) such as the beta-adrenergic receptor kinase (betaARK). This grant is requested to support a program in basic research directed at elucidating at a molecular and physiological level the mechanisms of regulation of adrenergic and other G protein-coupled receptors by these receptor kinases as well as by a recently identified unique phosphatase. These enzymes mediate, respectively, rapid agonist-promoted desensitization and resensitization of receptor signaling. Moreover, abnormal function of cardiac betaARK has recently been implicated as potentially contributing to the pathophysiology of heart failure. Accordingly, this research proposal has three closely linked goals, all of which involve a primary focus on the regulation of adrenergic receptor function by the G protein- coupled receptor kinases as an approach to gaining increased understanding of the normal and abnormal hormonal control of circulatory function. These goals are: 1) To determine the specificity of GRK action on adrenergic and other G protein-coupled receptors. 2) To define the physiological role of GRK's in vivo by "knocking out" their genes or by overexpressing the GRKs or specific inhibitors in transgenic animals. These experiments will allow us to assess the feasibility of altering cardiac function in vivo by manipulation of the betaARK system. 3) To determine the nature, properties and regulation of the phosphatases which reverse the action of GRKs. These studies will produce a comprehensive picture of the regulation of G protein-coupled receptors in which detailed information about molecular and cellular mechanisms provides the basis for understanding physiological consequences in the intact animal. Moreover, the results have the potential to point the way toward novel therapeutic strategies for cardiovascular diseases.

DESCRIPTION (provided by the applicant): G protein-coupled receptors (GPCRs) including those for catecholamines and angiotensin II regulate vascular reactivity, including vasoconstriction and vasodilation, as well as vascular smooth muscle (VSM) cell mitogenesis and migration. Vascular reactivity may be perturbed in hypertension, whereas altered VSM mitogenesis and migration characterize pathological intimal hyperplasia following surgical bypass or restenosis after arterial angioplasty. Following GPCR activation, GPCRs are phosphorylated by one of seven GPCR kinases (GRKs) and then one of two isoforms of b-arrestin is recruited to the receptor. b-arrestin binding sterically interdicts further signaling to G proteins, leading to receptor desensitization and attenuation of signaling. b-arrestins also play positive roles in signaling, serving as adapters and scaffolds to organize GPCR- mediated activation of MAP kinase cascades, such as the extracellular signal regulated kinases (ERK 1/2). These MAP kinases regulate mitogenesis and migration of VSM cells and other cell types. Our group has developed mice in which the GRKs and b-arrestins have been individually knocked out. We will utilize these animals, and VSM cells from them, to test the central hypothesis that regulation of GPCR signaling by b-arrestins and GRKs is critical for normal vascular homeostasis. Our specific aims are 1) To elucidate the vascular phenotype of b-arrestin and GRK knockout mice by analyzing conscious and anesthetized blood pressure responses and vascular reactivity using isolated aortic rings; 2) To elucidate the roles of b-arrestins and GRKs in signaling via endogenous GPCRs in isolated arterial and venous VSM cells from wild type and knockout mice by determining both A) the specificity of b-arrestins and GRKs in desensitizing second messenger signaling via endogenous GPCRs and B) the roles of b-arrestins and GRKs in GPCR stimulated ERK activation, proliferation, and migration of VSM cells; and 3) To determine if the loss of specific b-arrestins or GRKs alters in vivo proliferative intimal hyperplasia following mouse vein-graft surgery or arterial injury. These experiments have the potential to lead to the development of new strategies for limiting vein graft failure and restenosis.

PROJECT SUMMARY/ABSTRACT MOLECULAR REGULATION OF CARDIOVASCULAR SEVEN TRANSMEMBRANE RECEPTORS. All aspects of cardiovascular function are regulated by receptors of the seven transmembrane receptor (7TMR or GPCR) family, and they are the commonest target of therapeutic drugs. A universal mechanism regulating these receptors is desensitization of heterotrimeric G protein signaling. Classically, this is mediated by a two- step process in which activated receptors are phosphorylated by G protein-coupled receptor kinases, leading to the binding of a ?-arrestin (?arr) molecule which sterically interdicts further activation of the G protein. More recently it has become clear that ?arrs can also serve as multifunctional adaptors which act as signal transducers in their own right. Moreover, for many receptors ligands can be found which disproportionately activate either G protein- or ?arr-mediated signaling?i.e., biased ligands which may possess greater specificity of action and fewer side effects. Several such drugs, including one for decompensated congestive heart failure which targets the angiotensin II type 1 receptor (AT1R), have reached clinical trials. Accordingly, this proposal has three closely linked aims which involve developing a molecular- and atomic-level understanding of how such ?arr-mediated signaling is generated using as model systems two receptors of great cardiovascular significance, the ?2-adrenergic receptor (?2AR) and the AT1R. These aims are: 1) To discover novel allosteric stabilizers (nanobodies and small molecules) for biased conformations of the AT1R and ?2AR. 2) To delineate the structural basis for biased conformations of the AT1R by X-ray crystallography and DEER. 3) To determine the structure of GPCR-?arr complexes by cryo-electron microscopy. The insights which we will generate have the potential to guide the design of powerful new cardiovascular drugs and will further our understanding of the conserved signaling mechanisms of the greater GPCR family.

[unreadable] DESCRIPTION (provided by applicant): P-ARRESTINS AND GRKs IN CARDIOVASCULAR FUNCTION. G protein coupled receptors (GPCRs) such as those for catecholamines and angiotensin II regulate cardiovascular functions including vasoconstriction and vasodilation; vascular smooth muscle cell (VSM) mitogenesis and migration; and cardiac inotropy and chronotropy. These functions may be perturbed in hypertension, in pathological intimal hyperplasia as after bypass surgery or angioplasty, or in heart failure. Following GPCR activation, the receptors are phosphorylated by one of several G protein coupled receptor kinases (GRKs) and then bind one of the two isoforms of p-arrestin. p-arrestin binding sterically interdicts further signaling to G proteins leading to receptor desensitization and attenuation of second messenger generation, p-arrestins also play positive roles in signaling, serving as adaptors and scaffolds to organize receptor-mediated activation of MAP kinase cascades and other pathways. These MAP kinases regulate mitogenesis and migration of vascular smooth muscle cells. Recently we have found that receptor ligands may vary dramatically in their ability to stimulate G protein versus p-arrestin-mediated signaling. However, the cellular and physiological consequences of p-arrestin/GRK mediated signaling in cardiovascular (or other) systems are largely unknown. Accordingly, we will use several approaches to test our central hypothesis that p-arrestin/GRK- mediated signaling contributes significantly to the short and long-term regulation of cardiovascular function. Our approaches will include the use of mutant receptors and specific agonists which can selectively activate p-arrestin but not G protein signaling; siRNA to p-arrestins and GRKs; knock-out mice which we have previously developed lacking each of the p-arrestins and GRKs; and cells derived there from. Utilizing the p-adrenergic receptors and angiotensin IMA receptor as our models our specific aims are to determine: 1) the specificity of individual p-arrestins and GRKs in desensitizing G protein mediated second messenger generation and to compare this with their roles in mediating signaling, as for example through ERK activation. 2) The physiological effects of P-arrestin-mediated signaling, a) In vitro on cardiac myocytes and vascular smooth muscle cells, and b) In vivo on cardiac inotropy and chronotropy and on systemic blood pressure, c) The in vivo consequences of cardiac signaling by mutant p-adrenergic receptors and angiotensin II1A receptors which are uncoupled from G proteins but signal through p-arrestins. 3) The chronic effects of an ang analog (SII ang) which signals only through p-arrestins, on the development and course of CHF in a transgenic mouse overexpressing calsequestrin in the heart. These experiments have the potential to delineate an entirely novel and general mode of receptor-mediated signaling and to point the way to the development of novel therapeutics which target this recently discovered signaling mechanism. [unreadable] [unreadable]

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