1989 — 1990 |
Liggett, Stephen B |
K11Activity Code Description: Undocumented code - click on the grant title for more information. |
Molecular Basis of Adrenergic Receptor Function
The long term objective of this project is to relate adrenergic receptor (AR) molecular structure to hormone/drug interaction and receptor function. These receptors play key roles in regulating a host of physiologic processes in man. We propose to determine the structural components of the beta AR molecule which are involved in desensitization as deduced from site directed mutagenesis of the human beta2 AR gene. The sites chosen for alteration of the beta AR are located at intracellular regions of the molecule, and are suspected of being involved in one or more components of desensitization based on preliminary biochemical studies and their conserved nature throughout the family of G- protein coupled receptors. The "uncoupling" of the agonist occupied phosphorylated form of the beta-adrenergic receptor from Gs appears to be augmented by an as yet undefined protein. Such a protein (arresting) has been found in the analogous rhodopsin transducing photoreceptor system. Thus, a second specific aim of this proposal is to identify and characterize this putative regulatory protein in the beta-adrenergic receptor system. Both the delineation of key structural domains of the beta AR associated with desensitization and the characterization of this new regulatory protein may afford the means of altering the clinically relevant phenomenon of desensitization. The alpha2 AR appears to have two or possibly three subtypes based on Southern blot hybridization studies using the platelet alpha2 AR gene as a probe. We propose to clone and sequence this third subtype and to express the receptor in stable cell lines. This will be followed by pharmacologic and biochemical characterization with the ultimate goal of providing a means of differentiating the alpha2 AR subtypes by ligand binding and functional studies. These structure-function relationships could offer the basis for development of therapeutic subtype specific agonists and antagonists. Specific aims one and three will be carried out in this Physician Scientist Program during Phase I which also includes formal classroom studies at the Duke University Graduate School, while the second specific aim as listed above will be undertaken during Phase II.
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0.97 |
1990 — 2018 |
Liggett, Stephen B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Molecular Properties of B-Adrenergic Receptors in Asthma @ University of Cincinnati
Asthma is a prevalent chronic inflammatory disease characterized by constriction of the airways. Beta-adrenergic receptors (Beta2AR) on bronchial smooth muscle relax this constriction and are the targets of beta-agonists used for treatment bronchospasm. However, there is a high degree of inter-individual variation in beta2AR function due to factors that include receptor regulation by the asthmatic milieu, beta-agonist treatment, and polymorphisms of the receptor. Thus the most commonly utilized class of drugs for the treatment of asthma is likely suboptimal because of deficiencies in our understanding of target signal regulation. The long-term objective is to understand how beta2AR signaling is regulated by genetic factors, agonist structure and modulation of post- receptor transduction elements. In Specific Aim 1, the combinations of polymorphisms of the beta2AR gene found in the human population, arranged as haplotypes, will be studied within the context of expression and agonist regulation. In Specific Aim 2, the molecular basis of pleiotropic responses to agonist will be delineated. Here, we will delineate specific properties of agonists with respect to coupling to Gs, Gi and MAP kinase, sequestration, phosphorylation. and down- regulation using a multiply recombinant approach. In Specific Aim 3, the role of Gi expression in beta2AR signaling in the airway will be determined. Two transgenic mice will be created over-expressing Gi or a Gi inhibitor in airway smooth muscle. Since an increase in Gi has been reported to be an important component of beta2AR and bronchial hyper- reactivity in asthma, we will be able to directly assess this potential mechanism in isolation of other processes. The approach includes biochemical, ex vivo, and in vivo studies of beta2AR function in cells, trachea and intact mice so as to merge the in vitro results with relevant physiological function. In Specific Aim 4, the rate limiting step of beta2AR signal transduction will be explored by generating two transgenic mice, over-expressing adenylyl cyclase type VI or a peptide inhibitor, in airway smooth muscle. Again, a comprehensive approach linking signaling events and physiology will be utilized. These studies will further our understanding of beta2AR regulation and provide new insights into asthma pathophysiology and treatment.
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1 |
1994 |
Liggett, Stephen B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Properties of B Adrenergic Receptors in Asthma @ University of Cincinnati
Activation of beta-adrenergic receptors causes relaxation of bronchial smooth muscle and these receptors are the targets of the most effective therapy for asthma. Several lines of investigation suggest that defects in the beta-adrenergic receptor/G-protein/adenylyl cyclase system may be a contributory factor in asthma. The long term goals of this proposal are to determine the role of beta-adrenergic receptors in the pathogenesis of asthma, to elucidate the mechanisms and pathways of receptor regulation by various factors, and to establish relationships between receptor structure and function. First, the molecular structure of any additional beta- adrenergic receptor subtypes in lung will be determined by screening human lung cDNA libraries using probes from the known sequences of beta1-,beta2-, and beta3- adrenergic receptors and subsequent cloning. To assess whether a defect in the receptor structure is present in asthma, a large cohort of asthmatic patients will be studied. Genomic DNA will be isolated from blood samples and beta-adrenergic receptor sequences amplified by the polymerase chain reaction. The resulting receptor genes will be sequenced in their entirety. When matched with controls, this will allow for delineation of any unique abnormality in primary beta-adrenergic receptor structure present in asthmatics. to further explore the receptor- transduction system in human subjects, lung tissue from bronchoscopy, surgery or autopsy will be utilized. Receptor density on various pertinent cells (smooth muscle, endothelium, mucous glands, epithelium, macrophages) will be assessed with radioligand binding and autoradiography, and will be correlated with receptor mRNA levels using in situ hybridization and Northern blots. Under these conditions the expression of receptor at pertinent cells can be compared between normal and asthmatic lungs, and the relationship between expression and mRNA levels determined. It is also planned to establish permanent cell lines from explants of lungs from normal and asthmatic subjects which will allow for even further pharmacologic characterization of the receptor and various transduction pathways. To further define the pathways involved in regulation of beta- adrenergic receptor expression and function, it is planned to transfect the human beta-adrenergic gene into cells normally devoid of receptors, thus establishing permanent cell lines expressing the receptor. By site directed mutagenesis, receptors with altered regions thought to be involved in receptor function will be constructed and expressed. This will allow for a molecular assignment of determinants of several key regulatory events: homologous and heterologous desensitization, phosphorylation by PKA and the beta-adrenergic receptor kinase, agonist induced downregulation, receptor fatty acid acylation and G-protein coupling.
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1 |
1995 — 1998 |
Liggett, Stephen B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanisms of Alpha2 Adrenergic Receptor Function @ University of Cincinnati
Alpha-2-adrenergic receptors (alpha-2-AR) are cell surface receptors which upon binding catecholamines signal to the interior of the cell via G proteins. Alpha-2-AR are expressed in virtually every organ system and are known to play important roles in cardiovascular, pulmonary, renal, hepatic, metabolic, and central nervous system functions. Alpha-2-ARs have also been implicated in a number of pathologic processes and are the targets for pharmacologic agents in the treatment of a number of diseases. The long-term objective of this project is to understand the relationships between the molecular structures of these receptors and their functions. These goals will be carried out primarily by site-directed mutagenesis of the cDNAs encoding for the wild-type alpha-2-AR subtypes (alpha-2C10, alpha-2-C4, and alpha-2-C2), followed by recombinant expression in mammalian cells. This allows for directly comparing a given function between wild-type and mutated receptor and delineating a structure/function relationship. In Specific Aim 1, the role of G protein coupled receptor kinases in the phosphorylation of alpha-2-ARs during short-term agonist promoted desensitization will be studied. In Specific Aim 2, the phosphorylation domains of the alpha-2-AR will be mapped by assessing functional short-term agonist promoted desensitization and receptor phosphorylation in wild-type and alpha-2-ARs with mutated residues in regions that we suspect are sites for phosphorylation. In Specific Aim 3, the molecular features of the alpha- 2-AR subtypes which are responsible for the differences in agonist- promoted desensitization and phosphorylation observed between the three subtypes will be determined. Specific Aim 4 explores the mechanism of receptor sequestration (internalization) and downregulation, two key events which occur during long-term agonist promoted regulation of alpha- 2-ARs. Here, the subcellular events of receptor trafficking and processing will be examined using immunoelectron microscopy. In Specific Aim 5, the molecular determinants of alpha-2-AR sequestration and downregulation will be studied using site-directed mutagenesis of regions suspected to be involved in these processes including sites for palmitoylation, glycosylation, and phosphorylation, and regions involved with G protein coupling. In Specific Aim 6, the domains of the alpha-2- AR responsible for coupling to G1 and G2 will be delineated by both deletion and chimeric substitution mutagenesis. In Specific Aim 7 the molecular determinants of the observed differences in G2 coupling between the three alpha-2-AR subtypes will be determined. The results of these studies will help to determine at a fundamental level how alpha-2-AR carry out their signal transduction, how they are regulated, and how they can be modulated by therapeutic agents.
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1 |
1996 — 2000 |
Liggett, Stephen |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Molecular Biology of Cardiopulmonary Adrenergic Receptors @ University of Cincinnati
Adrenergic receptors (AR) are cell surface proteins that are the receptors for the endogenous catecholamines epinephrine and norepinephrine. These receptors are widely distributed throughout the cardiovascular and pulmonary systems and subserve a number of key functions, including modulation of cardiac inotropy and chronotropy, systemic vascular resistance, and bronchial smooth muscle tone. Adrenergic receptors are the targets for many drugs which act as agonists or antagonists ina the treatment of diseases such as hemodynamic shock, congestive heart failure, angina, hypertension, asthma. The focus of this proposal is on the betaARclass (composed of Beta1AR, beta2AR, and beta3AR subtypes) and the alpha2AR (composed of the alpha2C10, alpha2C4, and alpha2C2 subtypes) which have opposites effects on the effector enzyme adenylyl cyclase. With site- directed mutagenesis and recombinant expression studies, we now know many of the structural determinants required for certain properties of these receptors. However, their relevance within the context of heart and lung function in normal or diseased states is not known. Thus one objective of this research is to develop and study mice expressing mutated adrenergic receptors which lack single components of the transduction process. We have also recently found that the beta2AR is highly polymorphic in the normal population, and some polymorphisms are overrepresented in cohorts of patients with asthma and congestive heart failure. in some cases the polymorphisms which result in changes in the encoded amino acid significantly alter receptor function as assessed in in vitro recombinant expression systems. However, the physiologic significance of such polymorphisms for heart and lung function is not known. Thus the second objective is to develop and study mice expressing the known polymorphic forms of the beta1AR and beta2AR in heart and lung. In both Aims 1 and 2, a gene replacement strategy will be utilized to eliminate the background of endogenous wild-type receptor and to maintain physiologic levels of receptor expression. The third objective relates to mechanisms which are involved in the expression of the adrenergic receptors. Despite their importance in heart and lung function, little is known about how expression is regulated or what factors are critical for tissue specific expression. Thus this third objective will be to delineate critical regions of the genes for these receptors which are important for expression and to determine the transcription factors involved in cell-type specific expression.
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0.915 |
1999 — 2002 |
Liggett, Stephen B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Mechanisms of Beta Receptor Dysfunction in Heart Failure @ University of Cincinnati
Heart failure affects 5 million individuals in the U.S. and has a mortality rate of approximately 50%. The underlying basis of the common forms of heart failure, and the factors the lead to its progression are not well understood, and this ultimately affects our abilities to design innovative preventative and treatment strategies. With the advancing decline in function, the failing heart develops a significant diminution in a principal mechanism for increasing cardiac output: activation of the cardiac beta-adrenergic receptor (betaAR) signal transduction system. Whether this serves an entirely protective purpose, or whether certain aspects of this response contribute to the pathophysiology of heart failure is not known. The overall goals of this project are to delineate the contributions of dysfunctional beta1AR and beta2R signalling to the progressive contractile dysfunction in this syndrome, to determine the mechanisms of this alteration at the molecular and genetic levels, and to attempt approaches for rescue of heart failure that further confirm mechanism and may lead to novel therapeutics. These studies will be carried out in transgenic mouse models of hypertrophy and failure and include elements that provide for correlations between biochemical, molecular, and structural abnormalities and physiologic function of the intact heart. The specific areas are: 1) To determine the mechanisms of beta1AR and beta2R regulation in heart failure in transgenic mice engineered to express critical components of the hypertrophy/failure program. 2) To ascertain the relative contributions of the defects in receptor-G protein-effector signalling to the specific characteristics of hypertrophy/failure found in specific aim 1, by experiments aimed at rescuing the transduction pathway via targeted transgenesis. 3) To delineate the relevance of genetic variations (mutations/polymorphisms) of the beta1AR and beta2AR found in the human population to ventricular function within the context of hypertrophy and failure. These studies will be carried out by transgenically expressing these variants in the hearts of mouse models with ventricular dysfunction.
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1 |
2000 — 2002 |
Liggett, Stephen B |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Beta Adrenergic Receptor Variants in Heart Failure @ University of Cincinnati
Beta1-adrenergic and beta2-adrenergic receptors (betaAR) expressed in the heart and vasculature play key roles in regulating cardiac inotropy and chronotropy and represent targets for therapeutic agonists and antagonists in the treatment of heart failure. Heart failure exhibits extensive inter- individual variability in its clinical course and response to therapy. We hypothesize that betaAR polymorphisms, acting as disease modifiers, represent a substantial genetic component of this variability. Indeed, significant genetic variability in the structure of these receptors due to polymorphisms of their coding sequences has been identified. When expressed in transfected cells, a number of these polymorphic receptors display altered expression, agonist or antagonist binding affinities, activation of adenylyl cyclase, or agonist-promoted regulation. Having identified beta1AR and beta2AR polymorphisms of >1% in the population, and having determined their biochemical significance in cells, we now plan to focus on their relevance to heart failure. The broad their biochemical significance in cells, we plan to focus on their relevance to heart failure. The broad long-term objectives are thus to determine the disease modifying effects at the clinical, physiological and biochemical levels of betaAR polymorphisms in heart failure. Studies are designed to delineate their roles in: the development of left-ventricular hypertrophy and heart failure, the clinical prognosis and progression of failure, regulation of cardiovascular responsiveness to endogenous and exogenous activation, and modification of the clinical response to beta-blocker therapy. In addition, polymorphisms will be studied in gene targeted (tag- and-replace) mice so that more extensive biochemical and physiologic characterization during controlled cardiac modeling can be undertaken. These studies will delineate how genetic variability of betaAR alters cardiac function and impacts the clinical course and characteristics of heart failure. Such results may thus provide for important prognostic markers and lead to development of new heart failure treatments based on receptor genotype.
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1 |
2003 — 2013 |
Liggett, Stephen B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Basis of Variability of Lung Gpcr Signaling @ University of Maryland Baltimore
DESCRIPTION (provided by applicant): G-protein coupled receptors (GPCRs) are expressed throughout the lung, mediating homeostatic, adaptive, and pathogenic events, and are targets for many therapeutic agents. Human airway smooth muscle (HASM) express hundreds of GPCRs regulating contraction, relaxation, immune response, and growth, with direct relevance to asthma and COPD. However, substantial inter-individual variability in function, heterogeneity of signaling, and paradoxical responses of GPCRs are frequently found in clinical, ex vivo, and in vitro studies. Defining the molecular basis of this variability has been the broad long-term goal of this grant, and is critical for understanding disease pathobiology and heterogeneity, development of new drugs, and pharmacogenomics. During the past 5 years, polymorphisms of individual HASM GPCR genes were identified in a reference population and functionally characterized. These studies identified one mechanism of signaling variability in HASM, but also showed that the variability cannot be entirely explained by receptor polymorphisms. Three novel mechanisms of lung signaling variability were uncovered: alternative splicing, heterodimer formation, and genetic variation of G-protein coupled receptor kinases (GRKs). Thus the HASM receptorome is much more complex than previously recognized. In Aim 1, the structural and signaling effects of alternatively spliced GPCRs expressed in HASM will be determined. We have recently found that ~50% of GPCRs express multiple receptor "isoforms" in HASM due to alternative pre-mRNA splicing, which is subject to inter-individual variation. These isoforms will be cloned, expressed, and characterized in model-cell systems, and the signaling phenotypes confirmed in HASM. In Aim 2, heterodimer formation from selected GPCR pairs relevant to obstructive airway disease will be ascertained and their function determined. We find that HASM GPCRs can form heterodimers, often between very different receptors, that act as distinct signal-transduction units and alter airway responsiveness. Resonance energy transfer techniques will be utilized for detection and characterization of heterodimers, as well as studies of intracellular signaling events related to Gs, Gi, and Gq-coupled receptors in transfected cells and HASM. In Aim 3, the signaling impact of a GRK5 variant will be determined for selected GPCRs relevant to obstructive airway disease. GRKs regulate agonist-promoted function for most GPCRs. We have found a polymorphism of GRK5 that substantially alters 2AR function compared to WT GRK5, thus indicating a mechanism of variability at a hierarchic point above an individual receptor. Selected GPCRs will be studied in recombinant systems and HASM to ascertain the GRK5 variant phenotype in regards to receptor phosphorylation, internalization, desensitization, and GRK/-arrestin signaling. Collectively, results from these studies will define mechanisms of inter-individual variability and heterogeneity of HASM GPCRs, providing a basis for the diversity of phenotypes and drug-responses in obstructive lung disease. PUBLIC HEALTH RELEVANCE: Asthma and chronic obstructive pulmonary disease represent major national health problems. The cause of the airway constriction, and its relief by certain medications, is due to activation of receptors on the airway muscle. But, there are large differences in the extent of constriction, and its relief, from person to person that is not understood. This between-person variability limits our ability to understand more about airway constriction in these diseases and how to block or relieve it. This grant addresses the molecular basis for these differences so that we will understand more about these diseases and how to treat them.
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1 |
2005 — 2006 |
Liggett, Stephen B |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
A2-/B-Adrenergic Receptor Polymorphisms in Heart Failure @ University of Cincinnati
Adrenergic receptor (AR) function is critical for homeostasis in heart failure. Presynaptic alpha-2-AR control norepinephrine release from sympathetic nerves, while beta-1-and beta-2-AR expressed on cardiomyocytes increase inotropy and chronotropy. However, the expression and function of ARs, clinical progression, and response to beta-blockers is highly variable in heart failure, and the basis of this interindividual variability remains unknown. A role for common genetic variants in susceptibility, progression and therapeutic response is suggested by familial clustering of phenotypes, reduced penetrance in familial cardiomyopathies, and the existence of functionally significant polymorphisms of the three alpha2AR and two betaAR subtypes. The overall goal of this project is to define the relationships between AR polymorphisms and heart failure phenotypes, and to determine the mechanism by which they affect heart failure in the intact human, which will lead to personalized prognosis and treatment, based on receptor genotype. In Aim 1 we will complete polymorphism discovery in these intronless genes, assemble haplotypes and carry out in vitro studies to assess the consequences of genetic variation on receptor expression, function or regulation. In Aim 2, we will carry out an association and sibling study of 2-gene haplotypes of the alpha2c and beta1AR to ascertain heart failure risk. In an initial study of individual alpha2c and beta1AR polymorphisms, we found a 10-fold risk for heart failure in African-Americans. The use of extended haplotypes will potentially provide greater discrimination and precisely define the gene-gene and gene-environment interactions. In Aim 3 the functional status of cardiac beta1AR in patients with early and late failure in the absence of beta-blocker treatment, stratified by homozygous beta1AR haplotypes, will be ascertained. From transgenic mice, we have found that polymorphic beta1AR undergo phenotypic switching during the course of failure, which implies that there are "windows" of opportunity for therapeutic intervention. Studies will involve invasive hemodynamic testing of cardiac function in response to the beta1AR agonist dobutamine, the nonreceptor inotrope milrinone, and exercise. In retrospective studies we have shown that a beta1AR polymorphism may be associated with treatment response to beta-blocker. To further examine this, in Aim 4 we will carry out a prospective, double-blind, long-term, study of patients with homozygous beta1AR haplotypes to ascertain the effect of beta1AR genetic variability on carvedilol response. If the relationship holds, this would be the first pharmacogenetic test to predict who will be most likely to respond, and not respond, to this class of therapeutics.
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1 |
2007 — 2009 |
Liggett, Stephen B |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Alpha 2- and B-Adrenergic Receptor Polymorphisms in Heart Failure @ University of Cincinnati
Adrenergic receptor (AR) function is critical for homeostasis in heart failure. Presynaptic alpha-2-AR control norepinephrine release from sympathetic nerves, while beta-1-and beta-2-AR expressed on cardiomyocytes increase inotropy and chronotropy. However, the expression and function of ARs, clinical progression, and response to beta-blockers is highly variable in heart failure, and the basis of this interindividual variability remains unknown. A role for common genetic variants in susceptibility, progression and therapeutic response is suggested by familial clustering of phenotypes, reduced penetrance in familial cardiomyopathies, and the existence of functionally significant polymorphisms of the three alpha2AR and two betaAR subtypes. The overall goal of this project is to define the relationships between AR polymorphisms and heart failure phenotypes, and to determine the mechanism by which they affect heart failure in the intact human, which will lead to personalized prognosis and treatment, based on receptor genotype. In Aim 1 we will complete polymorphism discovery in these intronless genes, assemble haplotypes and carry out in vitro studies to assess the consequences of genetic variation on receptor expression, function or regulation. In Aim 2, we will carry out an association and sibling study of 2-gene haplotypes of the alpha2c and beta1AR to ascertain heart failure risk. In an initial study of individual alpha2c and beta1AR polymorphisms, we found a 10-fold risk for heart failure in African-Americans. The use of extended haplotypes will potentially provide greater discrimination and precisely define the gene-gene and gene-environment interactions. In Aim 3 the functional status of cardiac beta1AR in patients with early and late failure in the absence of beta-blocker treatment, stratified by homozygous beta1AR haplotypes, will be ascertained. From transgenic mice, we have found that polymorphic beta1AR undergo phenotypic switching during the course of failure, which implies that there are "windows" of opportunity for therapeutic intervention. Studies will involve invasive hemodynamic testing of cardiac function in response to the beta1AR agonist dobutamine, the nonreceptor inotrope milrinone, and exercise. In retrospective studies we have shown that a beta1AR polymorphism may be associated with treatment response to beta-blocker. To further examine this, in Aim 4 we will carry out a prospective, double-blind, long-term, study of patients with homozygous beta1AR haplotypes to ascertain the effect of beta1AR genetic variability on carvedilol response. If the relationship holds, this would be the first pharmacogenetic test to predict who will be most likely to respond, and not respond, to this class of therapeutics.
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1 |
2010 — 2013 |
Liggett, Stephen B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Lung Hrv: G-Protein Coupled Signaling Interactions in Asthma @ University of Maryland Baltimore
DESCRIPTION (provided by applicant): Human rhinovirus (HRV) infection causes at least 50% of asthma exacerbations. Airway epithelial cell (AEC) infection evokes the release of inflammatory, growth and bronchospastic factors, and other autocrine/paracrine mediators leading to generalized AEC and airway smooth muscle (ASM) "pro-exacerbation" pathology. This includes altered AEC lining fluid/ion content, ASM hyperreactivity, and ASM resistance to relaxation by (-agonists, which are controlled by G-protein coupled receptors (GPCRs) on these cells. The heterogeneity of these asthmatic responses is, in part, thought to be dependent on the HRV strain (serotype). There are over 100 HRV strains, yet little is known about how genomic differences in strains impact asthma exacerbation phenotypes. We have very recently completed sequencing the genomes of all 99 reference HRV-A and -B serotypes from a banked historical repository. This revealed previously unknown aspects of HRV RNA and protein structure, phylogenetic relationships, recombination, and extensive diversity among the canonical serotypes. It also provided structure-based sequence alignments which are a scaffold for integration of additional HRVs into the phylogenetic tree. The broad long-term objectives of this revised proposal are to ascertain the genomic features of modern HRV strains that contribute to specific asthmatic airway GPCR phenotypes and their heterogeneity. This will be accomplished by three aims. In Aim 1, we will determine the complete genome sequences of HRVs from 200 modern clinical isolates using massively parallel sequencing methods. In Aim 2, this data will be integrated into our structure-based reference genomic scaffold so as to define genomic regions that are similar and dissimilar amongst the strains, providing a rigorous mechanism to select the HRVs for in vitro functional studies. In Aim 3, GPCR signaling phenotypes of HRVs in the context of AEC and ASM will be ascertained in cell culture models using high-throughput methods (Sub Aim 1). And, these signaling phenotypes will be correlated to HRV genome features using Bayesian techniques with internal and external validations (Sub Aim 2). Such studies will provide a genomic basis for those HRVs that do, and do not, evoke AEC and ASM phenotypic traits, and thus establish some of the mechanisms of heterogeneity of viral induced asthma exacerbations. These findings may provide diagnostic and prognostic information, and pharmacologic strategies, for managing the most common cause of asthma exacerbations. PUBLIC HEALTH RELEVANCE: Human rhinovirus (HRV) infection causes about 50% of asthma attacks (and COPD exacerbations). There is, though, substantial variability in the nature and severity of the clinical features of asthma attacks from HRV infections, for reasons that are not known. However, it is now clear that there are many distinct HRV strains, and this proposal will define which HRVs, and which parts of their genomes, impose changes in airway receptor function that contribute to the pathology and symptoms of asthmatic attacks during HRV infection.
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0.955 |
2013 — 2017 |
Liggett, Stephen B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Airway Smooth Muscle Bitter Taste Receptors as Targets For Novel Bronchodilators @ University of Pennsylvania
PROJECT SUMMARY (See Instructions): Bronchodilators are used in the treatment of asthma for acute relief of bronchospasm and for long-term control. Currently, only one class of direct bronchodilators, p-agonists acting at airway smooth muscle P2- adrenergic receptors, are in use. With many asthmatics not achieving adequate disease control, there is a need for additional therapeutics, including direct bronchodilators acting by novel mechanisms. We have discovered multiple bitter taste receptor (TAS2R) subtypes on human airway smooth muscle (HASM). Activation of TAS2Rs by bitter ligands causes marked airway smooth muscle relaxation in vitro and in vivo, and in various models of asthma. TAS2R-mediated bronchodilation is as efficacious as p-agonists, and is via a completely different mechanism involving specialized Ca^^ signaling. There are thousands of potential TAS2R agonists derived from plants or synthesized for other purposes, yet prior to our findings these were not considered for asthma therapy. In this Project, we will examine the properties of HASM TAS2Rs with an eye towards agonists being a new class of bronchodilators for treating asthma. In Aim 1, we will focus on the potential for acute desensitization, which can limit therapeutic efficacy. Agonist-promoted desensitization of the 3 most abundant HASM TAS2Rs will be examined in HASM cells, bronchi from precision-cut human lung slices and recombinantly expressing model cells. G-protein coupling, phosphorylation, internalization, B arrestin interactions, and biased ligand properties will be established. The influence ofthe asthma diathesis on these properties will also be determined using 2 models, so that applicability to asthma treatment can be ascertained. In Aim 2, we will establish the specific GRK phosphorylation sites for the relevant HASM TAS2RS, using site-directed mutagenesis and recombinant expression, so that a precise mechanism ofthe signal quenching is determined. Some TAS2Rs appear to undergo upregulation when exposed chronically to agonists, a phenotype that is opposite to P2-adrenergic receptors which downregulate upon chronic p-agonist treatment. TAS2R upregulation would be an extremely favorable profile, acting to limit tachyphylaxis. Aim 3 will determine which ofthe HASM TAS2Rs display upregulation, the molecular mechanism ofthe process, and the basis for biased ligands to promote the effect. Collectively, these studies will define the molecular and pharmacologic properties of HASM TAS2Rs required for future development of a novel class of bronchodilators for treating asthma.
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0.958 |
2019 — 2021 |
Liggett, Stephen B |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Project 2 - Airway Smooth Muscle Bitter Taste Receptors as Targets For Novel Bronchodilators @ Rutgers Biomedical/Health Sciences-Rbhs
Project Summary Bitter taste receptors (TAS2Rs) are expressed on human airway smooth muscle (HASM) and when activated markedly relax the muscle and dilate the airway. Utilization of this pathway, which is distinct from that of ?- agonists acting at ?2-adrenergic receptors (?2ARs), will provide a new class of direct bronchodilators for treating or preventing bronchospasm in asthma. The TAS2R14 subtype is highly expressed in HASM and is a prime target for developing a novel therapeutic agent. However, there are gaps in our knowledge about the molecular/cellular biology and physiology of HASM TAS2Rs, including how they couple to relaxation, the potential for tachyphylaxis due to short-term (receptor phosphorylation) and long-term (downregulation of receptor expression) events, and the potential to bias receptor signaling towards favorable signaling for asthma treatment. The broad, long-term objective of the Project is to improve our understanding of HASM TAS2R biology relevant to treating airway contraction in asthma. To fill these gaps in our knowledge, in Aim 1 we will define the mechanism by which TAS2Rs evoke relaxation, which we hypothesize is via inhibiting phosphorylation of the actin severing protein cofilin. Studies will be performed in cultured HASM cells derived from nonasthmatic as well as asthmatic donor lungs, the latter being important because of the potential for the disease to modify receptor function. Studies will include siRNA-based knockouts of cofilin, and the upstream components of the proposed pathway that link the receptor:G-protein:effector complex to cofilin. In Aim 2, agonist-prompted phosphorylation of TAS2R14 by GRKs will be studied using whole cell phosphorylation and receptor purification experiments. To define the precise residues phosphorylated by GRKs, TAS2R14 will be mutated to substitute potential Ser/Thr phospho-acceptor sites with Ala, thus defining a bar-code for ?arrestin binding. The consequences of phosphorylation on ?arrestin conformation and intracellular receptor signaling, and HASM relaxation, will then be determined. In Aim 3, a panel of TAS2R14 agonists will be utilized to determine the mechanisms by which a TAS2R agonist can be biased away from deleterious outcomes and towards advantageous outcomes in regards to asthma therapy. This endeavor will provide the basis for agonist-based ?tuning? of the receptor to be highly efficacious in bronchodilating and inhibiting HASM proliferation, but display little short- or long-term agonist-promoted desensitization or downregulation, such that clinical tachyphylaxis is not apparent. All three aims will utilize parallel physiological measurements of contraction and relaxation using nonasthmatic and asthmatic HASM cells, and an inflammatory precision-cut human lung slice model, in order to link biochemical events to clinically relevant physiological responses. Collectively, these studies will provide the basis for development of a novel class of direct bronchodilators which can be utilized alone, or in combination, with ?-agonists for the treatment of asthma.
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0.903 |
2021 |
Liggett, Stephen B |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Characterization of Biased Airway Smooth Muscle Tas2r Agonists For Treating Asthma @ University of South Florida
Asthma remains a major public health issue worldwide, affecting all ages and ethnicities, with 50% of patients experiencing inadequate control of the disease. Constricted airways from contraction of airway smooth muscle is the major cause of airflow obstruction, and morbidity and mortality, in asthma. Despite this key point for intervention, only one class of direct bronchodilators (?-agonists) are available for treatment, and they are suboptimal for many patients. We have discovered bitter taste receptors (TAS2Rs) on human airway smooth muscle (HASM) cells which signal by a unique mechanism. When activated, TAS2Rs markedly relax HASM and relieve airway obstruction that is otherwise resistant to ?-agonists. However, the known TAS2R agonists have low affinity and evoke desensitization (tachyphylaxis) of the relaxation response over time via ?-arrestin mechanisms. The overarching hypothesis is that TAS2R agonists with novel structures can be highly effective in relaxing airways in an asthmatic milieu and yet not evoke tachyphylaxis. The broad long-term objectives are to determine the 3D structures and binding sites of two TAS2Rs expressed on HASM (R5 and R14) and then use these structures to perform virtual docking of an agnostic ultra-large compound library to identify potential ligands. These hits will be examined in engineered model cells, but will be most intensely studied within the context of the physiology of HASM cells, and human airways, under asthmatic conditions to delineate novel ways to engage the receptor that are biased towards relaxation and away from desensitization. Aim 1 will computationally determine the structures and binding sites for R5 and R14 using methods that include 13 trillion combinations of the residues to determine favorable energy conformations of inactive and active states. The structures will be used to dock compounds from a highly diverse library. These will be studied in Aim 2 to determine potency and efficacy in genetically engineered model cells and in HASM cells from nonasthmatic and asthmatic lungs. In Aim 3, the most favorable compounds will be studied to ascertain ?-arrestin engagement and biasing away from desensitization in model cells, HASM cells, and human lungs under asthmatic conditions. Results from Aims2/3 will be fed back into Aim1 to further refine a model of biased agonists for these HASM TAS2Rs. Based on our preliminary data that support all three Aims, we will learn about the structural requirements for biasing for these receptors. And, we anticipate that multiple highly effective agonists with unexpected structures will be identified and that their bronchodilating properties in asthma will represent a new class of powerful non-desensitizing agents for treating and preventing bronchospasm.
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0.955 |