Duke University

Massachusetts Institute of Technology

Robert J Lefkowitz

Marc G Caron

Walter J Koch

Klim King

Henrik G Dohlman

Jonathan S Stamler

Claude A Piantadosi

Jeffrey L Benovic

Howard A Rockman

Erin J Whalen

Martin J Lohse

Carmelo A Milano

Per-Otto Hagen

Mark Firman Bear

Louis M Luttrell

Shahab A Akhter

Laura Jane Stoppel

Henrick G Dohlman

Laura M Bohn

Fang-Tsyr Lin

Benjamin David Auerbach

Howard A Royman

Susanna Cotecchia

Lee F Allen

Laura Wingler

Sudha Shenoy

Alem W Kahsai

Biswaranjan Pani

Seungkirl Ahn

Dean Staus

The disclosure is directed to a G-protein coupled receptor complex. The complex includes (i) a chimeric G protein-coupled receptor (GPCR) comprising a non-native amino acid sequence located within the C-terminus of the GPCR and a synthetic phosphopeptide ligated to the non-native amino acid sequence; and (ii) a β-arrestin (βarr) protein bound to the C-terminus of the GPCR. The disclosure also provides an in vitro method for producing the aforementioned complex, as well as methods for identifying compounds or ligands which bind to and modulate the activity of the complex. Positive allosteric modulators of the β2 adrenergic receptor identified by screening a DNA-encoded library potentiate the activity of β2 agonists and have application in the treatment of obstructive airway disease, bronchospasm, or pre-term labor.

System and method for homogenous GPCR phosphorylation and identification of beta-2 adrenergic receptor positive allosteric modulators

Disclosed herein are novel methodologies of treating fragile X syndrome and related disorders by inhibiting mGlu-relevant signaling pathways via the reduction of &#x3b2;-arrestin2 protein levels or the diminution of mGluand &#x3b2;-arrestin2 protein interactions.

Methods of treating fragile X syndrome and related disorders

The present invention relates to a modified arrestin which includes an arrestin molecule and a ubiquitin molecule. This modified arrestin has an increased affinity for a GPCR, and traffics with the GPCR into endosomes. The present invention further relates to a method of screening compounds and sample solutions for a GPCR agonist, antagonist, inverse agonist, or desensitization active compound. The modified arrestin is useful in the methods of the present invention.

Modified trafficking patterns for arrestin and G-protein-coupled receptors via arrestin-ubiquitin chimera

Disclosed is a transformed yeast cell containing a first heterologous DNA sequence which codes for a mammalian G protein-coupled receptor and a second heterologous DNA sequence which codes for a mammalian G protein &#x3b1; subunit (mammalian G). The first and second heterologous DNA sequences are capable of expression in the cell, but the cell is incapable of expressing an endogenous G protein &#x3b1;-subunit (yeast G). The cells are useful for screening compounds which affect the rate of dissociation of Gfrom Gin a cell. Also disclosed is a novel DNA expression vector useful for making cells as described above. The vector contains a first segment comprising at least a fragment of the extreme amino-terminal coding sequence of a yeast G protein-coupled receptor. A second segment is positioned downstream from the first segment (and in correct reading frame therewith), with the second segment comprising a DNA sequence encoding a heterologous G protein-coupled receptor.

Expression of G protein coupled receptors in yeast

The present invention deals with gene therapy for treating chronic heart failure and other cardiac disease states which are accompanied by a reduced number or functioning of myocardial beta-adrenergic receptors (&#x3b2;-AR). &#x3b2;-AR receptor function is augmented in transgenic animals by delivery and expression of a beta-2-adrenergic receptor gene or a gene encoding a beta adrenergic receptor kinase inhibitor, resulting in increased in vivo left ventricular function. The present invention includes recombinant plasmid vectors, alternative beta-adrenergic receptor gene delivery strategies, and transgenic mice carrying a &#x3b2;-AR transgene, a &#x3b2;-ARK transgene, or a &#x3b2;-ARK inhibitor transgene.

Use of exogenous &#x3b2;-adrenergic receptor and &#x3b2;-adrenergic receptor kinase gene constructs to enhance myocardial function

Desensitization of receptors that control disease is prevented by inhibiting G-protein receptor kinases. This has applicability, e.g., for patients with heart failure or on a left ventricular heart device or a heart pump after surgery or about to undergo surgery and at high risk for a cardiac event or on an opiate or addicted to opiate or with cystic fibrosis or rheumatoid arthritis.

Preventing desensitization of receptors

Disclosed is a transformed yeast cell containing a first heterologous DNA sequence which codes for a mammalian G protean coupled receptor and a second heterologous DNA sequence which codes for a mammalian G protein &#x3b1; subunit (mammalian G). The first and second heterologous DNA sequences are capable of expression in the cell, but the cell is incapable of expressing an endogenous G protein &#x3b1;-subunit (yeast G). The cells are useful for screening compounds which affect the rate of dissociation of Gfrom Gin a cell. Also disclosed is a novel DNA expression vector useful for making cells as described above. The vector contains a first segment comprising at least a fragment of the extreme amino-terminal coding sequence of a yeast G protein coupled receptor. A second segment is positioned downstream from the first segment (and in correct reading frame therewith), with the second segment comprising a DNA sequence encoding a heterologous G protein coupled receptor.

The present invention relates, in general, to vascular smooth muscle proliferation and, in particular, to a method of inhibiting arterial and venous smooth muscle proliferation resulting, for example, from arterial injury or vein grafting. The invention also relates to an expression construct encoding a G&bgr;&ggr; inhibitor suitable for use in such a method.

Method of inhibiting smooth muscle cell proliferation

The present invention relates, in general, to myocardial hypertrophy and, in particular, to agents that inhibit cardiac Gq-coupled receptor signaling and to methods of inhibiting myocardial hypertrophy using same.

Intracellular inhibitors of Gq protein signaling

Method of detecting inhibitors of agonist-specific desensitization

The present invention provides a &bgr;arrestin knockout mouse useful for screeening compounds for efficacy in controlling pain, methods of controlling pain in subjects by inhibiting binding of &bgr;arrestin to phosphorylated &mgr; opioid receptors, and methods of screening a compound for activity in potentiating &mgr; opioid receptor agonist activity (e.g., morphine activity) by determining whether or not said compound inhibits &bgr;arrestin binding to a phosphorylated &mgr; opioid receptor.

Inhibition of &bgr;arrestin mediated effects prolongs and potentiates opioid receptor-mediated analgesia

The present invention deals with gene therapy for treating chronic heart failure and other cardiac disease states which are accompanied by a reduced number or functioning of myocardial beta-adrenergic receptors (&bgr;-AR). &bgr;-AR receptor function is augmented in transgenic animals by delivery and expression of a beta-2-adrenergic receptor gene or a gene encoding a beta adrenergic receptor kinase inhibitor, resulting in increased in vivo left ventricular function. The present invention includes recombinant plasmid vectors, alternative beta-adrenergic receptor gene delivery strategies, and transgenic mice carrying a &bgr;-AR transgene, a &bgr;-ARK transgene, or a &bgr;-ARK inhibitor transgene.

Use of exogenous &bgr;-adrenergic receptor and &bgr;-adrenergic receptor kinase gene constructs to enhance myocardial function

Disclosed is a transformed yeast cell containing a first heterologous DNA sequence which codes for a mammalian G protein coupled receptor and a second heterologous DNA sequence which codes for a mammalian G protein &agr; subunit (mammalian G,). The first and second heterologous DNA sequences are capable of expression in the cell, but the cell is incapable of expressing an endogenous G protein &agr;-subunit (yeast G,) The cells are useful for screening compounds which affect the rate of dissociation of G,from G,in a cell. Also disclosed is a novel DNA expression vector useful for making cells as described above. The vector contains a first segment comprising at least a fragment of the extreme amino-terminal coding sequence of a yeast G protein coupled receptor. A second segment is positioned downstream from the first segment (and in correct reading frame therewith), with the second segment comprising a DNA sequence encoding a heterologous G protein coupled receptor.

The present invention relates to a method of inhibiting desensitization of a cell to the effects of a compound. The method comprises contacting the cell with an agent capable of inhibiting phosphorylation, by a protein kinase, of a receptor for the compound present on the surface of the cell. The present invention also relates to a method of screening a compound for its ability to inhibit desensitization. The method comprises: i) contacting a receptor specific kinase-containing sample with the compound under conditions such that interaction between receptor specific kinase present in the sample and the compound can occur, and

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Durham, NC, United States of America

Robert J Lefkowitz