Abstract

. The heterotrimeric G protein Gaq regulates platelet activation, blood pressure and cardiac function. While Gaq is best known for its ability to stimulate phospholipase Cp (PLC|3), it also binds to G protein-coupled receptor kinase 2 (GRK2), which competitively inhibits PLCp activation, and to p63RhoGEF, a Rho guanine nucleotide exchange factor that modulates cytoskeletal structure. We recently demonstrated that a functional Gaq chimera could be produced in amounts sufficient for the structure determination of the Gaq-GRK2-Gpy complex. The surprising arrangement of activated heterotrimeric G proteins in this assembly suggested that RGS proteins and receptors could also associate to form even higher order signaling complexes.
Aim 1 examines the ability of RGS proteins such as RGS2 to bind GRK2- bound Gaq using a flow-cytometry binding assay, size-exclusion chromatography and crystallographic studies of Gaq-RGS and RGS-Gaq-GRK2-Gpy complexes.
Aim 2 investigates changes in the orientation of activated Gaq at the membrane upon effector binding as well as the interactions of Gaq-GRK2-Gpv and RGS proteins with intact receptors or their cytoplasmic loops in a membrane environment. The Gaq chimera also opens the door to the structural analysis of other Gaq-effector interactions. To initiate these efforts, Aim 3 seeks to define the molecular basis for Gaq-mediated activation of p63RhoGEF through site-directed mutagenesis, fluorescence polarization nucleotide exchange assays and structural studies. Relevance. By focusing our proposal on two unique effectors of Gaq,GRK2 and p63RhoGEF, we seek to define general paradigms for heterotrimeric G protein signaling through Gaq. We have also focused onGaq, GRK2, RGS2 and p63RhoGEF because all are strongly linked to cardiovascular physiology and disease, and it is not unreasonable to expect that these proteins coordinate their activities, either directly or indirectly, in living cells. GRK2 and Gaq/n are essential for proper heart development and function, RGS2 regulates blood pressure via attenuation of Gaq signaling, and p63RhoGEF induces changes in myocytes that are characteristic of cardiachypertrophy.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL086865-04
Application #
7758792
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Adhikari, Bishow B
Project Start
2007-01-15
Project End
2010-12-31
Budget Start
2010-01-01
Budget End
2010-12-31
Support Year
4
Fiscal Year
2010
Total Cost
$369,600
Indirect Cost

  Institution

Name
University of Michigan Ann Arbor
Department
Pharmacology
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109

  Publications

Waldschmidt, Helen V; Homan, Kristoff T; Cato, Marilyn C et al. (2017) Structure-Based Design of Highly Selective and Potent G Protein-Coupled Receptor Kinase 2 Inhibitors Based on Paroxetine. J Med Chem 60:3052-3069
Waldschmidt, Helen V; Homan, Kristoff T; Cruz-Rodríguez, Osvaldo et al. (2016) Structure-Based Design, Synthesis, and Biological Evaluation of Highly Selective and Potent G Protein-Coupled Receptor Kinase 2 Inhibitors. J Med Chem 59:3793-807
Tesmer, John J G (2016) Hitchhiking on the heptahelical highway: structure and function of 7TM receptor complexes. Nat Rev Mol Cell Biol 17:439-50
Yang, Pei; Homan, Kristoff T; Li, Yaoxin et al. (2016) Effect of Lipid Composition on the Membrane Orientation of the G Protein-Coupled Receptor Kinase 2-G?1?2 Complex. Biochemistry 55:2841-8
Wang, Yan; Virtanen, Jouko; Xue, Zhidong et al. (2016) Using iterative fragment assembly and progressive sequence truncation to facilitate phasing and crystal structure determination of distantly related proteins. Acta Crystallogr D Struct Biol 72:616-28
Taylor, Veronica G; Bommarito, Paige A; Tesmer, John J G (2016) Structure of the Regulator of G Protein Signaling 8 (RGS8)-G?q Complex: MOLECULAR BASIS FOR G? SELECTIVITY. J Biol Chem 291:5138-45
Tesmer, John J G (2016) Crystallographic Pursuit of a Protein-RNA Aptamer Complex. Methods Mol Biol 1380:151-60
Cash, Jennifer N; Davis, Ellen M; Tesmer, John J G (2016) Structural and Biochemical Characterization of the Catalytic Core of the Metastatic Factor P-Rex1 and Its Regulation by PtdIns(3,4,5)P3. Structure 24:730-740
Homan, Kristoff T; Waldschmidt, Helen V; Glukhova, Alisa et al. (2015) Crystal Structure of G Protein-coupled Receptor Kinase 5 in Complex with a Rationally Designed Inhibitor. J Biol Chem 290:20649-59
Schumacher, Sarah M; Gao, Erhe; Zhu, Weizhong et al. (2015) Paroxetine-mediated GRK2 inhibition reverses cardiac dysfunction and remodeling after myocardial infarction. Sci Transl Med 7:277ra31

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