Beta-adrenergic receptor (bAR) signaling is one of the most powerful regulators of cardiac function. In human heart failure, diminished receptor numbers at the plasma membrane associated with impaired G-protein coupling (desensitization) results in reduced responsiveness to neuro-hormones. Receptor desensitization is initiated by the phosphorylation of agonist activated bARs by bAR kinase-1 (bARK1). b-arrestin binds to the phosphorylated receptor resulting in the loss of effector (adenylyl cyclase) signaling. The bAR complex is targeted for endocytosis resulting in dephosphorylation of the receptor in the endosomal compartment before being recycled back to the plasma membrane. Previously we have shown that bARK1 interacts with phosphoinositide 3-kinase (PI3K) to form a cytosolic complex targeting PI3K to the activated receptor where PI3K plays a role in receptor endocytosis. We have now uncovered a novel phenomenon of receptor resensitization at the plasma membrane in vivo in mice by cardiac overexpression of an inactive PI3K mutant. Furthermore, we have demonstrated that bAR resensitization is beneficial as it prevents deleterious cardiac remodeling through preservation of bAR function. These preliminary data are contrary to the current paradigm of receptor resensitization which articulates that phosphorylated-desensitized receptor has to undergo internalization to be dephosphorylated before being recycled back to the plasma membrane. The molecular mechanism regulating this novel phenomenon of plasma membrane receptor-resensitization is not known. We hypothesize that receptor targeted PI3K activity negatively regulates receptor resensitization at the plasma membrane. Therefore, inhibition of receptor localized PI3K activity results in receptor resensitization at the plasma membrane without the need for internalization. The following specific aims are proposed in this study: 1) To determine whether PI3K activity regulates bAR resensitization. Detailed analysis of receptor resensitization by G-protein coupling, receptor phosphorylation, adenylyl cyclase activity/cAMP levels will be performed using PI3K mutants containing protein or/and lipid kinase activity. 2) To delineate whether PI3K-mediated bAR resensitization occurs through regulation of protein phosphatase activity. Analysis on regulation of protein phosphatase activity (PP1, PP2A etc.,) (in vivo and in vitro) by PI3K mutants and inhibitors will be carried out to define the role of PI3K activity. 3) To determine the molecular mechanism underlying the regulation of PP2A activity by PI3K. In-depth studies on regulation of phosphatase activity by lipid binding or phosphorylation of PP2A subunits and inhibitors of protein phosphatases (I-PP2As) will be performed using lipid binding studies, metabolic labeling, RNAi knock down and the use of PP2A and I2-PP2a mutants. PROJECT NARRATIVE: b-adrenergic receptors (bARs) belong to the largest family of cell surface receptors and are one of the strongest regulators of cardiac function. Human heart failure is characterized by downregulation (loss from the cell surface) and chronic desensitization (inability of the receptor to signal) of bARs. While the phenomenon of desensitization is well studied, resensitization (a process by which receptors rejuvenate and become competent to signal) is not well understood. Studies in our grant proposal will delineate this novel mechanism. Elucidation of this mechanism will lead to identification of molecules that would allow us to develop novel therapeutic strategies for heart failure by targeting resensitization. This is critical because the majority of the current therapeutic strategies involve targeting the receptor to attenuate downregulation. Importantly, determining the mechanism of resensitization will have broad universality as it is applicable to other G-protein coupled receptors.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL089473-03
Application #
7792377
Study Section
Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
Program Officer
Adhikari, Bishow B
Project Start
2008-04-01
Project End
2013-03-31
Budget Start
2010-04-01
Budget End
2011-03-31
Support Year
3
Fiscal Year
2010
Total Cost
$386,250
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
135781701
City
Cleveland
State
OH
Country
United States
Zip Code
44195
Mohan, Maradumane L; Naga Prasad, Sathyamangla V (2017) Scaffolding Function of PI3Kgamma Emerges from Enzyme's Shadow. J Mol Biol 429:763-772
Mohan, Maradumane L; Chatterjee, Arunachal; Ganapathy, Swetha et al. (2017) Noncanonical regulation of insulin-mediated ERK activation by phosphoinositide 3-kinase ?. Mol Biol Cell 28:3112-3122
Kiranmayi, Malapaka; Chirasani, Venkat R; Allu, Prasanna K R et al. (2016) Catestatin Gly364Ser Variant Alters Systemic Blood Pressure and the Risk for Hypertension in Human Populations via Endothelial Nitric Oxide Pathway. Hypertension 68:334-47
Watson, Lewis J; Alexander, Kevin M; Mohan, Maradumane L et al. (2016) Phosphorylation of Src by phosphoinositide 3-kinase regulates beta-adrenergic receptor-mediated EGFR transactivation. Cell Signal 28:1580-92
Sysa-Shah, Polina; Tocchetti, Carlo G; Gupta, Manveen et al. (2016) Bidirectional cross-regulation between ErbB2 and ?-adrenergic signalling pathways. Cardiovasc Res 109:358-73
Gupta, Manveen K; Asosingh, Kewal; Aronica, Mark et al. (2015) Defective Resensitization in Human Airway Smooth Muscle Cells Evokes ?-Adrenergic Receptor Dysfunction in Severe Asthma. PLoS One 10:e0125803
Tirupula, Kalyan C; Ithychanda, Sujay S; Mohan, Maradumane L et al. (2015) G protein-coupled receptors directly bind filamin A with high affinity and promote filamin phosphorylation. Biochemistry 54:6673-83
Ithychanda, Sujay Subbayya; Fang, Xianyang; Mohan, Maradumane L et al. (2015) A mechanism of global shape-dependent recognition and phosphorylation of filamin by protein kinase A. J Biol Chem 290:8527-38
Mohan, Maradumane L; Jha, Babal K; Gupta, Manveen K et al. (2013) Phosphoinositide 3-kinase ? inhibits cardiac GSK-3 independently of Akt. Sci Signal 6:ra4
Gupta, Manveen K; Halley, Carmel; Duan, Zhong-Hui et al. (2013) miRNA-548c: a specific signature in circulating PBMCs from dilated cardiomyopathy patients. J Mol Cell Cardiol 62:131-41

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