Role of Gbeta 2 in the Nucleus of Angiotensin Receptor Activated Cells

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DESCRIPTION (provided by applicant): The rennin-angiotensin system hormone, angiotensin II (Ang II) is a master regulator of normal cardiovascular physiology. Normally, Ang II regulates acute changes in blood pressure and water-electrolyte balance. Under pathological conditions, Ang II modulates gene expression leading to a remodeling program in the target cells. Both types of Ang II actions are mediated by AT1 receptors. Antagonists of AT1 receptor are used in antihypertensive therapy and are currently in clinical trial for prevention of congestive heart failure and other types of end organ damage. These drugs reverse the remodeling gene expression changes in addition to controlling the acute actions of Ang II. Therefore, a mechanistic understanding of modulation of gene expression by AT1 receptor is essential. We approached this problem by tracking protein-protein interactions in the nuclear compartment of Ang II receptor activated cells. To identify novel protein complexes involved in the modulation of gene expression, we analyzed nuclear proteome of AT1 receptor-activated cells by high throughput mass-spectrometry. Among the candidate proteins that translocated to the nucleus, the G-protein beta2 (G(32) subunit polypeptide was a surprising finding. GP is a component of heterotrimeric G proteins which are vital transducers of GPCR signals. Activation of a GPCR, such as AT1 receptor upon Ang II binding, causes catalytic dissociation of G-proteins into Gpy and Got subunits which in turn activate their respective effectors. The GP and Gy subunits always remain in complex and they are generally tethered to the inner face of the plasma membrane by lipid modifications on Gy. Therefore, the GP has traditionally been thought to transduce signals at plasma membrane and has never been shown to be part of the nuclear proteome. We found that Gp2 translocated into the nucleus upon AT1 receptor activation and formed a complex with HDAC5 and alpha-actinin-4. Actinin-4 is a calcium binding protein and HDAC5 is a chromatin remodeling enzyme. Hence, we hypothesize that Ang II activated AT1 receptor stimulates nuclear translocation of GP and assembly of a calcium sensitive chromatin remodeling complex involving actinin-4 and HDAC5. The goals of this project are (i) to determine the specificity of interaction between these three molecules using mutagenesis and Biacore analysis and (ii) to establish the functional significance of this complex formation by microarray analysis of AT1 receptor-dependent gene expression in RNAi knock-down of Gbeta 2 in cells. Our proposed studies will demonstrate a novel mode of control of gene expression by a GPCR.