Precise regulation of heterotrimeric G-protein signaling is important for maintaining proper cardiovascular system function. Indeed, G-protein signaling is frequently upregulated during cardiovascular disease suggesting that identifying mechanisms for inhibiting G-protein signaling may be an effective therapeutic strategy for the treatment and prevention of disease. The work presented in this thesis is directed at two RGS proteins, RGS2 and RGS5, the two highest expressing RGS proteins in VSMCs. Despite the large number of studies published on them, there is still much to be learned about the specific G-protein pathways that each RGS protein controls. Using genetic and molecular models, we set out to identify novel regulatory pathways controlling RGS2 and RGS5 function. We hypothesize that characterizing the determinants and regulation of RGS protein function will provide a better understanding of the signaling that occurs within VSMCs under both physiologic and pathophysiologic conditions. Our work presented in the first three studies of this thesis, describes novel regulatory pathways that are involved in regulating RGS2 protein function. We describe the production of RGS2 protein isoforms that are the result of alternative translational start site usage. Interestingly, the expression pattern of these proteins is controlled by the signaling status of the cell. In the second two studies, we identify a functional consequence of RGS2-interaction with the plasma membrane. We show that this is dependent on the interaction between the amphipathic α-helix and anionic phospholipids present in the plasma membrane. We further show that disruptions in this interaction, as occurs in the human population, can lead to reduced RGS2 function and thus potentially hypertension. Finally, our last study focuses on the function and regulation of RGS5, the single highest expressing RGS protein in VSMCs. We show that the regulation of RGS5 is dependent, similar to other VSMC-specific genes, on the activity of SRF and myocardin. However, interestingly, RGS5 expression is further controlled by the extent of DNA methylation that occurs in its proximal promoter. We show that this is an important regulator of RGS5 expression both in development as well as during disease, specifically in-stent restenosis.

Precise regulation of heterotrimeric G-protein signaling is important for maintaining proper cardiovascular system function. Indeed, G-protein signaling is frequently upregulated during cardiovascular disease suggesting that identifying mechanisms for inhibiting G-protein signaling may be an effective therapeutic strategy for the treatment and prevention of disease. The work presented in this thesis is directed at two RGS proteins, RGS2 and RGS5, the two highest expressing RGS proteins in VSMCs. Despite the large number of studies published on them, there is still much to be learned about the specific G-protein pathways that each RGS protein controls. Using genetic and molecular models, we set out to identify novel regulatory pathways controlling RGS2 and RGS5 function. We hypothesize that characterizing the determinants and regulation of RGS protein function will provide a better understanding of the signaling that occurs within VSMCs under both physiologic and pathophysiologic conditions. Our work presented in the first three studies of this thesis, describes novel regulatory pathways that are involved in regulating RGS2 protein function. We describe the production of RGS2 protein isoforms that are the result of alternative translational start site usage. Interestingly, the expression pattern of these proteins is controlled by the signaling status of the cell. In the second two studies, we identify a functional consequence of RGS2-interaction with the plasma membrane. We show that this is dependent on the interaction between the amphipathic α-helix and anionic phospholipids present in the plasma membrane. We further show that disruptions in this interaction, as occurs in the human population, can lead to reduced RGS2 function and thus potentially hypertension. Finally, our last study focuses on the function and regulation of RGS5, the single highest expressing RGS protein in VSMCs. We show that the regulation of RGS5 is dependent, similar to other VSMC-specific genes, on the activity of SRF and myocardin. However, interestingly, RGS5 expression is further controlled by the extent of DNA methylation that occurs in its proximal promoter. We show that this is an important regulator of RGS5 expression both in development as well as during disease, specifically in-stent restenosis.

Transmembrane signal transduction by ligand-activated G protein-coupled receptors (GPCRs) controls virtually every aspect of mammalian physiology, and this receptor class is the target of 40-50% of currently marketed pharmaceuticals. In addition to the clinical use of direct GPCR agonists and antagonists, it is now believed that GPCR effectors and regulators may also be viable drug targets with improved therapeutic efficacy and specificity. The prototypic role of Regulator of G protein Signaling (RGS) proteins is inhibition of G protein signaling through acceleration of GTP hydrolysis by GΑ, which promotes re-association of GΑ and GΒΓ subunits with the receptor at the cell membrane. In this way, RGS proteins determine the magnitude and duration of the cellular response to GPCR stimulation. Though RGS protein biochemistry has been well elucidated in vitro, the physiological functions of each RGS family member remain largely unexplored. RGS6 belongs to the R7 subfamily of RGS proteins originally identified in brain. Our acquisition of an RGS6-/- mouse allowed us to survey RGS6 expression in all tissues of the body revealing the greatest expression of RGS6 in brain. Despite robust neural RGS6 expression, little is known regarding functional roles of RGS6 in the brain and spinal cord. In addition, we identified several novel, higher molecular weight RGS6 immunoreactive bands specifically present in the nervous system. The plan of this thesis work was multifaceted. We sought to elucidate novel GPCR signaling cascades modulated by RGS6 in brain while simultaneously characterizing the expression patterns and identity of the novel RGS6 species specifically detected in the nervous system. Considering the large diversity of RGS6 isoforms present in brain, the abundance of potential RGS6 binding partners, and the possibility of discovering new mechanisms involved in RGS6 regulation, elucidation of the novel RGS6 molecular species is of paramount importance. Utilizing RGS6-/- mice we identified RGS6 as a critical modulator of two GPCRs in brain. First, by inhibiting the serotonin receptor 1A (5-HT1AR)-adenylyl cyclase (AC) axis, RGS6 functions to promote anxiety- and depression-related behaviors in mice. As a result, RGS6-/- mice exhibit a robust anxiolytic and antidepressant phenotype remarkably similar to that of animals treated chronically with therapeutic doses of selective serotonin reuptake inhibitors (SSRIs). RGS6 also inhibits GABAB receptor (GABABR)-G protein- activated inwardly rectifying potassium (GIRK) channel current in cerebellar granule cells, and loss of RGS6 results in cerebellar ataxia and gait abnormalities reversible by GABABR blockade. Furthermore, evaluation of voluntary alcohol drinking behaviors in WT versus RGS6-/- mice revealed a striking reduction in alcohol intake resulting from RGS6 loss in both acute and chronic alcohol consumption paradigms due, at least in part, to potentiation of GABABR signaling. Thus, RGS6 inhibitors have potential clinical utility in the treatment of mood disorders and alcoholism. We have shown that one novel RGS6 immunoreactive band expressed in the brain and spinal cord is a phospho-protein sensitive to Λ phosphatase-mediated dephosphorylation. Further, new information acquired from PCR amplification of RGS6 mRNA species from human brain cDNA libraries has necessitated substantial revisions to the RGS6 splicing scheme devised by the Fisher laboratory in 2003. To the 36 isoforms generated from two alternate transcription start sites (RGS6L vs. RGS6), the inclusion or exclusion of exons 14 and 17, and variable splicing to one of 7 different 3' terminal exons, we have added the possible insertion of three novel internal exons (A1, A2, A3), a retained intron, and two new 3' terminal exons. As a result, the number of RGS6 mRNAs present in brain could be as many as 248 unique species, an astonishing diversity unprecedented in the RGS protein family.