The R7 family of regulators of G protein signaling (RGS) proteins, comprising RGS6, RGS7, RGS9, and RGS11, regulate neuronal G protein signaling pathways. All members of the R7 RGS form trimeric complexes with the atypical G protein ␤ subunit, G␤5, and membrane anchor R7BP or R9AP. Association with G␤5 and membrane anchors has been shown to be critical for maintaining proteolytic stability of the R7 RGS proteins. However, despite its functional importance, the mechanism of how R7 RGS forms complexes with G␤5 and membrane anchors remains poorly understood. Here, we used protein-protein interaction, co-localization, and protein stability assays to show that association of RGS9 with membrane anchors requires G␤5. We further establish that the recruitment of R7BP to the complex requires an intact interface between the N-terminal lobe of RGS9 and protein interaction surface of G␤5. Site-directed mutational analysis reveals that distinct molecular determinants in the interface between G␤5 and N-terminal Dishevelled, EGL-10, Pleckstrin/DEP Helical Extension (DEP/DHEY) domains are differentially involved in R7BP binding and proteolytic stabilization. On the basis of these findings, we conclude that G␤5 contributes to the formation of the binding site to the membrane anchors and thus is playing a central role in the assembly of the proteolytically stable trimeric complex and its correct localization in the cell. Regulators of G protein signaling (RGS)2 proteins are key components of G protein-coupled receptor (GPCR) signaling pathways (1). They limit the duration that heterotrimeric G proteins spend in their active state and thus serve as powerful negative regulators of GPCR signal transmission. Biochemically, RGS proteins function as GTPase-accelerating proteins that facilitate GTP hydrolysis on G protein ␣ subunits (G␣) thereby promoting their inactivation (2, 3). There are more than 30 RGS proteins that are grouped into six families according to the homology and structural features (3, 4). Cumulatively, RGS proteins are thought to be ubiquitously expressed and have been demonstrated to regulate a range of the vital physiological processes (5-8).The R7 family of RGS proteins comprises four members: RGS6, RGS7, RGS9, and RGS11 and plays prominent roles in the nervous and cardiovascular systems (9, 10). The best studied member of this family, RGS9, is involved in regulating diverse GPCR signaling cascades in mammals (11, 12). Its short splice isoform, RGS9-1, regulates phototransduction cascade in rod and cone photoreceptors of the retina and is essential for the high temporal resolution of our vision (13-15). The long splice isoform, RGS9-2, is predominantly expressed in the striatum, a region of the brain involved in movement control and reward behavior (16 -18). RGS9-2 is thought to control G protein signaling downstream from the -opioid and D2 dopamine receptors and has been implicated in drug addiction and movement disorders (19 -23).Both RGS9 splice isoforms exist as constitutive heterotrimers with two other protei...

The human sodium-dependent vitamin C transporter 1 (hSVCT1) contributes to cellular uptake of ascorbic acid (AA). Although different aspects of hSVCT1 cell biology have been extensively studied, nothing is currently known about the broader hSVCT1 interactome that modulates its role in cellular physiology. Here, we identify the enzyme human glyoxalate reductase/hydroxypyruvate reductase (hGR/HPR) as an hSVCT1 associated protein by yeast two-hybrid (Y2H) screening of a human liver cDNA library. The interaction between hSVCT1 and hGR/HPR was further confirmed by in vitro GST pull-down assay, in vivo coimmunoprecipitation and mammalian two-hybrid firefly luciferase assays. This interaction had functional significance as coexpression of hGR/HPR with hSVCT1 led to an increase in AA uptake. Reciprocally, siRNA-mediated knockdown of endogenous hGR/HPR led to an inhibition of AA uptake. Given that oxalate is a degradation product of vitamin C and hGR/HPR acts to limit cellular oxalate levels, this association physically couples two independent regulators of cellular oxalate production. Furthermore, confocal imaging of human liver HepG2 cells coexpressing GFP-hSVCT1 and hGR/HPR-mCherry demonstrated that these two proteins colocalize within a subpopulation of intracellular organelles. This provides a possible molecular basis for organellar AA transport and regulation of local glyoxylate/glycolate concentration in the vicinity of organelle membranes.

Thunderstorms in central Florida frequently halt outdoor activities, requiring that one wait some prescribed time after an assumed last flash before safely resuming activities. The goal of this research is to develop a high-skill probabilistic method that can be used in high pressure real-world operations to terminate lightning warnings more quickly while maintaining safety. Probabilistic guidance tools are created for isolated warm season storms in central Florida using dual-polarized radar data at 1-min intervals. The parameters examined are maximum reflectivity and graupel presence at the 0°, −5°, −10°, −15°, and −20°C levels as well as composite reflectivity. Random samples of the radar data are used to train a generalized linear model (GLM) to make a probabilistic prediction whether a given flash is the storm’s last flash. The most statistically significant predictors for lightning cessation are found to be the storm’s maximum reflectivity in the composite and the 0°C levels, along with graupel presence or absence at the −5°, −10°, −15°, and −20°C levels. Statistical verification is used to analyze the performance of the two GLMs at different probability thresholds (95.0%, 97.5%, and 99.0%). When applying the cessation guidance as though storms are occurring in real time, results showed ~99% of the storms produced no additional lightning after the GLM suggested cessation had already occurred. Although these results are encouraging, the procedure must be tested on much larger datasets having different convective modes and different areal coverages to prove its value compared to operational forecasters.