RGS9-2 rescues dopamine D2 receptor levels and signaling in DYT1 dystonia mouse models.

Dopamine D2 receptor signaling is central for striatal function and movement, while abnormal activity is associated with neurological disorders including the severe early-onset DYT1 dystonia. Nevertheless, the mechanisms that regulate D2 receptor signaling in health and disease remain poorly understood. Here, we identify a reduced D2 receptor binding, paralleled by an abrupt reduction in receptor protein level, in the striatum of juvenile Dyt1 mice. This occurs through increased lysosomal degradation, controlled by competition between ß-arrestin 2 and D2 receptor binding proteins. Accordingly, we found lower levels of striatal RGS9-2 and spinophilin. Further, we show that genetic depletion of RGS9-2 mimics the D2 receptor loss of DYT1 dystonia striatum, whereas RGS9-2 overexpression rescues both receptor levels and electrophysiological responses in Dyt1 striatal neurons. This work uncovers the molecular mechanism underlying D2 receptor downregulation in Dyt1 mice and in turn explains why dopaminergic drugs lack efficacy in DYT1 patients despite significant evidence for striatal D2 receptor dysfunction. Our data also open up novel avenues for disease-modifying therapeutics to this incurable neurological disorder.

Sequential Fractionation Strategy Identifies Three Missing Proteins in the Mitochondrial Proteome of Commonly Used Cell Lines.

Mitochondria are undeniably the cell powerhouse, directly affecting cell survival and fate. Growing evidence suggest that mitochondrial protein repertoire affects metabolic activity and plays an important role in determining cell proliferation/differentiation or quiescence shift. Consequently, the bioenergetic status of a cell is associated with the quality and abundance of the mitochondrial populations and proteomes. Mitochondrial morphology changes in the development of different cellular functions associated with metabolic switches. It is therefore reasonable to speculate that different cell lines do contain different mitochondrial-associated proteins, and the investigation of these pools may well represent a source for mining missing proteins (MPs). A very effective approach to increase the number of IDs through mass spectrometry consists of reducing the complexity of the biological samples by fractionation. The present study aims at investigating the mitochondrial proteome of five phenotypically different cell lines, possibly expressing some of the MPs, through an enrichment-fractionation approach at the organelle and protein level. We demonstrate a substantial increase in the proteome coverage, which, in turn, increases the likelihood of detecting low abundant proteins, often falling in the category of MPs, and resulting, for the present study, in the identification of METTL12, FAM163A, and RGS13. All MS data have been deposited to the MassIVE data repository ( https://massive.ucsd.edu ) with the data set identifier MSV000082409 and PXD010446.

Regulation of the regulator of G protein signaling 2 expression and cellular localization by PKA and PKC pathways in mouse granulosa cells.

G protein-coupled receptor (GPCR) activation-mediated PKA and PKC pathways have been recognized to be important in ovarian physiology. Expression of regulator of G-protein signaling 2 (RGS2) has been reported in ovarian granulosa cells. The detailed mechanisms in PKA- and PKC-regulated RGS2 expression and cellular translocation in granulosa cells remain mostly unclear. PKA activator 8-bromo-cAMP and PKC activator phorbol-12, 13-didecanoate appeared to rapidly elevate both protein and mRNA levels and promoter activation of RGS2 gene. Two consensus Sp1 elements within the shortest 78 bp fragment of RGS2 promoter sequence were essential for the full responsiveness to PKA and PKC. PKC activation appeared to increase the RGS2 translocation from nucleus to cytosol. PKA- and PKC-mediated RGS2 transcription in a Sp-1-dependent manner and a PKC-mediated RGS2 intracellular translocation were noted in granulosa cells.

RGS1 expression is associated with poor prognosis in multiple myeloma.

AIMS: Multiple myeloma (MM) is an invariably fatal disease with highly heterogeneous outcome. Because of this heterogeneity of MM, risk stratification is crucial for therapeutic decision-making. However, no immunohistochemical prognostic or predictive markers have been established yet. The expression of regulator of G-protein signalling (RGS) proteins, which desensitise G-protein-coupled receptor signalling, has been reported to be associated with the prognosis of various malignancies. Recently, our group demonstrated the importance of RGS1 in chemokine signalling in a human MM cell line and normal plasmablasts. In the present study, we explored the prognostic value of RGS1 expression in patients with MM using immunohistochemistry. METHODS: We evaluated RGS1 protein expression in 79 bone marrow biopsies obtained from patients with MM between 2008 and 2010 at Asan Medical Center. Correlations between RGS1 expression and clinicopathological factors were analysed. RESULTS: High RGS1 protein expression was significantly associated with poor overall survival (p=0.005). After an adjusted multivariable analysis, high RGS1 protein expression (p=0.010), high International Myeloma Working Group risk (p=0.003) and high serum lactate dehydrogenase levels (p=0.040) were significantly associated with poor outcomes. CONCLUSIONS: RGS1 expression may be a prognostic marker for risk stratification and a promising target for the development of a new MM therapy.

Pericytic mimicry in well-differentiated liposarcoma/atypical lipomatous tumor.

Pericytes are modified smooth muscle cells that closely enwrap small blood vessels, regulating and supporting the microvasculature through direct endothelial contact. Pericytes demonstrate a distinct immunohistochemical profile, including expression of smooth muscle actin, CD146, platelet-derived growth factor receptor ß, and regulator of G-protein signaling 5. Previously, pericyte-related antigens have been observed to be present among a group of soft tissue tumors with a perivascular growth pattern, including glomus tumor, myopericytoma, and angioleiomyoma. Similarly, malignant tumor cells have been shown to have a pericyte-like immunoprofile when present in a perivascular location, seen in malignant melanoma, glioblastoma, and adenocarcinoma. Here, we examine well-differentiated liposarcoma specimens, which showed some element of perivascular areas with the appearance of smooth muscle (n = 7 tumors). Immunohistochemical staining was performed for pericyte antigens, including smooth muscle actin, CD146, platelet-derived growth factor receptor ß, and regulator of G-protein signaling 5. Results showed consistent pericytic marker expression among liposarcoma tumor cells within a perivascular distribution. MDM2 immunohistochemistry and fluorescence in situ hybridization for MDM2 revealed that these perivascular cells were of tumor origin (7/7 tumors), whereas double immunohistochemical detection for CD31/CD146 ruled out an endothelial cell contribution. These findings further support the concept of pericytic mimicry, already established in diverse malignancies, and its presence in well-differentiated liposarcoma. The extent to which pericytic mimicry has prognostic significance in liposarcoma is as yet unknown.

The pericyte antigen RGS5 in perivascular soft tissue tumors.

Perivascular soft tissue tumors are relatively uncommon neoplasms of unclear lineage of differentiation, although most are presumed to originate from or differentiate to pericytes or a modified perivascular cell. Among these, glomus tumor, myopericytoma, and angioleiomyoma share a spectrum of histologic findings and a perivascular growth pattern. In contrast, solitary fibrous tumor was once hypothesized to have pericytic differentiation--although little bona fide evidence of pericytic differentiation exists. Likewise the perivascular epithelioid cell tumor (PEComa) family shares a perivascular growth pattern, but with distinctive dual myoid-melanocytic differentiation. RGS5, regulator of G-protein signaling 5, is a novel pericyte antigen with increasing use in animal models. Here, we describe the immunohistochemical expression patterns of RGS5 across perivascular soft tissue tumors, including glomus tumor (n = 6), malignant glomus tumor (n = 4), myopericytoma (n = 3), angioleiomyoma (n = 9), myofibroma (n = 4), solitary fibrous tumor (n = 10), and PEComa (n = 19). Immunohistochemical staining and semi-quantification was performed, and compared to αSMA (smooth muscle actin) expression. Results showed that glomus tumor (including malignant glomus tumor), myopericytoma, and angioleiomyoma shared a similar diffuse immunoreactivity for RGS5 and αSMA across all tumors examined. In contrast, myofibroma, solitary fibrous tumor and PEComa showed predominantly focal to absent RGS5 immunoreactivity. These findings further support a common pericytic lineage of differentiation in glomus tumors, myopericytoma and angioleiomyoma. The pericyte marker RGS5 may be of future clinical utility for the evaluation of pericytic differentiation in soft tissue tumors.

Cellular deficiency in the RGS10 protein facilitates chemoresistant ovarian cancer.

More than 30 regulators of G protein signaling (RGS) proteins encompass the RGS protein superfamily of critical regulators essential to cellular homeostasis. There is enormous structural and functional diversity among the RGS superfamily, and as such they serve a wide range of functions in regulating cell biology and physiology. Recent evidence has suggested roles for multiple RGS proteins in cancer initiation and progression, which has prompted research toward the potential modulation of these proteins as a new approach in cancer therapy. This article will discuss basic RGS molecular pharmacology, summarize the cellular functions and epigenetic regulation of RGS10, review ovarian cancer chemotherapy and describe the role of RGS10 in ovarian cancer survival signaling.

Identification of extracellularly phosphorylated membrane proteins.

Ecto-protein kinases phosphorylate extracellular membrane proteins and exhibit similarities to casein kinases and protein kinases A and C. However, the identification of their protein substrates still remains a challenge because a clear separation from intracellular phosphoproteins is difficult. Here, we describe a straightforward method for the identification of extracellularly phosphorylated membrane proteins in human umbilical vein endothelial cells (HUVECs) and K562 cells which used the protease bromelain to selectively remove ectoproteins from intact cells and combined this with the subsequent analysis using IMAC and LC-MS/MS. A "false-positive" strategy in which cells without protease treatment served as controls was applied. Using this approach we identified novel phosphorylation sites on five ectophosphoproteins (NOTCH1, otopetrin 1, regulator of G-protein signalling 13 (RGS13), protein tyrosine phosphatase receptor type D isoform 3 (PTPRD), usherin isoform B (USH2A)). Use of bromelain appears to be a reliable technique for the further identification of phosphorylated surface-exposed peptides when extracellular adenosine-5'-triphosphate is elevated during purinergic signalling.

RGS9-2 modulates sensory and mood related symptoms of neuropathic pain.

The signal transduction modulator Rgs9-2 (Regulator of G protein signaling 9-2) plays a key role in dopaminergic and opioidergic transmission in the striatum. Rgs9-2 is a potent modulator of opiate reward and analgesia, but its role in chronic pain remains unknown. Here, we use the spared nerve injury model (SNI), to evaluate the influence of Rgs9-2 in sensory symptoms, as well as in anxiety and depression-like behaviors observed under neuropathic pain conditions. Our data demonstrate that knockout of the Rgs9 gene reduces the intensity of thermal hyperalgesia and mechanical allodynia the first few days after nerve injury. This small, but significant effect is only observed at early time points after nerve injury, whereas after the first week of SNI, Rgs9 knockout (Rgs9KO) and Rgs9 wildtype (Rgs9WT) mice show similar levels of mechanical allodynia and thermal hyperalgesia. Furthermore, Rgs9-2 deletion exacerbates anxiety and depression like behaviors several weeks after the emergence of the neuropathic pain symptoms. Our findings also reveal a temporal and regional regulation of Rgs9-2 protein expression by neuropathic pain, as Rgs9-2 levels are reduced in the spinal cord a few days after nerve injury, whereas decreased Rgs9-2 levels in the Nucleus Accumbens (NAc) are only observed several weeks after nerve injury. Thus, adaptations in Rgs9-2 activity in the spinal cord and in the NAc may contribute to sensory and affective components of neuropathic pain.

Decreased RGS6 expression is associated with poor prognosis in pancreatic cancer patients.

Regulator of G-protein signaling 6 (RGS6), a member of a family of RGS proteins, has been reported to involve in multiple processes during tumor development. However, its role in pancreatic cancer has not been studied yet. In this study, we aimed to investigate the expression of RGS6 in pancreatic cancer and its role in predicting outcomes of patients with pancreatic cancer. We first measured the expression of RGS6 mRNA in 20 cases of tumor tissues and matched adjacent non-tumorous tissues by quantitative real-time PCR and examined RGS6 protein by immunohistochemistry in tissue microarrays containing 90 tumor and 90 paired adjacent non-tumor tissues. Decreased RGS6 mRNA detected in primary tumor, compared with their non-tumor counterparts. In addition, decreased RGS6 protein expression was associated with tumor differentiation (P = 0.027), pT classification (P = 0.034), smoking status (P = 0.041) and a poor survival (P = 0.007). Cox proportional hazards regression modeling analysis revealed that lymph node metastasis (P = 0.001; hazard ratio, 2.347, 95% CI, 1.387-3.972), tumor differentiation (P = 0.015; hazard ratio, 0.505, 95% CI, 0.291-0.876) and RGS6 expression (P = 0.048; hazard ratio, 0.567, 95% CI, 0.324-0.994) were three independent prognostic factors. Taken together, these date demonstrate that RGS6 decreases in tumor tissue and may serve as a novel biomarker for outcomes in pancreatic cancer patients and be a potential therapeutic target potential therapeutic target.

The GABAB receptor associates with regulators of G-protein signaling 4 protein in the mouse prefrontal cortex and hypothalamus.

Regulators of G-protein signaling (RGS) proteins regulate certain G-protein-coupled receptor (GPCR)-mediated signaling pathways. The GABA(B) receptor (GABA(B)R) is a GPCR that plays a role in the stress response. Previous studies indicate that acute immobilization stress (AIS) decreases RGS4 in the prefrontal cortex (PFC) and hypothalamus (HY) and suggest the possibility of a signal complex composed of RGS4 and GABA(B)R. Therefore, in the present study, we tested whether RGS4 associates with GABA(B)R in these brain regions. We found the co-localization of RGS4 and GABA(B)R subtypes in the PFC and HY using double immunohistochemistry and confirmed a direct association between GABA(B2)R and RGS4 proteins using co-immunoprecipitation. Furthermore, we found that AIS decreased the amount of RGS4 bound to GABA(B2)R and the number of double-positive cells. These results indicate that GABA(B)R forms a signal complex with RGS4 and suggests that RGS4 is a regulator of GABA(B)R.

Relationship between RGS5 expression and differentiation and angiogenesis of gastric carcinoma.

AIM: To explore the regulator of G-protein signaling 5 (RGS5) expression in gastric carcinoma and its association with differentiation and microvascular density (MVD). METHODS: Expression of RGS5 and CD34 were examined in 76 cases of gastric carcinoma, including 22 cases with lymph node metastasis and 54 cases without lymph node metastasis determined by immunohistochemistry (IHC). MVD was assessed using CD34 monoclonal antibody. The presence of RGS5 and CD34 was analyzed by IHC using the Envision technique. RESULTS: The RGS5 expression in gastric carcinoma was positively correlated with the differentiation of the tumor (r = 0.345, P < 0.001), but not related with age, gender, tumor size, clinical stage and lymph node metastasis (P > 0.05). The average MVD in the group with lymph node metastasis was significantly higher than that in the group without lymph node metastasis (P < 0.05). RGS5 expression was negatively correlated with the average MVD (P < 0.05). CONCLUSION: RGS5 expression level in gastric carcinoma is associated with the differentiation and MVD of the tumor, and may be used as an important parameter for determining the prognosis of gastric carcinoma patients.

RGS19 enhances cell proliferation through its C-terminal PDZ motif.

Regulator of G protein signaling 19 (RGS19), also known as Galpha-interacting protein (GAIP), is a GTPase activating protein (GAP) for Galpha(i) subunits. Apart from its GAP function, RGS19 has been implicated in growth factor signaling through binding to GAIP-interacting protein C-terminus (GIPC) via its C-terminal PDZ-binding motif. To gain additional insight on its function, we have stably expressed RGS19 in a number of mammalian cell lines and examined its effect on cell proliferation. Interestingly, overexpression of RGS19 stimulated the growth of HEK293, PC12, Caco2, and NIH3T3 cells. This growth promoting effect was not shared by other RGS proteins including RGS4, RGS10 and RGS20. Despite its ability to stimulate cell proliferation, RGS19 failed to induce neoplastic transformation in NIH3T3 cells as determined by focus formation and soft-agar assays, and it did not induce tumor growth in athymic nude mice. Deletion mutants of RGS19 lacking the PDZ-binding motif failed to complex with GIPC and did not exhibit any growth promoting effect. Overexpression of GIPC alone in HEK293 cells stimulated cell proliferation whereas its knockdown in H1299 non-small cell lung carcinomas suppressed cell proliferation. This study demonstrates that RGS19, in addition to acting as a GAP, is able to stimulate cell proliferation in a GIPC-dependent manner.

Regulator of g protein signaling 3 modulates wnt5b calcium dynamics and somite patterning.

Vertebrate development requires communication among cells of the embryo in order to define the body axis, and the Wnt-signaling network plays a key role in axis formation as well as in a vast array of other cellular processes. One arm of the Wnt-signaling network, the non-canonical Wnt pathway, mediates intracellular calcium release via activation of heterotrimeric G proteins. Regulator of G protein Signaling (RGS) proteins can accelerate inactivation of G proteins by acting as G protein GTPase-activating proteins (GAPs), however, the possible role of RGS proteins in non-canonical Wnt signaling and development is not known. Here, we identify rgs3 as having an overlapping expression pattern with wnt5b in zebrafish and reveal that individual knockdown of either rgs3 or wnt5b gene function produces similar somite patterning defects. Additionally, we describe endogenous calcium release dynamics in developing zebrafish somites and determine that both rgs3 and wnt5b function are required for appropriate frequency and amplitude of calcium release activity. Using rescue of gene knockdown and in vivo calcium imaging assays, we demonstrate that the activity of Rgs3 requires its ability to interact with Galpha subunits and function as a G protein GAP. Thus, Rgs3 function is necessary for appropriate frequency and amplitude of calcium release during somitogenesis and is downstream of Wnt5 activity. These results provide the first evidence for an essential developmental role of RGS proteins in modulating the duration of non-canonical Wnt signaling.

Non-canonical functions of RGS proteins.

Regulators of G protein signalling (RGS) proteins are united into a family by the presence of the RGS domain which serves as a GTPase-activating protein (GAP) for various Galpha subunits of heterotrimeric G proteins. Through this mechanism, RGS proteins regulate signalling of numerous G protein-coupled receptors. In addition to the RGS domains, RGS proteins contain diverse regions of various lengths that regulate intracellular localization, GAP activity or receptor selectivity of RGS proteins, often through interaction with other partners. However, it is becoming increasingly appreciated that through these non-RGS regions, RGS proteins can serve non-canonical functions distinct from inactivation of Galpha subunits. This review summarizes the data implicating RGS proteins in the (i) regulation of G protein signalling by non-canonical mechanisms, (ii) regulation of non-G protein signalling, (iii) signal transduction from receptors not coupled to G proteins, (iv) activation of mitogen-activated protein kinases, and (v) non-canonical functions in the nucleus.

RGS14 is a multifunctional scaffold that integrates G protein and Ras/Raf MAPkinase signalling pathways.

MAPkinase signalling is essential for cell growth, differentiation and cell physiology. G proteins and tyrosine kinase receptors each modulate MAPkinase signalling through distinct pathways. We report here that RGS14 is an integrator of G protein and MAPKinase signalling pathways. RGS14 contains a GPR/GoLoco (GL) domain that forms a stable complex with inactive Gialpha1/3-GDP, and a tandem (R1, R2) Ras binding domain (RBD). We find that RGS14 binds and regulates the subcellular localization and activities of H-Ras and Raf kinases in cells. Activated H-Ras binds RGS14 at the R1 RBD to form a stable complex at cell membranes. RGS14 also co-localizes with and forms a complex with Raf kinases in cells. The regulatory region of Raf-1 binds the RBD region of RGS14, and H-Ras and Raf each facilitate one another's binding to RGS14. RGS14 selectively inhibits PDGF-, but not EGF- or serum-stimulated Erk phosphorylation. This inhibition is dependent on H-Ras binding to RGS14 and is reversed by co-expression of Gialpha1, which binds and recruits RGS14 to the plasma membrane. Gialpha1 binding to RGS14 inhibits Raf binding, indicating that Gialpha1 and Raf binding to RGS14 are mutually exclusive. Taken together, these findings indicate that RGS14 is a newly appreciated integrator of G protein and Ras/Raf signalling pathways.

RGS17, an overexpressed gene in human lung and prostate cancer, induces tumor cell proliferation through the cyclic AMP-PKA-CREB pathway.

We have identified RGS17 as a commonly induced gene in lung and prostate tumors. Through microarray and gene expression analysis, we show that expression of RGS17 is up-regulated in 80% of lung tumors, and also up-regulated in prostate tumors. Through knockdown and overexpression of RGS17 in tumor cells, we show that RGS17 confers a proliferative phenotype and is required for the maintenance of the proliferative potential of tumor cells. We show through exon microarray, transcript analysis, and functional assays that RGS17 promotes cyclic AMP (cAMP)-responsive element binding protein (CREB)-responsive gene expression, increases cAMP levels, and enhances forskolin-mediated cAMP production. Furthermore, inhibition of cAMP-dependent kinase prevents tumor cell proliferation, and proliferation is partially rescued by RGS17 overexpression. In the present study, we show a role for RGS17 in the maintenance of tumor cell proliferation through induction of cAMP signaling and CREB phosphorylation. The prevalence of the induction of RGS17 in tumor tissues of various types further implicates its importance in the maintenance of tumor growth.

Regulator of G protein signaling 2 mediates cardiac compensation to pressure overload and antihypertrophic effects of PDE5 inhibition in mice.

The heart initially compensates for hypertension-mediated pressure overload by enhancing its contractile force and developing hypertrophy without dilation. Gq protein-coupled receptor pathways become activated and can depress function, leading to cardiac failure. Initial adaptation mechanisms to reduce cardiac damage during such stimulation remain largely unknown. Here we have shown that this initial adaptation requires regulator of G protein signaling 2 (RGS2). Mice lacking RGS2 had a normal basal cardiac phenotype, yet responded rapidly to pressure overload, with increased myocardial Gq signaling, marked cardiac hypertrophy and failure, and early mortality. Swimming exercise, which is not accompanied by Gq activation, induced a normal cardiac response, while Rgs2 deletion in Galphaq-overexpressing hearts exacerbated hypertrophy and dilation. In vascular smooth muscle, RGS2 is activated by cGMP-dependent protein kinase (PKG), suppressing Gq-stimulated vascular contraction. In normal mice, but not Rgs2-/- mice, PKG activation by the chronic inhibition of cGMP-selective phosphodiesterase 5 (PDE5) suppressed maladaptive cardiac hypertrophy, inhibiting Gq-coupled stimuli. Importantly, PKG was similarly activated by PDE5 inhibition in myocardium from both genotypes, but PKG plasma membrane translocation was more transient in Rgs2-/- myocytes than in controls and was unaffected by PDE5 inhibition. Thus, RGS2 is required for early myocardial compensation to pressure overload and mediates the initial antihypertrophic and cardioprotective effects of PDE5 inhibitors.

Novel role for RGS1 in melanoma progression.

RGS1 (regulator of G protein signaling 1) encodes a member of the regulator of G protein family. Recently, RGS1 was found to be overexpressed in gene expression-profiling studies of melanoma. However, no analyses have been reported of its expression at the protein level in melanoma. In this study, the potential impact of RGS1 as a molecular prognostic marker for melanoma was assessed using immunohistochemical analysis of a melanoma tissue microarray containing primary cutaneous melanomas from 301 patients. High RGS1 expression was significantly correlated with increased tumor thickness (P=0.0083), mitotic rate (P=0.04), and presence of vascular involvement (P<0.02). Kaplan-Meier analysis demonstrated a significant association between increasing RGS1 expression and reduced relapse-free survival (P=0.0032) as well as disease-specific survival (DSS) (P=0.018) survival. Logistic regression analysis showed RGS1 overexpression to be significantly correlated to sentinel lymph node metastasis (P=0.04). Multivariate Cox regression analysis showed that increasing RGS1 immunostaining had an independent impact on the relapse-free survival (P=0.0069) and DSS (P=0.0077) of this melanoma cohort. In the analysis of DSS, RGS1 expression level was the most powerful factor predicting DSS. RGS1 immunostaining retained independent prognostic impact even when sentinel lymph node status was included in the prognostic model (P=0.0039). These results validate the role of RGS1 as a novel prognostic marker for melanoma given its impact on the survival associated with melanoma.

Microfabricated channel array electrophoresis for characterization and screening of enzymes using RGS-G protein interactions as a model system.

A microfluidic chip consisting of parallel channels designed for rapid electrophoretic enzyme assays was developed. Radial arrangement of channels and a common waste channel allowed chips with 16 and 36 electrophoresis units to be fabricated on a 7.62 x 7.62 cm(2) glass substrate. Fluorescence detection was achieved using a Xe arc lamp source and commercial charge-coupled device (CCD) camera to image migrating analyte zones in individual channels. Chip performance was evaluated by performing electrophoretic assays for G protein GTPase activity on chip using BODIPY-GTP as enzyme substrate. A 16-channel design proved to be useful in extracting kinetic information by allowing serial electrophoretic assays from 16 different enzyme reaction mixtures at 20 s intervals in parallel. This system was used to rapidly determine enzyme concentrations, optimal enzymatic reaction conditions, and Michaelis-Menten constants. A chip with 36 channels was used for screening for modulators of the G protein-RGS protein interaction by assaying the amount of product formed in enzyme reaction mixtures that contained test compounds. Thirty-six electrophoretic assays were performed in 30 s suggesting the potential throughput up to 4320 assays/h with appropriate sample handling procedures. Both designs showed excellent reproducibility of peak migration time and peak area. Relative standard deviations of normalized peak area of enzymatic product BODIPY-GDP were 5% and 11%, respectively, in the 16- and 36-channel designs.