Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis by G␣ subunits and thus facilitate termination of signaling initiated by G protein-coupled receptors (GPCRs). RGS proteins hold great promise as disease intervention points, given their signature role as negative regulators of GPCRs-receptors to which the largest fraction of approved medications are currently directed. RGS proteins share a hallmark RGS domain that interacts most avidly with G␣ when in its transition state for GTP hydrolysis; by binding and stabilizing switch regions I and II of G␣, RGS domain binding consequently accelerates G␣-mediated GTP hydrolysis. The human genome encodes more than three dozen RGS domaincontaining proteins with varied G␣ substrate specificities. To facilitate their exploitation as drug-discovery targets, we have taken a systematic structural biology approach toward cataloging the structural diversity present among RGS domains and identifying molecular determinants of their differential G␣ selectivities. Here, we determined 14 structures derived from NMR and x-ray crystallography of members of the R4, R7, R12, and RZ subfamilies of RGS proteins, including 10 uncomplexed RGS domains and 4 RGS domain/G␣ complexes. Heterogeneity observed in the structural architecture of the RGS domain, as well as in engagement of switch III and the all-helical domain of the G␣ substrate, suggests that unique structural determinants specific to particular RGS protein/G␣ pairings exist and could be used to achieve selective inhibition by small molecules.GTPase-accelerating proteins ͉ NMR structure ͉ RGS proteins ͉ x-ray crystallography G protein-coupled receptors (GPCRs) are critical for many physiological processes including vision, olfaction, neurotransmission, and the actions of many hormones (1). As such, GPCRs are the largest fraction of the ''druggable proteome,'' and their ligand-binding and signaling properties remain of considerable interest to academia and industry (2). GPCRs catalyze activation of heterotrimeric G proteins comprising a guanine nucleotide-binding G␣ subunit and an obligate G␤␥ dimer (3). Receptor-promoted activation of G␣␤␥ causes exchange of GDP for GTP by G␣ and resultant dissociation of G␤␥. GTP-bound G␣ and freed G␤␥ then regulate intracellular effectors such as adenylyl cyclase, phospholipase C, ion channels, RhoGEFs, and phosphodiesterases (1, 4). This ''G protein cycle'' is reset by the intrinsic GTP hydrolysis activity of G␣, producing G␣⅐GDP that favors heterotrimer reformation and, consequently, signal termination. Thus, a major determinant of the duration and magnitude of GPCR signaling is the lifetime of G␣ in the GTP-bound state.Regulators of G protein signaling are GTPase-accelerating proteins (GAPs) for G␣ subunits and thus facilitate GPCR signal termination (5). GAP activity is conferred by an RGS domain present in one or more copies within members of this protein superfamily (5). The archetypal RGS domain is composed of nine ␣-helices (6) and binds most avidly to G␣ in the transi...

Claspin is an essential protein for the ATR-dependent activation of the DNA replication checkpoint response in Xenopus and human cells. Here we describe the purification and characterization of human Claspin. The protein has a ring-like structure and binds with high affinity to branched DNA molecules. These findings suggest that Claspin may be a component of the replication ensemble and plays a role in the replication checkpoint by directly associating with replication forks and with the various branched DNA structures likely to form at stalled replication forks because of DNA damage.

"Regulator of G-protein signaling" (RGS) proteins facilitate the termination of G protein-coupled receptor (GPCR) signaling via their ability to increase the intrinsic GTP hydrolysis rate of G␣ subunits (known as GTPase-accelerating protein or "GAP" activity). RGS2 is unique in its in vitro potency and selectivity as a GAP for G␣ q subunits. As many vasoconstrictive hormones signal via G q heterotrimer-coupled receptors, it is perhaps not surprising that RGS2-deficient mice exhibit constitutive hypertension. However, to date the particular structural features within RGS2 determining its selectivity for G␣ q over G␣ i/o substrates have not been completely characterized. Here, we examine a trio of point mutations to RGS2 that elicits G␣ i -directed binding and GAP activities without perturbing its association with G␣ q . Using x-ray crystallography, we determined a model of the triple mutant RGS2 in complex with a transition state mimetic form of G␣ i at 2.8-Å resolution. Structural comparison with unliganded, wild type RGS2 and of other RGS domain/G␣ complexes highlighted the roles of these residues in wild type RGS2 that weaken G␣ i subunit association. Moreover, these three amino acids are seen to be evolutionarily conserved among organisms with modern cardiovascular systems, suggesting that RGS2 arose from the R4-subfamily of RGS proteins to have specialized activity as a potent and selective G␣ q GAP that modulates cardiovascular function. G protein-coupled receptors (GPCRs)4 form an interface between extracellular and intracellular physiology, as they convert hormonal signals into changes in intracellular metabolism and ultimately cell phenotype and function (1-3). GPCRs are coupled to their underlying second messenger systems by heterotrimeric guanine nucleotide-binding protein ("G-proteins") composed of three subunits: G␣, G␤, and G␥. Four general classes of G␣ subunits have been defined based on functional couplings (in the GTP-bound state) to various effector proteins. G s subfamily G␣ subunits are stimulatory to membrane-bound adenylyl cyclases that generate the second messenger 3Ј,5Ј-cyclic adenosine monophosphate (cAMP); conversely, G i subfamily G␣ subunits are generally inhibitory to adenylyl cyclases (4). G 12/13 subfamily G␣ subunits activate the small G-protein RhoA through stimulation of the GEF subfamily of RGS proteins, namely p115-RhoGEF, LARG, and PDZ-RhoGEF (5). G q subfamily G␣ subunits are potent activators of phospholipase-C␤ enzymes that generate the second messengers diacylglycerol and inositol triphosphate (6); more recently, two additional G␣ q effector proteins have been described: the receptor kinase GRK2 and the RhoA nucleotide exchange factor p63RhoGEF (7,8).The duration of GPCR signaling is controlled by the time G␣ remains bound to GTP before its hydrolysis to GDP. RGS proteins are key modulators of GPCR signaling by virtue of their ability to accelerate the intrinsic GTP hydrolysis activity of G␣ subunits (reviewed in Refs. 9 and 10). RGS2/G0S8, one of the first mammalian RGS ...

RGS proteins are critical modulators of G-protein-coupled receptor (GPCR) signaling given their ability to deactivate Gα subunits via GTPase-accelerating protein (GAP) activity. Their selectivity for specific GPCRs makes them attractive therapeutic targets. However, measuring GAP activity is complicated by slow guanosine diphosphate (GDP) release from Gα and lack of solution phase assays for detecting free GDP in the presence of excess guanosine triphosphate (GTP). To overcome these hurdles, the authors developed a Gα i1 mutant with increased GDP dissociation and decreased GTP hydrolysis rates, enabling detection of GAP activity using steady-state GTP hydrolysis. Gα i1 (R178M/A326S) GTPase activity was stimulated 6-to 12-fold by RGS proteins known to act on Gα i subunits and not affected by those unable to act on Gα i , demonstrating that the Gα/RGS domain interaction selectivity was not altered by mutation. The selectivity and affinity of Gα i1 (R178M/A326S) interaction with RGS proteins was confirmed by molecular binding studies. To enable nonradioactive, homogeneous detection of RGS protein effects on Gα i1 (R178M/A326S), the authors developed a Transcreener  fluorescence polarization immunoassay based on a monoclonal antibody that recognizes GDP with greater than 100-fold selectivity over GTP. Combining Gα i1 (R178M/A326S) with a homogeneous, fluorescence-based GDP detection assay provides a facile means to explore the targeting of RGS proteins as a new approach for selective modulation of GPCR signaling. (Journal of Biomolecular

The human DDB1 and DDB2 genes encode the 127 and 48 kDa subunits, respectively, of the damage-specific DNA-binding protein (DDB). Mutations in the DDB2 gene have been correlated with the hereditary disease xeroderma pigmentosum group E. We have investigated the proximal promoters of the DDB genes, both of which are G/C-rich and do not contain a TATA box. Transient expression analysis in HeLa cells using a luciferase reporter system indicated the presence of core promoters located within 292 bp (DDB1) and 220 bp (DDB2) upstream of the putative transcription initiation sites. Both core promoters contain multiple active Sp1 sites, with those of DDB1 at -123 to -115 and of DDB2 at -29 to -22 being critical determinants of promoter activity. In addition, an N-myc site at -56 to -51 for DDB1 is an essential transcription element, and mutations in a DDB1 NF-1 site at -104 to -92, a DDB2 NF-1 site at -68 to -56 and a DDB2 E2F site at +36 to +43 also reduce promoter activity. Taken together, these results suggest a regulation of basal transcription typical of cell cycle-regulated genes, and therefore support conjectures that the DDB heterodimer and/or its subunits have functions other than direct involvement in DNA repair.