The 3 3 5-exonucleases process DNA ends in many DNA repair pathways of human cells. Determination of the human TREX2 structure is the first of a dimeric 3-deoxyribonuclease and indicates how this highly efficient nonprocessive enzyme removes nucleotides at DNA 3 termini. Symmetry in the TREX2 dimer positions the active sites at opposite outer edges providing open access for the DNA. Adjacent to each active site is a flexible region containing three arginines positioned appropriately to bind DNA and to control its entry into the active site. Mutation of these three arginines to alanines reduces the DNA binding capacity by ϳ100-fold with no effect on catalysis. The human TREX2 catalytic residues overlay with the bacterial DnaQ family of 3-exonucleases confirming the structural conservation of the catalytic sites despite limited sequence identity, and mutations of these residues decrease the still measurable activity by ϳ10 5 -fold, confirming their catalytic role.During the multistep processes of DNA replication, repair, and recombination DNA ends are often remodeled by enzymes containing 3Ј 3 5Ј-exonuclease activities to remove mispaired, modified, fragmented, and normal nucleotides from DNA 3Ј termini. The major 3Ј 3 5Ј-exonuclease activity detected in mammalian cell extracts is catalyzed by the TREX1 1 enzyme (1-3). The gene encoding TREX1 and the closely related TREX2 gene were cloned (4, 5), and the 3Ј 3 5Ј-exonuclease activities of the recombinant proteins confirmed the catalytically robust nature of these enzymes (6, 7). In addition to the TREX enzymes, several other proteins containing 3Ј 3 5Ј-exonuclease activities have recently been identified in human cells (for recent reviews, see Refs. 8 and 9). The sequences and structures of these enzymes indicate a diverse collection that includes multidomain proteins such as the Werner syndrome (WRN) (10 -12) and p53 proteins (13) and the "proofreading" DNA polymerases ␦, ⑀, and ␥. The 3Ј 3 5Ј-exonuclease activities have been detected in the hMRE11 subunit of a doublestrand break DNA repair complex (14, 15) and the base excision repair apurinic/apyrimidinic endonuclease, .The presence of 3Ј 3 5Ј-exonuclease activities in a multitude of proteins likely reflects the importance of this activity in the proper maintenance of DNA 3Ј-ends.Defective 3Ј 3 5Ј-exonucleases impair critical DNA metabolic pathways and elicit devastating consequences in cells and animals. The Trex1Ϫ/Ϫ mice exhibit dramatically reduced survival resulting from inflammatory myocarditis leading to cardiomyopathy and circulatory failure (21). Deficiencies in the WRN protein result in the premature aging associated with Werner syndrome patients (22), and mice deficient in p53 or in the proofreading exonuclease of DNA polymerase ␦ show high incidences of cancers (23-25). Eliminating both APE alleles in mice results in early embryonic lethality (26), and defects in the hMRE11 complex have been associated with an ataxiatelangiectasia-like disorder and the Nijmegen breakage syndrome characterized by chrom...

β-arrestins are critical signalling molecules that regulate many fundamental physiological functions including the maintenance of euglycemia and peripheral insulin sensitivity. Here we show that inactivation of the β-arrestin-2 gene, barr2, in β-cells of adult mice greatly impairs insulin release and glucose tolerance in mice fed with a calorie-rich diet. Both glucose and KCl-induced insulin secretion and calcium responses were profoundly reduced in β-arrestin-2 (barr2) deficient β-cells. In human β-cells, barr2 knockdown abolished glucose-induced insulin secretion. We also show that the presence of barr2 is essential for proper CAMKII function in β-cells. Importantly, overexpression of barr2 in β-cells greatly ameliorates the metabolic deficits displayed by mice consuming a high-fat diet. Thus, our data identify barr2 as an important regulator of β-cell function, which may serve as a new target to improve β-cell function.

Class A G protein-coupled receptors (GPCRs) are known to form dimers and/or oligomeric arrays in vitro and in vivo. These complexes are thought to play important roles in modulating class A GPCR function. Many studies suggest that residues located on the "outer" (lipid-facing) surface of the transmembrane (TM) receptor core are critically involved in the formation of class A receptor dimers (oligomers). However, no clear consensus has emerged regarding the identity of the TM helices or TM subsegments involved in this process. To shed light on this issue, we have used the M 3 muscarinic acetylcholine receptor (M3R), a prototypic class A GPCR, as a model system. Using a comprehensive and unbiased approach, we subjected all outward-facing residues (70 amino acids total) of the TM helical bundle (TM1-7) of the M3R to systematic alanine substitution mutagenesis. We then characterized the resulting mutant receptors in radioligand binding and functional studies and determined their ability to form dimers (oligomers) in bioluminescence resonance energy transfer saturation assays. We found that M3R/M3R interactions are not dependent on the presence of one specific structural motif but involve the outer surfaces of multiple TM subsegments (TM1-5 and -7) located within the central and endofacial portions of the TM receptor core. Moreover, we demonstrated that the outward-facing surfaces of most TM helices play critical roles in proper receptor folding and/or function. Guided by the bioluminescence resonance energy transfer data, molecular modeling studies suggested the existence of multiple dimeric/oligomeric M3R arrangements, which may exist in a dynamic equilibrium. Given the high structural homology found among all class A GPCRs, our results should be of considerable general relevance.The superfamily of G protein-coupled receptors (GPCRs) 3 represents the largest group of cell surface receptors found in nature (1, 2). Following activation by extracellular ligands such as neurotransmitters, hormones, or sensory stimuli, GPCRs regulate an extraordinarily large number of important physiological responses, reflecting the fact that nearly 50% of drugs in current clinical use target specific GPCRs (3). A characteristic structural feature of all GPCRs is a transmembrane (TM) core consisting of a bundle of seven TM helices (TM1-7). The TM receptor core plays a critical role in propagating the ligandinduced conformational changes to the cytoplasmic receptor surface, enabling the receptor to interact with specific classes of heterotrimeric G proteins (2, 4).Accumulating evidence suggests that GPCRs are able to form dimers and/or higher order oligomeric complexes (5-10). Most convincingly, studies with class C GPCRs, exemplified by the ␥-aminobutyric acid, type B, and metabotropic glutamate receptors, have demonstrated that dimer formation is required for agonist-induced G protein activation, at least in this subclass of GPCRs (for recent reviews see Refs. 7,8). Class C GPCRs form stable dimeric interactions through well character...

No abstract

G protein-coupled receptors (GPCRs) comprise one of the largest protein families found in nature. Here we describe a new experimental strategy that allows rapid identification of functionally critical amino acids in the rat M(3) muscarinic acetylcholine receptor (M3R), a prototypic class I GPCR. This approach involves low-frequency random mutagenesis of the entire M3R coding sequence, followed by the application of a new yeast genetic screen that allows the recovery of inactivating M3R single point mutations. The vast majority of recovered mutant M3Rs also showed substantial functional impairments in transfected mammalian (COS-7) cells. A subset of mutant receptors, however, behaved differently in yeast and mammalian cells, probably because of the specific features of the yeast expression system used. The screening strategy described here should be applicable to all GPCRs that can be expressed functionally in yeast.

To explore the structural mechanisms underlying the assembly and activation of family A GPCR dimers, we used the rat M(3) muscarinic acetylcholine receptor (M3R) as a model system. Studies with Cys-substituted mutant M3Rs expressed in COS-7 cells led to the identification of several mutant M3Rs that exclusively existed as cross-linked dimers under oxidizing conditions. The cross-linked residues were located at the bottom of transmembrane domain 5 (TM5) and within the N-terminal portion of the third intracellular loop (i3 loop). Studies with urea-stripped membranes demonstrated that M3R disulfide cross-linking did not require the presence of heterotrimeric G proteins. Molecular modeling studies indicated that the cross-linking data were in excellent agreement with the existence of a low-energy M3R dimer characterized by a TM5-TM5 interface. [(35)S]GTPγS binding/Gα(q/11) immunoprecipitation assays revealed that an M3R dimer that was cross-linked within the N-terminal portion of the i3 loop (264C) was functionally severely impaired (∼50% reduction in receptor-G-protein coupling, as compared to control M3R). These data support the novel concept that agonist-induced activation of M3R dimers requires a conformational change of the N-terminal segment of the i3 loop. Given the high degree of structural homology among family A GPCRs, these findings should be of broad significance.

The M 3 muscarinic acetylcholine receptor (M3R) regulates many fundamental physiological functions. To identify novel M3R-interacting proteins, we used a recently developed yeast two-hybrid screen (split ubiquitin method) to detect interactions among membrane proteins. This screen led to the identification of many novel M3R-associated proteins, including the putative membrane protein transmembrane protein 147 (Tmem147). The amino acid sequence of Tmem147 is highly conserved among mammals, but its physiological roles are unknown at present. We initially demonstrated that Tmem147 could be coimmunoprecipitated with M3Rs in cotransfected mammalian cells (COS-7 cells). Confocal imaging studies showed that Tmem147 was localized to endoplasmic reticulum (ER) membranes and that the Tmem147/ M3R interaction occurred in the ER of cotransfected COS-7 cells, resulting in impaired trafficking of the M3R to the cell surface. To study the role of Tmem147 in modulating M3R function in a more physiologically relevant setting, we carried out studies with H508 human colon cancer cells that endogenously express M3Rs and Tmem147. Treatment of H508 cells with carbachol, a hydrolytically stable acetylcholine analog, promoted H508 cell proliferation and activation of the mitogenic kinase, p90RSK. Small interfering RNA-mediated knockdown of Tmem147 expression significantly augmented the stimulatory effects of carbachol on H508 cell proliferation and p90RSK activation. These effects were associated with an increase in the density of cell surface M3Rs. Our data clearly indicate that Tmem147 represents a potent negative regulator of M3R function, most likely by interacting with M3Rs in an intracellular compartment (ER). These findings may lead to new strategies aimed at modulating M3R activity for therapeutic purposes.

Increased hepatic glucose production is a key pathophysiological feature of type 2 diabetes. Like all other cell types, hepatocytes express many G protein-coupled receptors (GPCRs) that are linked to different functional classes of heterotrimeric G proteins. The important physiological functions mediated by G(s)-coupled hepatic glucagon receptors are well-documented. In contrast, little is known about the in vivo physiological roles of hepatocyte GPCRs that are linked to G proteins of the G(q) family. To address this issue, we established a transgenic mouse line (Hep-Rq mice) that expressed a G(q)-linked designer receptor (Rq) in a hepatocyte-selective fashion. Importantly, Rq could no longer bind endogenous ligands but could be selectively activated by a synthetic drug, clozapine-N-oxide. Clozapine-N-oxide treatment of Hep-Rq mice enabled us to determine the metabolic consequences caused by selective activation of a G(q)-coupled GPCR in hepatocytes in vivo. We found that acute Rq activation in vivo led to pronounced increases in blood glucose levels, resulting from increased rates of glycogen breakdown and gluconeogenesis. We also demonstrated that the expression of the V(1b) vasopressin receptor, a G(q)-coupled receptor expressed by hepatocytes, was drastically increased in livers of ob/ob mice, a mouse model of diabetes. Strikingly, treatment of ob/ob mice with a selective V(1b) receptor antagonist led to reduced glucose excursions in a pyruvate challenge test. Taken together, these findings underscore the importance of G(q)-coupled receptors in regulating hepatic glucose fluxes and suggest novel receptor targets for the treatment of type 2 diabetes.