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...

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