The activity of G protein-coupled receptors is regulated via hyperphosphorylation following agonist stimulation. Despite the universal nature of this regulatory process, the physiological impact of receptor phosphorylation remains poorly studied. To address this question, we have generated a knock-in mouse strain that expresses a phosphorylation-deficient mutant of the M 3 -muscarinic receptor, a prototypical G q/11 -coupled receptor. This mutant mouse strain was used here to investigate the role of M 3 -muscarinic receptor phosphorylation in the regulation of insulin secretion from pancreatic islets. Importantly, the phosphorylation deficient receptor coupled to G q/11 -signaling pathways but was uncoupled from phosphorylation-dependent processes, such as receptor internalization and β-arrestin recruitment. The knock-in mice showed impaired glucose tolerance and insulin secretion, indicating that M 3 -muscarinic receptors expressed on pancreatic islets regulate glucose homeostasis via receptor phosphorylation-/arrestin-dependent signaling. The mechanism centers on the activation of protein kinase D1, which operates downstream of the recruitment of β-arrestin to the phosphorylated M 3 -muscarinic receptor. In conclusion, our findings support the unique concept that M 3 -muscarinic receptor-mediated augmentation of sustained insulin release is largely independent of G protein-coupling but involves phosphorylation-/ arrestin-dependent coupling of the receptor to protein kinase D1. G-protein coupled receptor | ligand biasT he vast majority of G protein-coupled receptors (GPCRs) respond to agonist occupation by becoming rapidly hyperphosphorylated within intracellular domains (1-3). This process not only leads to the uncoupling of the receptor from its cognate G proteins, but also allows for the activation of G proteinindependent signaling, a process that is driven largely by the recruitment of β-arrestin adaptor proteins (4-7). As a consequence, GPCRs regulate an extensive array of signaling pathways and biological responses (3). G protein-independent signaling pathways have mostly been studied in recombinant systems. However, the current challenge is to understand to what extent these processes are involved in the regulation of key physiological responses.In the present study, we examined the in vivo role of GPCR phosphorylation by generating a knock-in mouse strain expressing a phosphorylation-deficient GPCR. Specifically, we used the M 3 -muscarinic acetylcholine receptor, a prototypic G q/11 -coupled GPCR, as a model system (8, 9). We and others have previously demonstrated that the M 3 -muscarinic receptor is rapidly phosphorylated on agonist occupation by a range of protein kinases that include members of the GPCR kinase (GRK) family, as well as casein kinase 1α and protein kinase CK2 (10-13). To define the potential physiological role of M 3 -muscarinic receptor phosphorylation, we generated a mouse strain where the wild-type M 3 -muscarinic receptor gene had been replaced by a phosphorylationdeficient mutan...

Supplementation with vitamin C had little effect on cellular levels in this group of healthy individuals, suggesting their diets were replete in vitamin C. The dose range of vitamin C used did not affect oxidative damage in PBMC DNA.

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a 'targeting' vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

The lambda phage Red recombination system can mediate efficient homologous recombination in Escherichia coli, which is the basis of the DNA engineering technique termed recombineering. Red mediated insertion of DNA requires DNA replication, involves a single-stranded DNA intermediate and is more efficient on the lagging strand of the replication fork. Lagging strand recombination has also been postulated to explain the Red mediated repair of gapped plasmids by an Okazaki fragment gap filling model. Here, we demonstrate that gap repair involves a different strand independent mechanism. Gap repair assays examining the strand asymmetry of recombination did not show a lagging strand bias. Directly testing an ssDNA plasmid showed lagging strand recombination is possible but dsDNA plasmids did not employ this mechanism. Insertional recombination combined with gap repair also did not demonstrate preferential lagging strand bias, supporting a different gap repair mechanism. The predominant recombination route involved concerted insertion and subcloning though other routes also operated at lower frequencies. Simultaneous insertion of DNA resulted in modification of both strands and was unaffected by mutations to DNA polymerase I, responsible for Okazaki fragment maturation. The lower efficiency of an alternate Red mediated ends-in recombination pathway and the apparent lack of a Holliday junction intermediate suggested that gap repair does not involve a different Red recombination pathway. Our results may be explained by a novel replicative intermediate in gap repair that does not involve a replication fork. We exploited these observations by developing a new recombineering application based on concerted insertion and gap repair, termed SPI (subcloning plus insertion). SPI selected against empty vector background and selected for correct gap repair recombinants. We used SPI to simultaneously insert up to four different gene cassettes in a single recombineering reaction. Consequently, our findings have important implications for the understanding of E. coli replication and Red recombination.