Agonist mediated internalization of M(2 )mAChR is β-arrestin-dependent

BACKGROUND: Muscarinic acetylcholine receptors (mAChRs) undergo agonist-promoted internalization, but evidence suggesting that the mechanism of internalization is β-arrestin dependent has been contradictory and unclear. Previous studies using heterologous over-expression of wild type or dominant-negative forms of β-arrestins have reported that agonist-promoted internalization of M(2 )mAChRs is a β-arrestin- and clathrin-independent phenomenon. In order to circumvent the complications associated with the presence of endogenous β-arrestin that may have existed in these earlier studies, we examined agonist-promoted internalization of the M(2 )mAChR in mouse embryonic fibroblasts (MEFs) derived from β-arrestin knockout mice that lack expression of either one or both isoforms of β-arrestin (β-arrestin 1 and 2). RESULTS: In wild type MEF cells transiently expressing M(2 )mAChRs, 40% of surface M(2 )mAChRs underwent internalization and sorted into intracellular compartments following agonist stimulation. In contrast, M(2 )mAChRs failed to undergo internalization and sorting into intracellular compartments in MEF β-arrestin double knockout cells following agonist stimulation. In double knockout cells, expression of either β-arrestin 1 or 2 isoforms resulted in rescue of agonist-promoted internalization. Stimulation of M(2 )mAChRs led to a stable co-localization with GFP-tagged β-arrestin within endocytic structures in multiple cell lines; the compartment to which β-arrestin localized was determined to be the early endosome. Agonist-promoted internalization of M(2 )mAChRs was moderately rescued in MEF β-arrestin 1 and 2 double knockout cells expressing exogenous arrestin mutants that were selectively defective in interactions with clathrin (β-arrestin 2 ΔLIELD), AP-2 (β-arrestin 2-F391A), or both clathrin/AP-2. Expression of a truncated carboxy-terminal region of β-arrestin 1 (319–418) completely abrogated agonist-promoted internalization of M(2 )mAChRs in wild type MEF cells. CONCLUSION: In summary, this study demonstrates that agonist-promoted internalization of M(2 )mAChRs is β-arrestin- and clathrin-dependent, and that the receptor stably co-localizes with β-arrestin in early endosomal vesicles

Discrimination, mastery, and depressive symptoms among African American men

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/88130/1/watkins_hudson_etal2011.pd

Effects of macrofauna on acoustic backscatter from the seabed: Field manipulations in West Sound, Orcas Island, Washington, U.S.A.

Previous observations with a bottom-mounted, radially scanning sonar (BAMS) at 40 kHz suggested that macrofaunal activities influence low-angle, acoustic backscatter from seafloor sediments. In order to test that possibility experimentally, we measured and modeled time series of backscatter strength at both 40 and 300 kHz prior to manipulation and then introduced several macrofaunal species at known abundances to randomly selected locations within the ensonified area. We worked in West Sound, Orcas Island, Washington, at a water depth of 20.4 m and for the more frequently recorded 40-kHz series extracted effects by the time-series method known as intervention analysis, wherein the intervention was the experimental alteration. We observed increased backscatter from patches of the small protobranch bivalve Acila castrensis, and of the cockle Clinocardium nuttali, from bait used as chum for fishes and crabs, and from tethered crabs (Cancer magister); other treatments showed no significant change. All of the effective treatments involved increased backscatter at 300 kHz from animals that have obvious hard parts or air bladders. Power calculations for intervention analysis and geoacoustic modeling suggest that failure of other treatments to show significant effects on backscatter strength stems from the small size of the organisms and structures used relative to the 40-kHz wavelength (3.7 cm) and to low sound-speed contrasts between surficial sediments at this site and overlying water (at both frequencies), producing low backscatter levels from both volume heterogeneity and surface microtopography. This experiment demonstrates, however, that low-angle acoustic backscatter can be used to observe at least some populations of benthic animals over a large area (ca. 8000 m2) and that intervention analysis can be a useful tool where logistics permit repeated observation but few or no spatial replicates—frequently the case in ecological manipulations