Biomedical subjects

Alicia Gates

Publications and source records attributed to Alicia Gates.

4 recordsLinked to original sources

PubMed2006

Agonist mediated internalization of M2 mAChR is beta-arrestin-dependent.

BACKGROUND: Muscarinic acetylcholine receptors (mAChRs) undergo agonist-promoted internalization, but evidence suggesting that the mechanism of internalization is beta-arrestin dependent has been contradictory and unclear. Previous studies using heterologous over-expression of wild type or dominant-negative forms of beta-arrestins have reported that agonist-promoted internalization of M2 mAChRs is a beta-arrestin- and clathrin-independent phenomenon. In order to circumvent the complications associated with the presence of endogenous beta-arrestin that may have existed in these earlier studies, we examined agonist-promoted internalization of the M2 mAChR in mouse embryonic fibroblasts (MEFs) derived from beta-arrestin knockout mice that lack expression of either one or both isoforms of beta-arrestin (beta-arrestin 1 and 2). RESULTS: In wild type MEF cells transiently expressing M2 mAChRs, 40% of surface M2 mAChRs underwent internalization and sorted into intracellular compartments following agonist stimulation. In contrast, M2 mAChRs failed to undergo internalization and sorting into intracellular compartments in MEF beta-arrestin double knockout cells following agonist stimulation. In double knockout cells, expression of either beta-arrestin 1 or 2 isoforms resulted in rescue of agonist-promoted internalization. Stimulation of M2 mAChRs led to a stable co-localization with GFP-tagged beta-arrestin within endocytic structures in multiple cell lines; the compartment to which beta-arrestin localized was determined to be the early endosome. Agonist-promoted internalization of M2 mAChRs was moderately rescued in MEF beta-arrestin 1 and 2 double knockout cells expressing exogenous arrestin mutants that were selectively defective in interactions with clathrin (beta-arrestin 2 DeltaLIELD), AP-2 (beta-arrestin 2-F391A), or both clathrin/AP-2. Expression of a truncated carboxy-terminal region of beta-arrestin 1 (319-418) completely abrogated agonist-promoted internalization of M2 mAChRs in wild type MEF cells. CONCLUSION: In summary, this study demonstrates that agonist-promoted internalization of M2 mAChRs is beta-arrestin- and clathrin-dependent, and that the receptor stably co-localizes with beta-arrestin in early endosomal vesicles.

Journal Article↗

PubMed2006

Expression Analysis Systematic Explorer (EASE) analysis reveals differential gene expression in permanent and transient focal stroke rat models.

To gain greater insight on the molecular mechanisms that underlie ischemic stroke, we compared gene expression profiles in transient (tMCAO) and permanent middle cerebral artery occlusion (pMCAO) stroke models using Expression Analysis Systematic Explorer (EASE) pathway analysis software. Many transcripts were induced in both stroke models, including genes associated with transcriptional pathways, cell death, stress responses and metabolism. However, EASE analysis of the regulated genes indicated molecular functions and biological processes unique to each model. Pathways associated with tMCAO included inflammation, apoptosis and cell cycle, while pMCAO was associated with the induction of genes encoding neurotransmitter receptors, ion channels, growth factors and signaling molecules. An intriguing finding was the involvement of tyrosine kinases and phosphatases following pMCAO. These results provide evidence that neuronal death following tMCAO and pMCAO involves distinct mechanisms. These findings may give new insight to the molecular mechanisms involved in stroke and may lead to novel neuroprotective strategies.

Animals↗

PubMed2005

Transient hypoxia induces sequestration of M1 and M2 muscarinic acetylcholine receptors.

Oxidative stress has been implicated in impairing muscarinic acetylcholine receptor (mAChR) signaling activity. It remains unclear, however, whether alterations in the cell surface distribution of mAChRs following oxidative stress contribute to the diminished mAChR signaling activity. We report here that M1 and M2 mAChRs, stably expressed in Chinese hamster ovary cells, undergo sequestration following transient hypoxic-induced oxidative stress (2% O2). Sequestration of M1 and M2 mAChRs following transient hypoxia was associated with an increase in phosphorylation of these receptors. Over-expression of a catalytically inactive G protein-coupled receptor kinase 2 (GRK2 K220R) blocked the increased phosphorylation and sequestration of the M2, but not M1, mAChRs following transient hypoxia. Hypoxia induced phosphorylation and sequestration of the M1 mAChR was, however, blocked by over-expression of a catalytically inactive casein kinase 1 alpha (CK1alpha K46R). These results are the first demonstration that M1 and M2 mAChRs undergo sequestration following transient hypoxia. The data suggest that increased phosphorylation of M1 and M2 mAChRs underlies the mechanism responsible for sequestration of these receptors following transient hypoxia. We report here that distinct pathways involving CK1alpha and GRK2 mediated sequestration of M1 and M2 mAChRs following transient hypoxic-induced oxidative stress.

Animals↗

PubMed2005

Neuroprotection by neuregulin-1 following focal stroke is associated with the attenuation of ischemia-induced pro-inflammatory and stress gene expression.

Neuregulins are a family of growth factors with potent neuroprotective properties. We recently demonstrated that neuregulin-1 blocked delayed neuronal death following focal ischemic stroke in the rat. Focal ischemia results in the release of pro-inflammatory cytokines that produce profound changes in gene expression and contribute to cell death associated with stroke. Inflammatory and stress mediators are involved in the pathogenesis of focal ischemic brain damage. We examined whether neuregulin-1 can influence inflammatory and stress gene expression in the rat brain following transient middle cerebral artery occlusion (MCAO). In this study, we compared gene expression profiles in animals treated with neuregulin-1beta (NRG-1) or vehicle followed by MCAO. We used the Affymetrix GeneChip system to analyze gene expression in focal ischemia of the rat brain. Several inflammatory and stress genes were significantly induced following MCAO compared to sham controls including heat shock protein-70 (HSP70), interleukin-1beta, and macrophage chemotattractant protein-1 (JE/MCP-1). Treatment with NRG-1 attenuated the expression of many of these genes by 50% or more. In vitro studies demonstrated that NRG-1 suppressed inflammatory gene expression in activated macrophages. NRG-1 also prevented neuronal death induced by oxygen-glucose deprivation in a rat neuroblastoma cell line, suggesting that NRG-1 may have both direct and indirect neuroprotective capacity. These results demonstrate that NRG-1 can regulate inflammatory and stress gene expression and may give new insight to the molecular mechanisms involved in the neuroprotective role of neuregulins in stroke.

Animals↗