Background: Glucagon-like peptide 1 agonists differ in chemical structure, duration of action and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. Methods: We randomly assigned patients with type 2 diabetes and cardiovascular disease to the addition of once-weekly subcutaneous injection of albiglutide (30 mg to 50 mg) or matching placebo to standard care. We hypothesized that albiglutide would be noninferior to placebo for the primary outcome of first occurrence of cardiovascular death, myocardial infarction, or stroke. If noninferiority was confirmed by an upper limit of the 95% confidence interval for the hazard ratio of less than 1.30, closed-testing for superiority was prespecified. Findings: Overall, 9463 participants were followed for a median of 1.6 years. The primary composite outcome occurred in 338 of 4731 patients (7.1%; 4.6 events per 100 person-years) in the albiglutide group and in 428 of 4732 patients (9.0%; 5.9 events per 100 person-years) in the placebo group (hazard ratio, 0.78; 95% confidence interval [CI ], 0.68 to 0.90), indicating that albiglutide, was superior to placebo (P<0.0001 for noninferiority, P=0.0006 for superiority). The incidence of acute pancreatitis (albiglutide 10 patients and placebo 7 patients), pancreatic cancer (6 and 5), medullary thyroid carcinoma (0 and 0), and other serious adverse events did not differ significantly between the two groups. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. (Funded by GlaxoSmithKline; Harmony Outcomes ClinicalTrials.gov number, NCT02465515.) noninferiority; P = 0.06 for superiority). There seems to be variation in the results of existing trials with GLP-1 receptor agonists, which if correct, might reflect drug structure or duration of action, patients studied, duration of follow-up or other factors.

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The epithelial-specific adaptor AP1B sorts basolateral proteins, but the trafficking routes where it performs its sorting role remain controversial. Here, we used an RNAi approach to knock down the medium subunit of AP1B (1B) in the prototype epithelial cell line Madin-Darby canine kidney (MDCK). 1B-knocked down MDCK cells displayed loss of polarity of several endogenous and exogenous basolateral markers transduced via adenovirus vectors, but exhibited normal polarity of apical markers. We chose two well characterized basolateral protein markers, the transferrin receptor (TfR) and the vesicular stomatitis virus G protein, to study the sorting role of AP1B. A surface-capture assay introduced here showed that 1B-knocked down MDCK cells plated on filters at confluency and cultured for 4.

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The interesting observation was made 20 years ago that psychotic manifestations in patients with systemic lupus erythematosus are associated with the production of antiribosomal-P protein (anti-P) autoantibodies. Since then, the pathogenic role of anti-P antibodies has attracted considerable attention, giving rise to long-term controversies as evidence has either contradicted or confirmed their clinical association with lupus psychosis. Furthermore, a plausible mechanism supporting an anti-P–mediated neuronal dysfunction is still lacking. We show that anti-P antibodies recognize a new integral membrane protein of the neuronal cell surface. In the brain, this neuronal surface P antigen (NSPA) is preferentially distributed in areas involved in memory, cognition, and emotion. When added to brain cellular cultures, anti-P antibodies caused a rapid and sustained increase in calcium influx in neurons, resulting in apoptotic cell death. In contrast, astrocytes, which do not express NSPA, were not affected. Injection of anti-P antibodies into the brain of living rats also triggered neuronal death by apoptosis. These results demonstrate a neuropathogenic potential of anti-P antibodies and contribute a mechanistic basis for psychiatric lupus. They also provide a molecular target for future exploration of this and other psychiatric diseases.

The epithelial-specific adaptor AP1B sorts basolateral plasma membrane (PM) proteins in both biosynthetic and recycling routes, but the site where it carries out this function remains incompletely defined. Here, we have investigated this topic in Fischer rat thyroid (FRT) epithelial cells using an antibody against the medium subunit 1〉. This antibody was suitable for immunofluorescence and blocked the function of AP1B in these cells. The antibody blocked the basolateral recycling of two basolateral PM markers, Transferrin receptor (TfR) and LDL receptor (LDLR), in a perinuclear compartment with marker and functional characteristics of recycling endosomes (RE). Live imaging experiments demonstrated that in the presence of the antibody two newly synthesized GFP-tagged basolateral proteins (vesicular stomatitis virus G [VSVG] protein and TfR) exited the trans-Golgi network (TGN) normally but became blocked at the RE within 3-5 min.By contrast, the antibody did not block trafficking of green fluorescent protein (GFP)-LDLR from the TGN to the PM but stopped its recycling after internalization into RE in ϳ45 min. Our experiments conclusively demonstrate that 1) AP1B functions exclusively at RE; 2) TGN-to-RE transport is very fast and selective and is mediated by adaptors different from AP1B; and 3) the TGN and AP1B-containing RE cooperate in biosynthetic basolateral sorting. INTRODUCTIONEpithelial cells display an asymmetric distribution of plasma membrane (PM) proteins into apical and basolateral PM domains (Yeaman et al., 1999;Mostov et al., 2003; RodriguezBoulan et al., 2005). Early studies demonstrated that this polarity was achieved by sorting of newly synthesized proteins at the level of the trans-Golgi network (TGN; Rindler et al., 1984;Fuller et al., 1985;Griffiths and Simons, 1986) and of endocytosed proteins at the level of recycling endosomes (RE;Mostov and Cardone, 1995;Odorizzi and Trowbridge, 1997). Trafficking in both biosynthetic and recycling routes is controlled by apical sorting signals (e.g., N-and O-glycans, lipid anchors, and protein domains with affinity for lipid rafts), by microtubule motor determinants (Rodriguez-Boulan and Gonzalez, 1999;Schuck and Simons, 2004;Rodriguez-Boulan et al., 2005) and by basolateral sorting signals (e.g., tyrosine, dileucine, and monoleucine motifs, some similar to endocytic determinants, and noncanonic motifs not yet matching any consensus sequence; Rodriguez-Boulan et al., 2005). Tyrosine-based signals are recognized by a family of organelle-specific tetrameric AP (adaptor protein) adaptors via their subunit, whereas dileucine motifs may be recognized via large (␥ and ␦) and small ( 1, 3) subunits of AP adaptors acting together (Bonifacino and Lippincott-Schwartz, 2003;Janvier et al., 2003).The early paradigm of two separate sorting sites for proteins in biosynthetic or recycling pathways has, however, progressively shifted over the past decade to a different paradigm in which both TGN and RE share a sorting role in the biosynthetic route. This new paradigm emer...

Current models put forward that the epidermal growth factor receptor (EGFR) is efficiently internalized via clathrin-coated pits only in response to ligand-induced activation of its intrinsic tyrosine kinase and is subsequently directed into a lysosomal-proteasomal degradation pathway by mechanisms that include receptor tyrosine phosphorylation and ubiquitylation. Herein, we report a novel mechanism of EGFR internalization that does not require ligand binding, receptor kinase activity, or ubiquitylation and does not direct the receptor into a degradative pathway. Inhibition of basal protein kinase A (PKA) activity by H89 and the cell-permeable substrate peptide Myr-PKI induced internalization of 40 -60% unoccupied, inactive EGFR, and its accumulation into early endosomes without affecting endocytosis of transferrin and -opioid receptors. This effect was abrogated by interfering with clathrin function. Thus, the predominant distribution of inactive EGFR at the plasma membrane is not simply by default but involves a PKA-dependent restrictive condition resulting in receptor avoidance of endocytosis until it is stimulated by ligand. Furthermore, PKA inhibition may contribute to ligand-induced EGFR endocytosis because epidermal growth factor inhibited 26% of PKA basal activity. On the other hand, H89 did not alter ligand-induced internalization of EGFR but doubled its half-time of down-regulation by retarding its segregation into degradative compartments, seemingly due to a delay in the receptor tyrosine phosphorylation and ubiquitylation. Our results reveal that PKA basal activity controls EGFR function at two levels: 1) residence time of inactive EGFR at the cell surface by a process of "endocytic evasion," modulating the accessibility of receptors to stimuli; and 2) sorting events leading to the down-regulation pathway of ligand-activated EGFR, determining the length of its intracellular signaling. They add a new dimension to the fine-tuning of EGFR function in response to cellular demands and cross talk with other signaling receptors. INTRODUCTIONThe mechanisms that regulate the endocytic behavior of epidermal growth factor receptor (EGFR) have been a longstanding subject of intense research as a model system of regulated vesicular protein traffic associated to signaling, cellular demands, and cancer (Trowbridge et al., 1993;Di Fiore and Gill, 1999;Carpenter, 2000;Ceresa and Schmid, 2000;Schlessinger, 2000;Wiley and Burke, 2001). Endocytosis provides a pathway for gradual attenuation or desensitization of receptor signaling and also allows a single receptor to transmit different signals from different locations in the cell before degradation, thus enhancing the range of modulation and response variability (Di Fiore and Gill, 1999;Ceresa and Schmid, 2000;Wiley and Burke, 2001). Defects in internalization and/or degradation pathways, as well as distinct endocytic routing displayed by different EGFR family members, have been associated with cell transformation and oncogenesis (Di Fiore and Gill, 1999;Ceresa and Schm...

Research following introduction of the MDCK model system to study epithelial polarity (1978) led to an initial paradigm that posited independent roles of the trans Golgi network (TGN) and recycling endosomes (RE) in the generation of, respectively, biosynthetic and recycling routes of plasma membrane (PM) proteins to apical and basolateral PM domains. This model dominated the field for 20 years. However, studies over the past decade and the discovery of the involvement of clathrin and clathrin adaptors in protein trafficking to the basolateral PM has led to a new paradigm. TGN and RE are now believed to cooperate closely in both biosynthetic and recycling trafficking routes. Here, we critically review these recent advances and the questions that remain unanswered.