The gene product of the gp78/AMFR ubiquitin E3 ligase cDNA is selectively recognized by the 3F3A antibody within a subdomain of the endoplasmic reticulum

The receptor for the autocrine motility factor/phosphoglucose isomerase cytokine (gp78 or AMFR) has been extensively characterized using the 3F3A monoclonal antibody. Cloning of AMFR identified a seven-transmembrane domain G-protein-coupled receptor ubiquitin E3 ligase whose identity as AMFR was based on prior expression cloning with the 3F3A mAb that generated a truncated sequence. We show here that the gp78/AMFR gene product is indeed recognized by the 3F3A mAb. The FLAG-taggedAMFR immunoprecipitated with an anti-FLAG antibody was recognized by the 3F3A mAb in Western blot analysis and cells transfected with AMFR exhibit increased labeling with the 3F3A mAb. The 3F3A mAb does not however recognize higher molecular weight isoforms of AMFR. 3F3A labeling colocalizes with tagged AMFR in a peripheral ER network but does not recognize FLAG- or GFP-tagged AMFR localized to a perinuclear ER domain that likely corresponds to misfolded forms of the protein retained in the ER. These data indicate that 3F3A antibody binding is highly specific for a subpopulation of AMFR localized to an ER subdomain. Coexpression of AMFR-GFP and a lumenal ER-targeted RFP presented extensive colocalization in living cells andAMFR-GFP is concentrated in a basal ER network morphologically similar to that labeled by the 3F3A mAb in fixed cells. The3F3A anti-AMFR mAb therefore selectively recognizes a subpopulation of expressed AMFR localized to a subdomain of the ER.     

Coordinated action of NSF and PKC regulates GABAB receptor signaling efficacy

The obligatory heterodimerization of the GABAB receptor (GBR) raises fundamental questions about molecular mechanisms controlling its signaling efficacy. Here, we show that NEM sensitive fusion (NSF) protein interacts directly with the GBR heterodimer both in rat brain synaptosomes and in CHO cells, forming a ternary complex that can be regulated by agonist stimulation. Inhibition of NSF binding with a peptide derived from GBR2 (TAT-Pep-27) did not affect basal signaling activity but almost completely abolished agonist-promoted GBR desensitization in both CHO cells and hippocampal slices. Taken with the role of PKC in the desensitization process, our observation that TAT-Pep-27 prevented both agonist-promoted recruitment of PKC and receptor phosphorylation suggests that NSF is a priming factor required for GBR desensitization. Given that GBR desensitization does not involve receptor internalization, the NSF/PKC coordinated action revealed herein suggests that NSF can regulate GPCR signalling efficacy independently of its role in membrane trafficking. The functional interaction between three bona fide regulators of neurotransmitter release, such as GBR, NSF and PKC, could shed new light on the modulation of presynaptic GBR action.     

Efficient transmission of pandemic H1N1 influenza viruses with high-level oseltamivir resistance

The limited availability of approved influenza virus antivirals highlights the importance of studying the fitness and transmissibility of drug-resistant viruses. S247N is a novel, naturally occurring N1 neuraminidase mutation that reduces oseltamivir sensitivity and greatly potentiates oseltamivir resistance in the context of the H275Y mutation. Here we show that highly oseltamivir-resistant viruses containing both the S247N and H275Y mutations transmit efficiently in the guinea pig transmission model.     

Impedance responses reveal β₂-adrenergic receptor signaling pluridimensionality and allow classification of ligands with distinct signaling profiles

The discovery that drugs targeting a single G protein-coupled receptor (GPCR) can differentially modulate distinct subsets of the receptor signaling repertoire has created a challenge for drug discovery at these important therapeutic targets. Here, we demonstrate that a single label-free assay based on cellular impedance provides a real-time integration of multiple signaling events engaged upon GPCR activation. Stimulation of the β₂-adrenergic receptor (β₂AR) in living cells with the prototypical agonist isoproterenol generated a complex, multi-featured impedance response over time. Selective pharmacological inhibition of specific arms of the β₂AR signaling network revealed the differential contribution of G_s-, G_i- and Gβγ-dependent signaling events, including activation of the canonical cAMP and ERK1/2 pathways, to specific components of the impedance response. Further dissection revealed the essential role of intracellular Ca²⁺ in the impedance response and led to the discovery of a novel β₂AR-promoted Ca²⁺ mobilization event. Recognizing that impedance responses provide an integrative assessment of ligand activity, we screened a collection of β-adrenergic ligands to determine if differences in the signaling repertoire engaged by compounds would lead to distinct impedance signatures. An unsupervised clustering analysis of the impedance responses revealed the existence of 5 distinct compound classes, revealing a richer signaling texture than previously recognized for this receptor. Taken together, these data indicate that the pluridimensionality of GPCR signaling can be captured using integrative approaches to provide a comprehensive readout of drug activity.     

Lamin A/C Mutants Disturb Sumo1 Localization and Sumoylation in Vitro and in Vivo

A-type lamins A and C are nuclear intermediate filament proteins in which mutations have been implicated in multiple disease phenotypes commonly known as laminopathies. A few studies have implicated sumoylation in the regulation of A-type lamins. Sumoylation is a post-translational protein modification that regulates a wide range of cellular processes through the attachment of small ubiquitin-related modifier (sumo) to various substrates. Here we showed that laminopathy mutants result in the mislocalization of sumo1 both in vitro (C2C12 cells overexpressing mutant lamins A and C) and in vivo (primary myoblasts and myopathic muscle tissue from the Lmna^{H222P /H222P} mouse model). In C2C12 cells, we showed that the trapping of sumo1 in p.Asp192Gly, p.Gln353Lys, and p.Arg386Lys aggregates of lamin A/C correlated with an increased steady-state level of sumoylation. However, lamin A and C did not appear to be modified by sumo1. Our results suggest that mutant lamin A/C alters the dynamics of sumo1 and thus misregulation of sumoylation may be contributing to disease progression in laminopathies.     

Identification and purification of membrane and soluble forms of the major surface protein of Leishmania promastigotes

A major integral membrane glycoprotein of 63 kDa (p63), present at 500,000 copies/cell, was found on the surface of Leishmania major LEM 513 promastigotes. This protein was labeled either by surface iodination of the cells or by metabolic incorporation of [35S]methionine. Peptide maps of the proteins labeled by the two procedures were identical. Protein p63 was purified in three steps: extraction and phase separation in the nonionic detergent Triton X-114, chromatography on DEAE-cellulose, and finally chromatography on a Mono-Q column. The carbohydrate content as well as the concanavalin A receptor activity were characterized. A hydrophilic form of p63 was generated during the purification of the protein. This form was not derived by proteolysis from the amphiphilic protein found in the membrane, but may have been generated by the hydrolysis of a lipid containing myristyl residue(s) anchoring the protein in the membrane.     

Phosphorylation of the rev gene product of human immunodeficiency virus type 1.

Replication of human immunodeficiency virus type 1 requires the functional expression in trans of the virally encoded rev gene product (previously called art/trs). Here we demonstrate that this protein can be metabolically labeled with 32Pi. The phosphate receptor in the rev protein is shown to be exclusively serine. Treatment of rev-expressing cells with phorbol ester, a specific activator of protein kinase C, led to significant but transient enhancement of the level of rev phosphorylation. These results indicate that the rev protein is posttranslationally modified in vivo and suggest that the level of this modification is subject to modulation by extracellular stimuli.     

The induced circular dichroism of bilirubin complexed with the alpha-helix form of poly(L-lysine)

Binding of bilirubin by the alpha-helix conformation of poly(L-lysine) in water induces optical activity. The bisignate circular dichroism spectrum exhibits exciton bands centred at 444 nm, negative, and at 525 nm, positive. The magnitude of the induced circular dichroism depends on the concentration of total bilirubin and total lysine residues, the molar ratio of total lysine residues-to-total bilirubin molecules, the pH and the degree of polymerization of poly(L-lysine). Although bilirubin binds to the random coil conformation of poly(L-lysine), as evidence by the absorption spectrum, the complex is optically inactive. The results suggest that bilirubin binds to the poly(L-lysine) in the form of dimers and oligomers.