PubMed Health⌕ Search

Biomedical subjects

Oussama El Far

Publications and source records attributed to Oussama El Far.

7 recordsLinked to original sources
PubMed2005

Pias1 interaction and sumoylation of metabotropic glutamate receptor 8.

Group III presynaptic metabotropic glutamate receptors (mGluRs) play a central role in regulating presynaptic activity through G-protein effects on ion channels and signal transducing enzymes. Like all Class C G-protein-coupled receptors, mGluR8 has an extended intracellular C-terminal domain (CTD) presumed to allow for modulation of downstream signaling. In a yeast two-hybrid screen of an adult rat brain cDNA library with the CTDs of mGluR8a and 8b (mGluR8-C) as baits, we identified sumo1 and four different components of the sumoylation cascade (ube2a, Pias1, Piasgamma, Piasxbeta) as interacting proteins. Binding assays using recombinant GST fusion proteins confirmed that Pias1 interacts not only with mGluR8-C but also with all group III mGluR CTDs. Pias1 binding to mGluR8-C required a region N-terminal to a consensus sumoylation motif and was not affected by arginine substitution of the conserved lysine 882 within this motif. Co-transfection of fluorescently tagged mGluR8a-C, sumo1, and enzymes of the sumoylation cascade into HEK293 cells showed that mGluR8a-C can be sumoylated in vivo. Arginine substitution of lysine 882 within the consensus sumoylation motif, but not other conserved lysines within the CTD, abolished in vivo sumoylation. Our results are consistent with post-translational sumoylation providing a novel mechanism of group III mGluR regulation.

Amino Acid Motifs↗
PubMed2004

The neuronal glycine transporter 2 interacts with the PDZ domain protein syntenin-1.

The glycine transporter subtype 2 (GlyT2) is localized at glycinergic axon terminals where it mediates the re-uptake of glycine from the extracellular space. In this study, we used the yeast two-hybrid system to search for proteins that interact with the cytoplasmic carboxy terminal tail region of GlyT2. Screening of a rat brain cDNA library identified the PDZ domain protein syntenin-1 as an intracellular binding partner of GlyT2. In pull-down experiments, the interaction between GlyT2 and syntenin-1 was found to involve the C-terminal amino acid residues of GlyT2 and the PDZ2 domain of syntenin-1. Syntenin-1 is widely expressed in brain and co-localizes with GlyT2 in brainstem sections. Furthermore, syntenin-1 binds syntaxin 1A, which is known to regulate the plasma membrane insertion of GlyT2. Thus, syntenin-1 may be an in vivo binding partner of GlyT2 that regulates its trafficking and/or presynaptic localization in glycinergic neurons.

Amino Acid Sequence↗
PubMed2002

G-protein-coupled receptors for neurotransmitter amino acids: C-terminal tails, crowded signalosomes.

G-protein-coupled receptors (GPCRs) represent a superfamily of highly diverse integral membrane proteins that transduce external signals to different subcellular compartments, including nuclei, via trimeric G-proteins. By differential activation of diffusible G(alpha) and membrane-bound G(beta)gamma subunits, GPCRs might act on both cytoplasmic/intracellular and plasma-membrane-bound effector systems. The coupling efficiency and the plasma membrane localization of GPCRs are regulated by a variety of interacting proteins. In this review, we discuss recently disclosed protein interactions found with the cytoplasmic C-terminal tail regions of two types of presynaptic neurotransmitter receptors, the group III metabotropic glutamate receptors and the gamma-aminobutyric acid type-B receptors (GABA(B)Rs). Calmodulin binding to mGluR7 and other group III mGluRs may provide a Ca(2+)-dependent switch for unidirectional (G(alpha)) versus bidirectional (G(alpha) and G(beta)gamma) signalling to downstream effector proteins. In addition, clustering of mGluR7 by PICK1 (protein interacting with C-kinase 1), a polyspecific PDZ (PSD-95/Dlg1/ZO-1) domain containing synaptic organizer protein, sheds light on how higher-order receptor complexes with regulatory enzymes (or 'signalosomes') could be formed. The interaction of GABA(B)Rs with the adaptor protein 14-3-3 and the transcription factor ATF4 (activating transcription factor 4) suggests novel regulatory pathways for G-protein signalling, cytoskeletal reorganization and nuclear gene expression: processes that may all contribute to synaptic plasticity.

Amino Acid Sequence↗
PubMed2002

Gephyrin interacts with Dynein light chains 1 and 2, components of motor protein complexes.

The clustering of glycine receptors and major subtypes of GABA(A) receptors at inhibitory synapses is mediated by the tubulin-binding protein gephyrin. In an attempt to identify additional components of inhibitory postsynaptic specializations, we performed a yeast two-hybrid screen using gephyrin as bait. Multiple positive clones encoded either the dynein light chain-1 (Dlc-1), also known as dynein LC8 and protein inhibitor of neuronal nitric oxide synthase, or its homolog Dlc-2. Dlc-1 protein bound efficiently to gephyrin in in vitro binding assays and colocalized with gephyrin during coexpression in HEK293 cells. The binding site for Dlc was mapped to a fragment of 63 amino acids within the central linker domain of gephyrin. In hippocampal neurons, endogenous Dlc protein was enriched at synaptic sites identified by synaptophysin and gephyrin immunostaining. Immunoelectron microscopy in spinal cord sections revealed Dlc immunoreactivity at the edges of postsynaptic differentiations, in close contact with cytoskeletal structures and at the periphery of the Golgi apparatus. Because Dlc-1 and Dlc-2 have been described as stoichiometric components of cytoplasmic dynein and myosin-Va complexes, our results suggest that motor proteins are involved in the subcellular localization of gephyrin.

Amino Acid Sequence↗
PubMed2002

Interaction of the C-terminal region of the rat serotonin transporter with MacMARCKS modulates 5-HT uptake regulation by protein kinase C.

The serotonin transporter (SERT) mediates the re-uptake of released serotonin into presynaptic nerve terminals. Its activity is regulated by different mechanisms including protein kinase C (PKC) triggered internalization. Here, we used yeast 2-hybrid screening and cotransfection into 293 cells to identify a homologue of the myristoylated alanine-rich C kinase substrate (MARCKS), MacMARCKS, as a C-terminally interacting protein of SERT. Upon cotransfection with SERT, MacMARCKS caused a reduction in the maximal rate of [(3)H]serotonin uptake and reduced its down-regulation elicited by activation of PKC. Our data are consistent with MARCKS proteins regulating the plasma membrane dynamics of neurotransmitter transporters.

Animals↗
PubMed2002

Interaction of synaptophysin with the AP-1 adaptor protein gamma-adaptin.

Synaptophysin is one of the most abundant proteins of the synaptic vesicle membrane. Here, we selected the cytoplasmic carboxyterminal region of synaptophysin to search for interacting proteins by using the yeast two-hybrid system. This identified gamma-adaptin, a component of the AP-1 adaptor complex, as a synaptophysin binding protein. An anti-synaptophysin antibody coimmunoprecipitated gamma-adaptin from brain extracts, and immunocytochemistry disclosed a partial colocalization of synaptophysin and gamma-adaptin in the perinuclear region of cultured hippocampal neurons. Our results are consistent with synaptophysin serving as a docking site for AP-1 during clathrin-dependent vesicle budding and/or kinesin-based transport reactions.

Adaptor Protein Complex gamma Subunits↗