The γ-aminobutyric acid type A (GABA
            <sub>A</sub>
            ) receptor-associated protein (GABARAP) promotes GABA
            <sub>A</sub>
            receptor clustering and modulates the channel kinetics

Members of the erbB family receptor tyrosine kinases (erbB1, erbB2, erbB3, and erbB4) are overexpressed in a variety of human cancers and represent important targets for the structure-based drug design. Homo-and heterodimerization (oligomerization) of the erbB receptors are known to be critical events for receptor signaling. To block receptor self-associations, we have designed a series of peptides derived from potential dimerization surfaces in the extracellular subdomain IV of the erbB receptors (erbB peptides). In surface plasmon resonance (BIAcore) studies, the designed peptides have been shown to selectively bind to the erbB receptor ectodomains and isolated subdomain IV of erbB2 with submicromolar affinities and to inhibit heregulin-in- erbB2 (neu, HER2) is a member of the epidermal growth factor or HER family of tyrosine kinase receptors that also includes erbB1 (EGFR, 1 HER1), erbB3 (HER3), and erbB4 (HER4) (1-4). Overexpression of erbB receptors has been found in many types of human cancer raising the possibility that receptor-directed therapies may be useful as cancer management strategies. Greater expression of erbB2 on transformed cells than on normal epithelial tissues allows selective targeting of tumor cells using various approaches (5-13). A variety of strategies have also been developed for targeting the erbB1 receptor, including monoclonal antibodies, ligand-linked immunotoxins, tyrosine kinase inhibitors, and antisense approaches.Recently, we have reported the design of an anti-erbB2 peptide mimetic, AHNP, derived from the structure of the CDR-H3 loop of the anti-erbB2 monoclonal antibody 4D5 and demonstrated its in vitro and in vivo activities in disabling erbB2 tyrosine kinases similar to the monoclonal antibody (14 -16). We have argued that another interesting approach for disabling erbB receptor activity would be targeting protein-protein interaction surfaces. Because protein-protein interactions play a key role in various mechanisms of cellular growth and differentiation, and viral replication, inhibition of these interactions is a promising novel approach for rational drug design against a wide number of cellular and viral targets (17,18). Synthetic peptides that disrupt protein-protein interactions have been successfully shown to act as inhibitors of HIV-1 protease (19), HIV-1 reverse transcriptase (20), herpes simplex virus ribonucleotide reductase (21), and thymidilate synthase (22). Binding of polypeptide hormones, growth factors, or cytokines to cell surface receptors activates dimerization (oligomerization) of the receptors, which leads to the signal transduction to the interior of the cell (23). Although most of the receptor inhibitors developed to date have been focused on the blockade of receptor-ligand or enzyme-substrate interactions, repression of receptor-receptor interactions that accompany oligomerization might also represent an important target for disabling receptor functioning. This approach has been recently used for the design of peptidic estrogen receptor inhibitors ...

Section: Discussionmentioning

confidence: 77%

Disabling Receptor Ensembles with Rationally Designed Interface Peptidomimetics

Berezov

Chen

Liu

et al. 2002

“…In another series of experiments, we recorded GABA A single channel currents in cells expressing GABA A ␣ 1 and ␤ 1 subunits (no ␥ 2S ) together with full-length GABARAP. Because the GABARAP binding site is on the ␥ subunit, GABARAP would not bind to these receptors, and indeed there is no clustering of receptors under these conditions (8). In seven cell-attached patches on cells expressing ␣ 1 A, examples of high conductance channels activated with 1 M GABA in one of these outside-out patches.…”

Section: Outside-out Patchesmentioning

confidence: 99%

“…When co-expressed with GABA A subunits in QT-6 quail fibroblasts, GABARAP causes clustering of GABA A receptors accompanied by changes in whole cell current kinetics (8). Although this protein was not found in close proximity to mature native GABA A receptors in the plasmalemma of cortical neurons in a study using fluorescent antibodies to GABARAP (9), there is compelling evidence that co-expression of GABARAP with subunits of GABA A receptors does result in increased clustering of receptors in many cells (8). These observations suggest that GABARAP may be involved at an early stage in the clustering of GABA A receptors even though it appears not to be present as part of the anchoring mechanism for the receptors.…”

mentioning

confidence: 99%

Conductance of Recombinant GABA Channels Is Increased in Cells Co-expressing GABAA A Receptor-associated Protein

Everitt

Luu

Cromer

et al. 2004

Self Cite

High conductance ␥-aminobutyric acid type A (GABA A ) channels (>40 picosiemens (pS)) have been reported in some studies on GABA A channels in situ but not in others, whereas recombinant GABA A channels do not appear to display conductances above 40 pS. Furthermore, the conductance of some native GABA A channels can be increased by diazepam or pentobarbital, which are effects not reported for expressed GABA A channels. GABARAP, a protein associated with native GABA A channels, has been reported to cause clustering of GABA A receptors and changes in channel kinetics. We have recorded single channel currents activated by GABA in L929 cells expressing ␣ 1 , ␤ 1 , and ␥ 2S subunits of human GABA A receptors. Channel conductance was never higher than 40 pS and was not significantly increased by diazepam or pentobarbital, although open probability was increased. In contrast, in cells expressing the same three subunits together with GABARAP, channel conductance could be significantly higher than 40 pS, and channel conductance was increased by diazepam and pentobarbital. GABARAP caused clustering of receptors in L929 cells, and we suggest that there may be interactions between subunits of clustered GABA A receptors that make them open co-operatively to give high conductance "channels." Recombinant channels may require the influence of GABARAP and perhaps other intracellular proteins to adopt a fuller repertoire of properties of native channels.

“…MAP-1B was shown to link GABA C receptors to the cytoskeleton at retinal synapses (10). GABARAP is a 13.9-kDa microtubule-associated protein that binds specifically to the ␥2 subunit and promotes the clustering of GABA A receptors expressed in Qt-6 quail fibroblasts (11,12). Differences in functional properties were reported between the clustered and unclustered receptors (12).…”

mentioning

confidence: 99%

“…GABARAP is a 13.9-kDa microtubule-associated protein that binds specifically to the ␥2 subunit and promotes the clustering of GABA A receptors expressed in Qt-6 quail fibroblasts (11,12). Differences in functional properties were reported between the clustered and unclustered receptors (12). It has also recently been reported to mediate intracellular transport of GABA A receptors by virtue of its specific interaction with N-ethylmaleimide-sensitive factor (13).…”

mentioning

confidence: 99%

Identification, Molecular Cloning, and Characterization of a Novel GABAA Receptor-associated Protein, GRIF-1

Beck¹,

Brickley²,

Wilkinson³

et al. 2002

103

A novel 913-amino acid protein, ␥-aminobutyric acid type A (GABA A ) receptor interacting factor-1 (GRIF-1), has been cloned and identified as a GABA A receptorassociated protein by virtue of its specific interaction with the GABA A receptor ␤2 subunit intracellular loop in a yeast two-hybrid assay. GRIF-1 has no homology with proteins of known function, but it is the rat orthologue of the human ALS2CR3/KIAA0549 gene. GRIF-1 is expressed as two alternative splice forms, GRIF-1a and a C-terminally truncated form, GRIF-1b. GRIF-1 mRNA has a wide distribution with a major transcript size of 6.2 kb. GRIF-1a protein is only expressed in excitable tissues, i.e. brain, heart, and skeletal muscle major immunoreactive bands of M r ϳ 115 and 106 kDa and, in muscle and heart only, an additional 88-kDa species. When expressed in human embryonic kidney 293 cells, GRIF-1a yielded three immunoreactive bands with M r ϳ 115, 106, and 98 kDa. Co-expression of GRIF-1a and ␣1␤2␥2 GABA A receptors in mammalian cells revealed some co-localization in the cell cytoplasm. Anti-FLAGagarose specifically precipitated GRIF-1 FLAG and GABA A receptor ␤2 subunits from human embryonic kidney 293 cells co-transfected with GRIF-1a FLAG and ␤2 subunit clones. Further, immobilized GRIF-1-(8 -633) specifically precipitated in vitro GABA A receptor ␣1 and ␤2 subunit immunoreactivities from detergent extracts of adult rat brain. The respective GABA A receptor ␤2 subunit/GRIF-1 binding domains were mapped using the yeast two-hybrid reporter gene assays. A possible role for GRIF-1 as a GABA A receptor ␤2 subunit trafficking factor is proposed.