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

Abstract

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.

Trafficking Kinesin Protein (TRAK)-mediated Transport of Mitochondria in Axons of Hippocampal Neurons

“…The conservation in amino acid sequence between TRAK1 and TRAK2 and the similarity in their known functional properties (i.e. their direct binding to the cargo domains of KHC; their ability to co-distribute with mitochondria, resulting in the collapse of the mitochondrial network when overexpressed in cells; the redistribution of mitochondria to the tips of processes when either TRAK1 or TRAK2 are co-expressed with KHC; and the co-immunoprecipitation of Miro1 or Miro2 with either TRAK1 or TRAK2 following overexpression in mammalian cells) suggest that both proteins have similar functions (9,12,13,15,17). Yet despite the finding that TRAK1 and TRAK2 shRNAs had similar efficacies in heterologous expression, TRAK2 shRNAis had no effect on mitochondrial mobility.…”

“…Thus, TRAK1 and TRAK2 may participate in microtubule-based transport of early endosomes by acting as an adaptor linking Hrs-containing endosomes to kinesin (20,21). TRAKs have also been linked to the forward trafficking of inhibitory GABA A neurotransmitter receptors (9,32) and Kir2.1 inwardly rectifying potassium channels (33). TRAKs may therefore be promiscuous in terms of the cargo they transport.…”

“…pCMVTag4aTRAK2 (C-terminal FLAG-tagged TRAK2) was as in Ref. 9; pcDNAHisMaxKIF5C (N-terminal His tag), pCMVTRAK2(283-913) (N-terminal c-Myc tag), and pCIShumanTRAK1 (pCIShTRAK1) were as in Ref. 12; pDsRed1-Mito for visualization of mitochondria, pECFP-TRAK2, and pKIF5C-EYFP were as in Ref.…”

“…For double transfections, a 1:1 ratio for a total of 10 g of DNA was used, and for triple transfections, a 1:1:1 ratio was used again with 10 g of DNA/250-ml culture flask. Cells were harvested 24 -48 h post-transfection, and either cell homogenates were analyzed by immunoblotting, or transfected cell homogenates were 1% (v/v) Triton X-100 detergent-solubilized, and extracts were collected following centrifugation for 40 min at 4°C at 100,000 ϫ g (9).…”

“…There are two mammalian TRAKs, TRAK1 and TRAK2, that share ϳ58% amino acid homology (9,10). The TRAKs, like their Drosophila orthologue Milton (11), have been shown to function as kinesin adaptors linking kinesin heavy chain (KHC) to mitochondria by their association with Miro1/2.…”