Identification, molecular cloning, and characterization of a novel GABAA receptor-associated protein, GRIF-1

A novel 913-amino acid protein, gamma-aminobutyric acid type A (GABA(A)) receptor interacting factor-1 (GRIF-1), has been cloned and identified as a GABA(A) receptor-associated protein by virtue of its specific interaction with the GABA(A) receptor beta 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) approximately 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) approximately 115, 106, and 98 kDa. Co-expression of GRIF-1a and alpha 1 beta 2 gamma 2 GABA(A) receptors in mammalian cells revealed some co-localization in the cell cytoplasm. Anti-FLAG-agarose specifically precipitated GRIF-1(FLAG) and GABA(A) receptor beta 2 subunits from human embryonic kidney 293 cells co-transfected with GRIF-1a(FLAG) and beta 2 subunit clones. Further, immobilized GRIF-1-(8-633) specifically precipitated in vitro GABA(A) receptor alpha 1 and beta 2 subunit immunoreactivities from detergent extracts of adult rat brain. The respective GABA(A) receptor beta 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 beta 2 subunit trafficking factor is proposed.