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 beta2 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 similar to 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 similar to 115, 106, and 98 kDa. Co-expression of GRIF-1a and alpha1beta2gamma2 GABA(A) receptors in mammalian cells revealed some co-localization in the cell cytoplasm. Anti-FLAG-agarose specifically precipitated GRIF-1(FLAG) and GABAA receptor beta2 subunits from human embryonic kidney 293 cells co-transfected with GRIF-1a(FLAG) and beta2 subunit clones. Further, immobilized GRIF-1-(8-633) specifically precipitated in vitro GABA(A) receptor alpha1 and beta2 subunit immunoreactivities from detergent extracts of adult rat brain. The respective GABA(A) receptor beta2 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 beta2 subunit trafficking factor is proposed.