Purchase this article with an account.
X. Song, H. Qian; Human GABA rho1 Subunit Interacts with Cellular Retinoic Acid-binding Protein (CRABP) in Retina . Invest. Ophthalmol. Vis. Sci. 2003;44(13):5143.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: The GABAc receptor is a ligand-gated chloride channel that predominately expressed in retinal neurons. GABA rho subunits are thought to participate in forming the GABAc receptor. Each rho subunit contains a large intracellular domain that could interact with cellular proteins for receptor anchoring, clustering, and regulation. In this study, we used CytoTrop XR yeast 2-hybrid system to screen for retinal proteins that bind with human GABA rho1 subunit, and identified cellular retinoic acid-binding protein (CRABP) as a potential interactor. Methods: The large intracellular domain of human GABA rho1 subunit (amino acids 358 to 457) was cloned in frame into a bait vector (pSOS, Stratagene). The construct was used to screen a bovine retinal cDNA library (Stratagene) in a temperature-sensitive mutant yeast strain (cdc25H). A GST-His- human rho1 fusion protein was generated with pET42a vector and produced in BL21 bacteria. For immunoprecipitation experiments, the fusion proteins were mixed with baboon retinal proteins and pulled down by Ni-NTA coated agarose beads (Qiagen). The precipitated proteins were analyzed by Western-blot and detected with anti-mouse CRABP antibody (gift of Dr. Saari). Results: Approximately two million cDNA recombinants were screened with human rho1 subunit as bait. Two hundreds clones were obtained by growing the transfected yeast on SD/Galactose (-UL) selection medium at 37°C. Nucleotide sequencing of these clones indicate 18 of them contained a full open-reading frame encoding bovine CRABP. The interaction between GABA rho1 and CRABP were confirmed using retinal preparations. When baboon retinal proteins were mixed with the GST-His-human rho1 fusion protein, CRABP was found in the pellets pulled down by Ni-NTA beads. In control, GST-His fusion protein itself did not co-precipitate with CRABP. To determine the CRABP binding domain on human rho1 subunit, the intracellular loop was segmented and analyzed with CytoTrop XR yeast 2-hybrid system. No interaction was found between CRABP and the first 69 amino acid segment, suggesting the binding site may be lie in the last 31 amino acid segment of the intracellular loop. Conclusions: We identified CRABP as one of the cellular proteins that interact with GABA rho1 subunit in primate retina. CRABP is widely distributed in retinal cells, and is a vehicle to deliver RA into the nucleus. The interaction between rho subunit and CRABP would provide a novel mechanism to control gene regulation through GABAc receptor.
This PDF is available to Subscribers Only