Molecular neuroscienceThe GABA ρ1 subunit interacts with a cellular retinoic acid binding protein in mammalian retina
Section snippets
Yeast two-hybrid screening
A commercially available CytoTrap two-hybrid system (Stratagene) was used to identify GABAC receptor binding partners. The system is based on the translocation of active human SOS (hSOS, the human equivalent of Drosophila “Son Of Sevenless” protein) to its site of action at the inner leaflet of the plasma membrane. The bait protein is expressed as a fusion protein with hSOS, and the prey is targeted to the membrane via a myristoylation signal sequence. Interacting fusion proteins recruit SOS to
Interaction of human ρ1 subunit with CRABP in mammalian retina
The CytoTrap yeast two-hybrid system was used to screen a bovine retinal cDNA library (Stratagene) for new partners interacting with the large intracellular loop of the human GABAC receptor ρ1 subunit (ρ1). Approximately 1.3 million recombinants were screened with a pSOS-ρ1 construct. Two hundred yeast colonies, grown at 37°C, were isolated from the selection plate. Among them, 23 colonies contained an insert sequence code for bovine CRABP type I (CRABP I, CRABP for short) (Shubeita et al., 1987
Discussion
Ionotropic neurotransmitter receptors are traditionally thought of as mediating fast synaptic transmission by gating ion channels. Although controlling ion flux is considered the main function of these receptors, the transmembrane structure of the receptor subunits may also serve as an alternative signal transduction mechanism. Indeed, recent studies have demonstrated some “metabotropic” functions for certain types of ionotropic glutamate receptors (Schenk and Matteoli, 2004). For example, AMPA
Acknowledgments
We are grateful to Ruth Zelka for her assistance in neurite measurement, and Dr. David S. Weiss for providing the SH5p-ρ1 neuroblastoma cell line. This work was supported by grants from the National Eye Institute (EY-12028, EY-06516, EY 014557, and core grant EY-01792), and a Senior Scientific Investigator Award (H.R.) from Research to Prevent Blindness, Inc.
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