Elsevier

Neuroscience

Volume 136, Issue 2, 2005, Pages 467-475
Neuroscience

Molecular neuroscience
The GABA ρ1 subunit interacts with a cellular retinoic acid binding protein in mammalian retina

https://doi.org/10.1016/j.neuroscience.2005.08.018Get rights and content

Abstract

Interactions between the intracellular domain of ligand-gated membrane receptors and cytoplasmic proteins play important roles in their assembly, clustering, and function. In addition, protein–protein interactions may provide an alternative mechanism by which neurotransmitters activate intracellular pathways. In this study, we report a novel interaction between the GABA ρ1 subunit and cellular retinoic acid binding protein in mammalian retina that could serve as a link between the GABA signaling pathway and the control of gene expression in neurons. The interaction between the intracellular loop of the human GABA ρ subunit and cellular retinoic acid binding protein was identified using a CytoTrap XR yeast two-hybrid system, and was further confirmed by co-precipitation of the human GABA ρ subunit and cellular retinoic acid binding protein from baboon retinal samples. The cellular retinoic acid binding protein binding domain on the human ρ1 subunit was located to the C-terminal region of human GABA ρ subunit, and the interaction of the human GABA ρ subunit with cellular retinoic acid binding protein could be antagonized by a peptide derived from within the binding domain of the ρ1 subunit. Since cellular retinoic acid binding protein is a carrier protein for retinoic acid, we investigated the effect of GABA on retinoic acid activity in neuroblastoma cells containing endogenously expressed cellular retinoic acid binding protein. In the absence of the ρ1 receptor, these cells showed enhanced neurite outgrowth when exposed to retinoic acid and GABA had no effect on their response to retinoic acid. In contrast, cells stably transfected with the human ρ1 subunit showed a significantly reduced sensitivity to retinoic acid when exposed to GABA. These results suggest that the GABA receptor subunit effectively altered gene expression through its interaction with the cellular retinoic acid binding protein pathway.

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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