Molecular and Functional Interaction between Protocadherin-γC5 and GABA_AReceptors

Abstract

We have found that the γ2 subunit of the GABAA receptor (γ2-GABAAR) specifically interacts with protocadherin γ-C5 (Pcdh-γC5) in the rat brain. The interaction occurs between the large intracellular loop of the γ2-GABAAR and the cytoplasmic domain of Pcdh-γC5. In brain extracts, Pcdh-γC5 co-immunoprecipitates with GABAARs. In co-transfected HEK293 cells, Pcdh-γC5 promotes the transfer of γ2-GABAAR to the cell surface. We have previously shown that in cultured hippocampal neurons, endogenous Pcdh-γC5 forms clusters, some of which associate with GABAergic synapses. Overexpression of Pcdh-γC5 in hippocampal neurons increases the density ofγ2-GABAAR clusters but has no significant effect on the number of GABAergic contacts that these neurons receive, indicating that Pcdh-γC5 is not synaptogenic. Deletion of the cytoplasmic domain of Pcdh-γC5 enhanced its surface expression but decreased the association with both γ2-GABAAR clusters and presynaptic GABAergic contacts. Cultured hippocampal neurons from the Pcdh-γ triple C-type isoform knockout (TCKO) mouse (Pcdhgtcko/tcko) showed plenty of GABAergic synaptic contacts, although their density was reduced compared with sister cultures from wild type and heterozygous mice. Knocking down Pcdh-γC5 expression with shRNA decreased γ2-GABAAR cluster density and GABAergic innervation. The results indicate that although Pcdh-γC5 is not essential for GABAergic synapse formation or GABAAR clustering, i) Pcdh-γC5 regulates the surface expression of GABAARs via cis-cytoplasmic interaction with γ2-GABAAR; and ii) Pcdh-γC5 plays a role in the stabilization and maintenance of some GABAergic synapses.

Clustered protocadherins

“…Although synaptic defects were observed in Pcdhg mutants (Wang et al, 2002b;Weiner et al, 2005;Garrett and Weiner, 2009), the issue of whether Pcdhg proteins play a direct role in synaptic interactions remains to be elucidated. Suggestive evidence for this idea was provided by the observation that Pcdhgc5 specifically interacts with the γ-aminobutyric acid A (GABA A ) receptor, which is required for stabilization and maintenance of GABAergic synapses in cultured hippocampal neurons (Li et al, 2012).…”

Cadherins and catenins in dendrite and synapse morphogenesis

“…Several of these cadherin family members have been implicated in various aspects of neuronal development and function. [1][2][3] The classical cadherins are associated with a group of cytosolic proteins, collectively called the catenins. While the functional roles of the cadherincatenin cell adhesion complex have been extensively investigated in epithelial cells, it is now clear that components of the complex are well expressed in central neurons at different stages during development.…”

Ring Finger Protein 34 (RNF34) Interacts with and Promotes γ-Aminobutyric Acid Type-A Receptor Degradation via Ubiquitination of the γ2 Subunit

“…Yeast Two-hybrid (Y2H)-The detailed experimental procedure for Y2H has been described elsewhere (30,31,38). All vectors and yeast strains were from Dr. Roger Brent (University of California at San Francisco) or Origene Technologies (Rockville, MD).…”

“…shRNAs for RNF34 and Rescue mRNA-The procedure for making the shRNAs has been described elsewhere (34,36,38,40). Two shRNA constructs targeting RNF34 were generated (Fig.…”

“…Co-immunoprecipitation of RNF34 and GABA A Rs from Brain Membrane Extracts-The experimental procedure has been described elsewhere (38). Briefly, the rat forebrain membrane fraction was incubated with RIPA buffer (10 mM TrisHCl, 137 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% SDS, 1% Triton-X 100, 1% sodium deoxycholate, pH 7.4) containing 1 mM PMSF and a protease inhibitor mixture (10 g/ml trypsin inhibitor, type I-S, 10 g/ml trypsin inhibitor, type II-O, 1 mM benzamidine) for 1 h followed by centrifugation at 50,000 ϫ g for 1 h. The supernatant was used for immunoprecipitations.…”