Phospholipase C-related inactive protein is implicated in the constitutive internalization of GABA_A receptors mediated by clathrin and AP2 adaptor complex

A mechanism for regulating the strength of synaptic inhibition is enabled by altering the number of GABA_A receptors available at the cell surface. Clathrin and adaptor protein 2 (AP2) complex-mediated endocytosis is known to play a fundamental role in regulating cell surface GABA_A receptor numbers. Very recently, we have elucidated that phospholipase C-related catalytically inactive protein (PRIP) molecules are involved in the phosphorylation-dependent regulation of the internalization of GABA_A receptors through association with receptor β subunits and protein phosphatases. In this study, we examined the implications of PRIP molecules in clathrin-mediated constitutive GABA_A receptor endocytosis, independent of phospho-regulation. We performed a constitutive receptor internalization assay using human embryonic kidney 293 (HEK293) cells transiently expressed with GABA_A receptor α/β/γ subunits and PRIP. PRIP was internalized together with GABA_A receptors, and the process was inhibited by PRIP-binding peptide which blocks PRIP binding to β subunits. The clathrin heavy chain, μ2 and β2 subunits of AP2 and PRIP-1, were complexed with GABA_A receptor in brain extract as analyzed by co-immunoprecipitation assay using anti-PRIP-1 and anti-β2/3 GABA_A receptor antibody or by pull-down assay using β subunits of GABA_A receptor. These results indicate that PRIP is primarily implicated in the constitutive internalization of GABA_A receptor that requires clathrin and AP2 protein complex.