Proteasomal Degradation of γ-Aminobutyric AcidB Receptors Is Mediated by the Interaction of the GABAB2 C Terminus with the Proteasomal ATPase Rtp6 and Regulated by Neuronal Activity

Khaled Zemoura,Dietmar Benke

doi:10.1074/jbc.m113.541987

Abstract

Regulation of cell surface expression of neurotransmitter receptors is crucial for determining synaptic strength and plasticity, but the underlying mechanisms are not well understood. We previously showed that proteasomal degradation of GABAB receptors via the endoplasmic reticulum (ER)-associated protein degradation (ERAD) machinery determines the number of cell surface GABAB receptors and thereby GABAB receptor-mediated neuronal inhibition. Here, we show that proteasomal degradation of GABAB receptors requires the interaction of the GABAB2 C terminus with the proteasomal AAA-ATPase Rpt6. A mutant of Rpt6 lacking ATPase activity prevented degradation of GABAB receptors but not the removal of Lys(48)-linked ubiquitin from GABAB2. Blocking ERAD activity diminished the interaction of Rtp6 with GABAB receptors resulting in increased total as well as cell surface expression of GABAB receptors. Modulating neuronal activity affected proteasomal activity and correspondingly the interaction level of Rpt6 with GABAB2. This resulted in altered cell surface expression of the receptors. Thus, neuronal activity-dependent proteasomal degradation of GABAB receptors by the ERAD machinery is a potent mechanism regulating the number of GABAB receptors available for signaling and is expected to contribute to homeostatic neuronal plasticity.

Highlights

Introduction of Peptides into HEK293 Cells—Small synthetic peptides were introduced into HEK 293 cells as described in Ref. 20
GABAB2 Interacts with the Proteasomal AAA-ATPase Rpt6— We recently showed that the expression of total and cell surface GABAB receptors is regulated by proteasomes via the ER-associated protein degradation (ERAD) [12]
The co-localization of GABAB2 clusters with Rpt6 clusters was considerably increased (193 Ϯ 12% of control, Fig. 1C). These results suggest that interaction of Rpt6 with GABAB2 mediates proteasomal degradation of GABAB receptors

Summary

Introduction

Introduction of Peptides into HEK293 Cells—Small synthetic peptides were introduced into HEK 293 cells as described in Ref. 20. A peptide containing the same amino acids but in a random sequence was used as a control (RRRRRRR-RLGPHVRMFVSSVP, GenScript). Twenty-four hours after transfection with GABAB receptor and Rpt plasmids, the HEK 293 cells were washed with PBS and incubated for 5 min with 50 ␮M pyrenebutyric acid in PBS. Results: Proteasomal degradation of GABAB receptors is mediated by interaction with Rpt and modulated by neuronal activity. Modulating neuronal activity affected proteasomal activity and correspondingly the interaction level of Rpt with GABAB2. This resulted in altered cell surface expression of the receptors. Neuronal activity-dependent proteasomal degradation of GABAB receptors by the ERAD machinery is a potent mechanism regulating the number of GABAB receptors available for signaling and is expected to contribute to homeostatic neuronal plasticity

Methods

Results

Conclusion