Ubiquitin ligase RNF167 regulates AMPA receptor-mediated synaptic transmission

Abstract

AMPA receptors (AMPARs) mediate the majority of fast excitatory neurotransmission, and their density at postsynaptic sites determines synaptic strength. Ubiquitination is a posttranslational modification that dynamically regulates the synaptic expression of many proteins. However, very few of the ubiquitinating enzymes implicated in the process have been identified. In a screen to identify transmembrane RING domain-containing E3 ubiquitin ligases that regulate surface expression of AMPARs, we identified RNF167. Predominantly lysosomal, a subpopulation of RNF167 is located on the surface of cultured neurons. Using a RING mutant RNF167 or a specific shRNA to eliminate endogenous RNF167, we demonstrate that AMPAR surface expression increases in hippocampal neurons with disrupted RNF167 activity and that RNF167 is involved in activity-dependent ubiquitination of AMPARs. In addition, RNF167 regulates synaptic AMPAR currents, whereas synaptic NMDAR currents are unaffected. Therefore, our study identifies RNF167 as a selective regulator of AMPAR-mediated neurotransmission and expands our understanding of how ubiquitination dynamically regulates excitatory synapses.

Fig. 1.

RNF167 is an E3 ligase that ubiquitinates GluA2 and regulates its surface expression. (A) Schematic representation of the subset of RING-domain E3 ubiquitin ligases that localize to endosomes. Signal peptide, transmembrane domains, and RING domains are indicated. (B) Surface expression of GluA2 was evaluated by using FACS analysis of HEK293T cells coexpressing Flag-GluA2 and WT or RING domain mutant E3 ligases. Data are presented as the mean of GluA2 surface expression relative to WT (% ± SEM, n = 3 independent experiments). (C) WT, but not mutant, RNF167 ubiquitinates GluA2. HEK293T cells expressing Flag-GluA2 and RNF167 were lysed, and Flag-GluA2 was immunoprecipitated and immunoblotted for the presence of ubiquitin. The asterisk (*) represents a nonspecific band found in total cell lysates. (D) Immunoprecipitated HA-tagged WT RNF167 expressed in HEK293T cells was deglycosylated by using endoglycosydase H (endo H) or peptide-N-glycosydase F (PNGaseF) and immunoblotted by using RNF167 antibody. (E) Mobility profiles of RNF167 glycosylation mutants. Thirty micrograms of total proteins from HEK293T cells expressing RNF167 WT or mutants were analyzed by immunoblotting for RNF167. See also Fig. S1 A–C.

Fig. 2.

RNF167 is broadly expressed in vivo and localizes to lysosomes. (A) PCR analysis of human RNF167 mRNA tissue expression. Shown are the expression profile of RNF167 (Upper) and β-actin (Lower). (B) Crude synaptosomes from adult female rat brain tissues were immunoblotted with specific antibodies as indicated. A typical result is shown. (C–G) Subcellular distribution of RNF167 was analyzed by using confocal microscopy. HeLa cells expressing HA-tagged RNF167 were stained for RNF167 (red) and the early endosome marker GFP-Rab5 (C, green), late endosome markers GFP-Rab7 (D, green), or GFP-Rab9 (E, green). (Scale bar: 10 µm.) Boxed areas are shown at higher magnification and colocalization in yellow. (F) HeLa cells expressing RNF167-YFP (green) counterstained for endogenous Lamp-1 (red). Colocalization is shown (yellow) in the merge image. Boxed areas are shown at higher magnification. (G) (Left) A representative image of a hippocampal neuron (DIV15) expressing HA-tagged RNF167 (green) and stained for endogenous Lamp-2 (red) and the neuronal marker MAP2 (blue). (Scale bar: 10 µm.) The boxed area is shown at higher magnification (Right). Colocalization between HA-RNF167 and the lysosomal marker Lamp-2 is shown (yellow) in the merge image. (Scale bar: 5 µm.)

Fig. 3.

RNF167 regulates synaptic AMPARs. (A and B) Hippocampal neurons were transduced with WT or mutant RNF167 lentiviruses and the surface expression of AMPARs was evaluated by using confocal microscopy. Surface-expressed endogenous GluA2 was labeled under nonpermeabilized conditions. (A) Representative images are shown. (Scale bar: 5 µm.) (B) Quantitation of GluA2 surface expression in A. Bar graph shows that mutant but not WT RNF167 increases surface expression of GluA2 and is presented as mean ± SEM. Each group contains 30 cells from three independent experiments (*P < 0.05 and ***P < 0.005 using ANOVA one-way analysis and Tukey’s multiple comparison test). See also Fig. S2. (C–F) Scatter plots show amplitude of EPSCs for single pair (○) and mean (●). Representative superimposed traces are shown [black, control (CTL); green, WT or mutant RNF167]. (Scale bars: C and D, 20 pA and 10 ms; E and F, 20 pA and 50 ms.) Bar graphs show that expression of mutant RNF167 affects AMPAR currents (C; 77% increase, n = 10, *P = 0.037) but not NMDAR currents (E; 14.5% increase, n = 10, P = 0.80), and that WT RNF167 expression neither affects AMPAR (D; 5.4% increase, n = 10, P = 1.0) nor NMDAR currents (F; 1.6% increase, n = 10, P = 0.91). Paired-pulse ratios (PPR) show no differences when WT (G; P = 0.22) or mutant RNF167 (H; P = 0.65) is expressed. Errors bars represent mean ± SEM.

Fig. 4.

Reduced RNF167 expression in cultured neurons decreases the activity-dependent ubiquitination of GluA2 and increases surface expression of AMPARs subunits. (A) DIV17 cortical neurons expressing shCTL or shRNF167 were treated with bicuculline (Bic; 40 µM, 7 min) or its control (CTL, DMSO). GluA2 was immunoprecipitated from cell lysate and immunoblotted for ubiquitin. Total expression of GluA2 and RNF167 were monitored by using specific antibodies as indicated. The asterisk (*) represents a nonspecific band found in total cell lysate. See also Fig. S3. (B) At DIV17, cortical neurons expressing shCTL or shRNF167 were incubated with NHS-SS-biotin to label surface-expressed proteins. Biotinated proteins were precipitated from cell lysate and immunoblotted by using specific antibodies as indicated. Lysate represents 25% of input used for streptavidin precipitation. (C) Hippocampal neurons were transfected with shCTL or shRNF167 and stained live for endogenous surface GluA1 and GluA2 under nonpermeabilized conditions before being evaluated using confocal microscopy. Representative images show AMPAR surface expression and EGFP from transfected cells. (Scale bar: 5 µm.) (D) Quantitation of AMPAR surface expression in C. Bar graph shows that shRNF167 but not shCTL increases surface expression of GluA1 and GluA2 and is presented as mean ± SEM. Each group contains 44–45 cells from three independent experiments (*P < 0.0001 using Student’s unpaired t test). (E) Hippocampal neurons were transduced with shCTL or shRNF167 lentiviruses, transfected with Flag-AMPAR subunits, stained under nonpermeabilized (surface) and permeabilized (intracellular) conditions, and the expression of Flag-AMPARs was evaluated by using confocal microscopy. Representative images show surface and intracellular expression of AMPARs. (Scale bar: 5 µm.) Quantitation of AMPAR surface (F) and intracellular (G) expression. Bar graphs show that shRNF167 but not shCTL increases surface expression of Flag-AMPARs and are presented as mean ± SEM (each group contains 30–40 cells from three to four independent experiments, *P < 0.0001 using Student’s unpaired t test).

Fig. 5.

RNF167 knockdown affects AMPAR but not NMDAR currents in hippocampal neurons. Scatter plots show amplitude of EPSCs for single pairs (○) and the mean (●). Representative superimposed traces are shown [black, control (CTL); green, shRNF167 or shRNF167 + RNF167*]. (Scale bars: A and B, 20 pA and 20 ms; C and D, 20 pA and 50 ms.) Bar graphs show that expression of shRNF167 affects AMPAR currents (A; 105% increase, n = 8, *P = 0.04) but not NMDAR currents (C; 22.5% increase, n = 9, P = 0.91). The expression of shRNF167 and RNF167 resistant (RNF167*) rescued AMPAR currents to level similar to control (B; 29.1% increase, n = 9, P = 0.43) without affecting NMDAR currents (D; 12.2% increase, n = 9, P = 0.69). Errors bars represent mean ± SEM. See also Fig. S1D for rescue construct expression.