Figure 1: Shrm4 interaction with GABABRs modulates cell surface expression.
(a) Representations of Shrm4 and GABABR subunit 1a and 1b interacting domains. The PDZ domain of Shrm4 (1–91 aa) was used as bait for Y2H screening on adult human brain cDNA library. Twenty positive clones were isolated; six encoded for a 100 aa stretch of the GABABR subunit 1 C-terminal tail (both isoforms (1a and 1b)). The PDZ domain and ΔASD2 constructs interact with the C-terminal tail of GABAB1 (+++) while ΔPDZ truncated construct does not (−). TMD: Transmembrane domains. (b) Co-immunoprecipitation experiments on adult rat brain extracts using anti-Shrm4 antibody show Shrm4 and GABAB1 association (full blot in Supplementary Fig. 11). (c) (Left) dSTORM imaging of GABABR (red) and Shrm4 (green) on 14DIV rat cultured hippocampal neurons. (Right) Shrm4 and GABABR puncta co-cluster as evidenced by cross-correlation analysis. Error-bars are s.e.m.; number of regions=29, number of fields=3. Scale bar, 0.4 μm. (d) GST pull-down experiments on 18DIV rat cultured hippocampal neurons (1) and rat brain (2) extract show that Shrm4-PDZ domain (GST-PDZ; aas 1–91) pulls down both GABAB1 isoforms whereas (3) mutant GST-PDZ (AA) does not (full blot in Supplementary Fig. 11). (e) Co-immunoprecipitation experiments on HEK293 cells expressing HA-Shrm4 and GABAB2-Flag, showing that Shrm4 does not associate with GABAB2 in absence of GABAB1 (full blot in Supplementary Fig. 11). (f) Scheme of truncated GABABR constructs: deletion 1: Δ859–961; deletion 2: Δ922–961; and deletion 3: Δ870–961 with co-immunoprecipitation results from HEK293 cells expressing Shrm4-GFP and each of the GABAB1a constructs. GFP-Shrm4 immunoprecipitates full-length GABAB1a, deletion 2, and deletion 3, but not deletion 1 (full blot in Supplementary Fig. 11). (g) (Top) GST pull-down experiments using GST-PDZ or mutant GST-PDZ (AA) on lysates of HEK293 cells overexpressing GABAB1 and GABAB2 incubated with Tat-control peptide or Tat-859–870 peptide corresponding to the previously identified minimal GABAB1-Shrm4 binding region. (Bottom) Histograms showing GABAB1a mean intensity normalized to Tat-control peptide±s.e.m. n=3; **P=0.046; t-test (full blot in Supplementary Fig. 11). (h) Surface immunostaining for GABABR in rat cultured hippocampal neurons at 18DIV transfected with scrambled, shRNA#1, shRNA#2 or rescue constructs at 8DIV. Scale bar, 15 μm. Histograms show mean±s.e.m. n=5–15, Scrambled versus shRNA#1 *P=0.0153; Scrambled versus shRNA#2 *P=0.0153; One-way ANOVA-Mann–Whitney. (i) GABABR cell surface biotinylation from 18DIV rat cultured hippocampal neurons infected with scrambled#1 (Scr#1) or shRNA#1 (shRNA#1) at 8DIV and western blot. Shrm4 silencing reduces GABAB1 surface expression, but does not change total signal for GABAB1 or surface (and total) signal for GABAAR α1 subunit. Histograms show mean±s.e.m.; n=4; **P=0.0069; t-test (full blot in Supplementary Fig. 11).
