| Annotated protein: | Shroom family member 4. Gene symbol: SHROOM4. Taxonomy: Rattus norvegicus (Rat). Uniprot ID: F1LVL5 |
| antibody wiki: | |
| SynGO gene info: | SynGO data @ SHROOM4 |
| Ontology domain: | Biological Process |
| SynGO term: | regulation of postsynaptic membrane neurotransmitter receptor levels (GO:0099072) |
| Annotated paper: | Zapata J, et al. "Epilepsy and intellectual disability linked protein Shrm4 interaction with GABA(B)Rs shapes inhibitory neurotransmission" Nat Commun. 2017 Mar 6;8:14536 PMID:28262662 |
| Figure(s): | Fig. 1, Fig. 4, Fig. 5, Supplementary fig. 2, Supplementary fig. 10 |
| Annotation description: | Fig. 1a To explore the role(s) of Shrm4 in neurons, we searched for binding partners using yeast two-hybrid (Y2H) screening. The PDZ domain of Shrm4 (residues 1-91; Fig. 1a) was selected as the bait to screen against an adult human brain cDNA library, as this domain participates in protein-protein interactions. Twenty positive cDNA clones were isolated; six of these encoded a 100 amino acids stretch of the C-terminal tail of GABAB1 present in both splice variants B1a and B1b, which differ by the inclusion of two Sushi domains in the N terminus only in GABAB1a (refs 12,14) (Fig. 1a) Fig. 1h, Supplementary Fig. 2c We extended this approach to neurons, transfecting Scrambled, shRNA#1, shRNA#2 or Rescue constructs at 8DIV for later analysis of surface levels of GABAB1 at 18DIV. Interestingly, both shRNAs reduced surface levels of GABAB1 that can be rescued by the re-expression of Shrm4 (Fig. 1h, full neurons in Supplementary Fig. 2c). Fig. 1i We next used lentiviral delivery to corroborate our immunostaining results. For this we used cell surface biotinylation assays for GABABRs (Fig. 1i). Shrm4 knockdown reduced surface expression of GABABRs without affecting the total expression of GABABRs or the total and surface expression of GABAAR a1 subunits used as negative controls (Fig. 1i). Thus, Shrm4 is important for GABAB1 trafficking in neurons. Fig. 4d GABABRs activate G protein-coupled inwardly-rectifying K+ channels, generating slow inhibitory postsynaptic currents (IPSCs). As Shrm4 regulates GABABR dendritic cell surface number, we examined K+ currents induced by the GABABR agonist baclofen (10-100 mM) on 14DIV hippocampal neurons transfected at 7DIV with either GFP-coexpressing knockdown shRNA#1 or scrambled#1 or p150-cc1. Peak K+ current densities (pA/pF) were lower in Shrm4-silenced and dynein-inhibited neurons compared with scrambled controls (Fig. 4d), consistent with a reduced number of dendritic GABABRs. Fig. 4f Whole-cell Kþ currents evoked by baclofen in the CA1 of acute slices from injected animals were significantly reduced by Shrm4 knockdown compared with scrambled controls (Fig. 4f). The injection of AAV5-shRNA#2 induced similar reductions in K+ current confirming the specificity of our results. Thus, Shrm4 silencing reduces functional GABABR responses in vitro and in vivo, in accord with a reduced number of surface dendritic GABABRs. Fig. 5a, b, Supplementary Fig. 10e Shrm4 silencing in vivo affects hippocampal tonic inhibition For cells expressing Shrm4-shRNA#1, both current and noise variance were reduced compared with controls (Fig. 5b). By contrast, GABAAR-mediated mIPSCs in DGGCs were unaffected (Supplementary Fig. 10e), indicating that Shrm4 specifically regulates d subunit-containing GABAAR-mediated responses. Interestingly, co-immunoprecipitation using brain extracts and monoclonal anti-GABAAR d subunit revealed that GABABRs and d-subunit-containing GABAARs co-associate. By contrast, the synaptic GABAAR g2 subunit37 was absent (Fig. 5f). Thus, by controlling postsynaptic GABABRs, Shrm4 is also able to regulate tonic inhibition mediated by delta subunit-containing GABAARs. |
| Evidence tracking, Biological System: | Cultured neurons Intact tissue |
| Evidence tracking, Protein Targeting: | Antibody (detection) RNAi / shRNA |
| Evidence tracking, Experiment Assay: | Confocal Biochemical fractionation (generic) Y2H Whole-cell patch clamp |
| Annotator(s): | Chiara Verpelli (ORCID:0000-0003-2949-9725) Carlo Sala (ORCID:0000-0003-0662-9523) |
| Lab: | CNR Neuroscience Institute Milan and Dept. of Biotechnology and Translational Medicine, University of Milan, 20129 Milan, Italy |
| SynGO annotation ID: | 4665 |
| Dataset release (version): | 20231201 |
| View annotation as GO-CAM model: |  |