Fig 2: GK domain interactions are differentially regulated.For this figure, western blots shown on the left side are representative examples of three independent experiments; the corresponding quantification of coIP band intensities from these three experiments are shown in the dot plots on the right side indicating mean values ± SEM. (A) MYC-PSG, MYC-PSG R568A and MYC-GK were coexpressed with FLAG-GKAP. Following MYC IP, precipitated proteins were analysed by western blot. GKAP coprecipitated with PSG and GK domain constructs. The GK domain mutant PSG R568A was not able to bind GKAP. (B) Following coexpression of MYC-PSD-95 or MYC-PSD-95 R568A with FLAG-Gnb5, together with either CRIPT-derived PDZ3 or mutPDZ3 ligand, proteins were precipitated with aMYC-antibody and analysed by western blot. Gnb5 coIP with either PSD-95 or PSD-95 R568A was efficiently promoted by the presence of PDZ3-binding ligand, irrespective of the GK domain mutation R568A. (C) CoIP of MYC-PSD-95 and FLAG-GKAP together with either CRIPT-derived PDZ3 or mutPDZ3 ligand constructs and analysis of precipitated proteins by western blot with antibodies to the corresponding tags. The presence of CRIPT-derived PDZ3 ligands in the lysate had almost negligible effect on PSD-95 GKAP interaction.10.7554/eLife.41299.014Figure 5—source data 1.Source data for Figure 5.

Fig 3: The heterotrimeric G protein subunit Gnb5 is a novel PSD-95 interactor.(A) GST pull-down from crude synaptosomal proteins (comparable amounts of GST tagged proteins observable by Coomassie, upper panel) enabled comparison of Gnb5 binding to the GK domain alone versus the SH3-GK domain. Gnb5 is effectively enriched in the GST-GK pull-down compared to bead controls or GST-SH3-GK pull-downs, as observed by western blot with a commercially available aGnb5 antibody (lower panel). The GST pull-down shown on the left side is a representative example of three independent experiments; the corresponding quantification of copurified Gnb5 band intensities from these three experiments is shown in the dot plot on the right side indicating mean values ± SEM. (B) CoIP experiment of tagged Gnb5 (Gnb5-Clover) with tagged SH3-GK or GK (FLAG-SH3-GK or FLAG-GK). Immunoprecipitation of Gnb5-Clover with aGFP antibody efficiently copurified the GK-domain construct (observed via western blot with aFLAG antibodies, lower panel). The western blot shown (left side) is a representative example of three independent experiments; the corresponding quantification of coIP band intensities from these three experiments is shown in the dot plot on the right side indicating mean values ± SEM. (C) Cultures of rat hippocampal neurons (E18) were fixed at DIV21 and stained for Gnb5 together with the dendritic marker MAP2 (microtubule-associated protein 2) and either the postsynaptic protein PSD-95 (left panel) or the presynaptic marker Synapsin (right panel) and respective fluorescent secondary antibodies, and visualised by confocal microscopy. Scale bars: 5 µm.10.7554/eLife.41299.010Figure 3—source data 1.Source data for Figure 3A,B.

Fig 4: Gnb5 - PSD-95 complex formation is regulated by CRIPT-derived PDZ3 ligand binding.For this figure, western blots shown on the left side are representative examples of three independent experiments; the corresponding quantification of coIP band intensities from these three experiments are shown in the dot plots on the right side indicating mean values ± SEM. (A) MYC-PSD-95 and FLAG-Gnb5 or FLAG-shortGnb5 were coexpressed with either CRIPT-derived PDZ3 or mutPDZ3 ligand constructs. MYC-PSD-95 was precipitated and proteins were analysed by western blot with αFLAG antibodies. Coexpression of the CRIPT-derived PDZ3 ligand facilitated the coIP of PSD-95 and Gnb5, coIP with the shortGnb5 construct (N-terminal truncation) was much less efficient. In the presence of the CRIPT-derived mutPDZ3 ligand, coprecipitated proteins were not detectable. (B) CoIP of MYC-PSG and FLAG-Gnb5 together with either CRIPT-derived PDZ3 or mutPDZ3 ligand constructs. The presence of CRIPT-derived PDZ3 ligand constructs facilitated coprecipitation of PSG and Gnb5 (see comparative western blot with αFLAG antibodies, lower panel). (C) Coexpression of MYC-PSG or MYC-PSG L460P with FLAG-Gnb5 and subsequent MYC IP. PSG L460P IP efficiently copurifies Gnb5 (observed by western blot with αFLAG antibodies).10.7554/eLife.41299.012Figure 4—source data 1.Source data for Figure 4.

Fig 5: Graphical Summary.(A) PSD-95 C-terminal domains (PSG module) functionally cooperate and regulate homotypic and heterotypic complex formation. We propose that CRIPT-derived PDZ3 ligand binding to the PDZ3 domain induces a loosening of the intramolecular SH3-GK interaction. This ‘open’ conformation is then able to initiate subsequent oligomerisation and protein binding. (B) Model of CRIPT-derived PDZ3 ligand-dependent and ligand-independent binding to the PSD-95 C-terminal SH3-GK domain tandem. Ligand - PDZ3 domain binding facilitates association with Gnb5.