Figure 1.
Representative FP competition curves.
Binding of 1–512 µM GPCR C-tail peptides to a fixed concentration of PSD-95 PDZ1 (left panel), PDZ2 (middle panel) or PDZ3 (right panel) and Cy5-labeled probe. The data points are averages of three independent measurements, and the error bars represent the standard error of the mean. The solid lines are the fitted curves.
Table 1.
Ki values for GPCR C-tail interactions with the PSD-95 PDZ domains determined by FP.
Figure 2.
Time-resolved binding of GPCR C-tails to the PSD-95 PDZ domains.
Binding of GPCR C-tail peptides to immobilized PSD-95 PDZ1 (left panel), PDZ2 (middle panel), or PDZ3 (right panel) monitored by SPR. (A) Time-resolved binding of 500 µM GPCR C-tail peptides. (B) Steady-state responses and the corresponding fitted curves (solid lines). The curves are reference and blank subtracted.
Figure 3.
Domain preference and peptide selectivity.
Comparison of Ki values for binding of GPCR and reference (KIF1Bα, GluN2B, and CRIPT) C-tail peptides to PSD-95 PDZ2 and PDZ1 (A), PDZ2 and PDZ3 (B), PDZ1 and PDZ3 (C), and isolated PDZ2 and PDZ2 in the PDZ1-2 supramodule (D). The dashed lines represent identical Ki values for the two domains. The dotted lines in A and D are line fits to the data points; C-tails binding to only one of the domains were omitted from the fitting. NA, no affinity, defined as a Ki value above 1000 µM. Error bars represent fitting errors or the standard error of the mean (see Table 1).
Figure 4.
Validation of colocalization assay.
PSD-95 is visualized by fusion to GFP, the receptors by fusion to SNAP-tag and labeling with the fluorescent SNAP-tag substrate BG-647. Cells transfected only with PSD-95-GFP were stained with DiD to visualize the plasma membrane. (A) Cells transfected with PSD-95-GFP only. (B–F) Cells coexpressing PSD-95-GFP and SNAP-5-HTR2C (B), SNAP-β1AR (C), SNAP-κOR (D), or SNAP-β1AR-AAA (E). The graphs (right) show averaged line scans along the regions of interest indicated on the overlay images. The signal from SNAP-GPCRs and the membrane dye is shown in red; PSD-95-GFP is shown in green. Scale bars are 10 µm.
Figure 5.
GPCR–PSD-95 interactions identified by FP colocalize in cells.
PSD-95 is visualized by fusion to GFP, the receptors by fusion to SNAP-tag and labeling with BG-647. Cells coexpressing PSD-95-GFP and SNAP-hSSTR1 (A), SNAP-hSSTR1-AAA (B), SNAP-Y2 (C), SNAP-CXCR2 (D), or SNAP-β2AR (E). The graphs (right) show averaged line scans along the regions of interest indicated on the overlay images. The signal from SNAP-GPCRs is shown in red; PSD-95-GFP is shown in green. Scale bars are 10 µm.
Figure 6.
mSSTR1 and PSD-95 colocalize in primary hippocampal neurons.
Mouse neurons cultivated for 20 days in vitro were stained for mSSTR1 (red signal) and PSD-95 (green signal). Yellow color in the merged picture (right panels) indicates colocalization of mSSTR1 and PSD-95 and is particularly seen on dendritic spines. The lower panels show a magnification of the dashed region depicted in the overviews. Nuclei of primary neurons were counterstained with DAPI (left panels). Several neurons were not stained by the anti-SSTR1 antibody (indicated by arrowheads), indicating that the mSSTR1 staining is specific. Scale bars are 10 µm.