Figure 5
From: Scribble co-operatively binds multiple α1D-adrenergic receptor C-terminal PDZ ligands
Structure-function analysis of the α1D-CT:SCRIB PDZ4 interaction. (A) Dynamic mass redistribution assays quantifying phenylephrine efficacy in HEK293 cells stably expressing SNAP-α1D-AR alone, or transfected with SCRIB WT, PDZ4, or SCRIB containing only PDZ domains 1, 2 and 3 (ΔPDZ4). Data are the mean of 12 replicates ± SEM. (B) Cell surface expression of SNAP-α1D-AR in HEK293 cells transfected with vector control (pGlue), ΔPDZ4, PDZ4, or SCRIB WT (top panel, green); nuclear stain TO-PRO-3 was used to normalize for cell number (bottom panel, red). (C) Quantification of data from B (mean ± SEM, n = 3, 6 replicates; ***p < 0.001 from pGLUE, One-way ANOVA with Tukey’s post-hoc tests). (D) Molecular docking model of α1D-CT:SCRIB PDZ4 interaction (purple = PDZ4, green = α1D-CT, PDB ID = 4WYT used for model). (E) Sequence alignment of SCRIB PDZ domains (boxes indicate residues identified in D). (F) Biolayer interferometry (BLI) analysis of SCRIB mutations H1170A and R1110G on α1D-CT binding (mean ± SEM, n = 3). (G) X-ray crystallography structure of SCRIB PDZ4 R1110G (mutation highlighted in blue; PDB ID = 6EEY). (H) R1110G (orange) causes a 4.5 Å shift in carboxylate binding loop, as determined by superposition with WT PDZ4 (purple).
