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. 2019 Oct 1;9(1):14073.
doi: 10.1038/s41598-019-50671-6.

Scribble co-operatively binds multiple α1D-adrenergic receptor C-terminal PDZ ligands

Eric M Janezic  1 , Dorathy-Ann Harris  1 , Diana Dinh  1 , Kyung-Soon Lee  1 , Aaron Stewart  1 , Thomas R Hinds  1 , Peter L Hsu  1   2 , Ning Zheng  1   2 , Chris Hague  3
Affiliations

Scribble co-operatively binds multiple α1D-adrenergic receptor C-terminal PDZ ligands

Eric M Janezic et al. Sci Rep. .

Abstract

Many G protein-coupled receptors (GPCRs) are organized as dynamic macromolecular complexes in human cells. Unraveling the structural determinants of unique GPCR complexes may identify unique protein:protein interfaces to be exploited for drug development. We previously reported α1D-adrenergic receptors (α1D-ARs) - key regulators of cardiovascular and central nervous system function - form homodimeric, modular PDZ protein complexes with cell-type specificity. Towards mapping α1D-AR complex architecture, biolayer interferometry (BLI) revealed the α1D-AR C-terminal PDZ ligand selectively binds the PDZ protein scribble (SCRIB) with >8x higher affinity than known interactors syntrophin, CASK and DLG1. Complementary in situ and in vitro assays revealed SCRIB PDZ domains 1 and 4 to be high affinity α1D-AR PDZ ligand interaction sites. SNAP-GST pull-down assays demonstrate SCRIB binds multiple α1D-AR PDZ ligands via a co-operative mechanism. Structure-function analyses pinpoint R1110PDZ4 as a unique, critical residue dictating SCRIB:α1D-AR binding specificity. The crystal structure of SCRIB PDZ4 R1110G predicts spatial shifts in the SCRIB PDZ4 carboxylate binding loop dictate α1D-AR binding specificity. Thus, the findings herein identify SCRIB PDZ domains 1 and 4 as high affinity α1D-AR interaction sites, and potential drug targets to treat diseases associated with aberrant α1D-AR signaling.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
In situ affinity determination of α1D-adrenergic receptor C-terminal PDZ ligand:PDZ protein interactions. (A) Real-time biolayer interferometry (BLI) association/dissociation curve measuring binding of α1D C-terminus (α1D-CT) to purified scribble (SCRIB). Biotin-labeled α1D-CT was immobilized to streptavidin probes. Indicated concentrations of SCRIB were used as analytes. (Bio. = Biocytin, Diss. = Dissociation). (B–F) Quantified BLI binding data for biotin labeled α1D-CT binding to (B) SCRIB, (C) α1-syntrophin (SNTA), (D) human discs large MAGUK scaffold protein 1 (DLG1), (E) calcium/calmodulin dependent serine protein kinase (CASK), and (F) membrane palmitoylated protein 7 (MPP7). (G) Comparative analysis of BLI concentration-response curves for α1D-CT:PDZ protein association binding. (H) Reverse BLI assay of purified α1D-CT (analyte) bound to immobilized biotin-labeled SCRIB (probe). Data are presented as mean ± SEM, n = 3.
Figure 2
Figure 2
In situ and in vitro analysis of α1D-adrenergic receptor C-terminal PDZ ligand:SCRIB single PDZ domain interactions. (A–D) Biolayer interferometry (BLI) analyses of immobilized biotin-labeled α1D-CT binding to (A) SCRIB PDZ domain 1 (PDZ1), (B) SCRIB PDZ domain 2 (PDZ2), (C) SCRIB PDZ domain 3 (PDZ3) and (D) SCRIB PDZ domain 4. BLI data are presented as mean ± SEM, n = 3. (E) Top panel, PAGE NIR of BG-782 labeled SNAP-α1D-AR co-immunoprecipitated with TAP-SCRIB containing all 4 PDZ domains (WT), PDZ domain 1 (PDZ1), 2 (PDZ2), 3 (PDZ3) or 4 (PDZ4), or no PDZ domains (ΔPDZ) from HEK293 cell lysates. Bottom panel, Anti-HA western blot of upper gel for listed TAP-SCRIB constructs. ◄ indicates SNAP-α1D-AR monomer band.
Figure 3
Figure 3
In situ and in vitro analysis of α1D-adrenergic receptor C-terminal PDZ ligand:SCRIB truncation mutant interactions. (A–C). Biolayer interferometry (BLI) analyses of α1D-CT binding to SCRIB ΔPDZ4 (A), SCRIB ΔPDZ34 (B) and SCRIB PDZ34 (C). BLI data are presented as mean ± SEM, n = 3. (D) Co-immunoprecipitation of myc-α1D-AR with transfection vehicle (− vector), empty pGlue vector (+vector), TAP-SCRIB containing all 4 PDZ domains (WT), or sequentially truncated at the C-terminus (CT), PDZ domain 4 (ΔPDZ4), PDZ domain 3 (ΔPDZ34), PDZ domain 2 (PDZ1) or PDZ domain 1 (ΔPDZ) from HEK293 cell lysates. Shown are western blots of TAP-SCRIB constructs (top panel), myc-α1D-AR multimers (middle panel) and monomers (bottom panel). For full blots reference Supplemental Fig. 2.
Figure 4
Figure 4
SNAP-α1D-adrenergic receptor C-terminal PDZ ligand:GST-SCRIB pulldown assays indicate a co-operative binding model. (A) PAGE NIR of HEK293 cell lysates transfected with vehicle alone (− vector), pSNAP vector (SNAP), N-terminal SNAP-tagged α1D-AR C-terminus (α1D-CT), α1A, α1B and α1D-AR. (B) Coomassie stain of GST-SNAP-α1D-CT purification ± IPTG induction, unbound (FT), bound to beads (B), following TEV cleavage (+TEV) and anion exchange chromatography (AEX). (C) PAGE NIR of purified SNAP-α1D-CT pre-labeled with BG-782. (D) SNAP-α1D-CT standard curve plotting concentration of BG-782 labeled SNAP-α1D-CT versus fluorescence quantified at λ = 800 nm. (E–G) Representative PAGE NIR gels of SNAP-α1D-CT pulldowns with GST-SCRIB (E), GST-PDZ4 (F), or GST-PDZ1 (G). (H) Concentration-response curves quantifying SNAP-α1D-CT bound to GST-SCRIB, SCRIB truncated before PDZ domain (ΔPDZ4), before PDZ domain 3 (ΔPDZ34), SCRIB PDZ1, or SCRIB PDZ4 (mean ± SEM, n = 3–4). ***p < 0.001, One-way ANOVA with Tukey’s post-hoc test.
Figure 5
Figure 5
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).
Figure 6
Figure 6
Biolayer interferometry analysis of α1D-adrenergic receptor C-terminal PDZ ligand:SCRIB H793A/H1170A interactions. Biolayer interferometry (BLI) was used to quantify α1D-CT binding to full length SCRIB containing point mutations H1170A (A), H793A (B), or both H793A and H1170A (C). ▼ indicate the SCRIB PDZ domain harboring the denoted H → A mutation. Data are presented as mean ± SEM, n = 3. (D) Hypothetical model of the α1D-AR:SCRIB:DAPC macromolecular complex in human cells.
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References

    1. Overington JP, Al-Lazikani B, Hopkins AL. How many drug targets are there? Nat. Rev. Drug Discov. 2006;5:993–996. doi: 10.1038/nrd2199. - DOI - PubMed
    1. Shukla AK, Xiao K, Lefkowitz RJ. Emerging paradigms of β-arrestin-dependent seven transmembrane receptor signaling. Trends Biochem. Sci. 2011;36:457–469. doi: 10.1016/j.tibs.2011.06.003. - DOI - PMC - PubMed
    1. Romero G, von Zastrow M, Friedman PA. Role of PDZ proteins in regulating trafficking, signaling, and function of GPCRs: means, motif, and opportunity. Adv. Pharmacol. 2011;62:279–314. doi: 10.1016/B978-0-12-385952-5.00003-8. - DOI - PMC - PubMed
    1. Ritter SL, Hall RA. Fine-tuning of GPCR activity by receptor-interacting proteins. Nat. Rev. Mol. Cell Biol. 2009;10:819–830. doi: 10.1038/nrm2803. - DOI - PMC - PubMed
    1. Tsunoda S, et al. A multivalent PDZ-domain protein assembles signaling complexes in a G-protein-coupled cascade. Nature. 1997;388:243–249. doi: 10.1038/40805. - DOI - PubMed

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