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A molecular code for endosomal recycling of phosphorylated cargos by the SNX27–retromer complex

Abstract

Recycling of internalized receptors from endosomal compartments is essential for the receptors' cell-surface homeostasis. Sorting nexin 27 (SNX27) cooperates with the retromer complex in the recycling of proteins containing type I PSD95–Dlg–ZO1 (PDZ)-binding motifs. Here we define specific acidic amino acid sequences upstream of the PDZ-binding motif required for high-affinity engagement of the human SNX27 PDZ domain. However, a subset of SNX27 ligands, such as the β2 adrenergic receptor and N-methyl-D-aspartate (NMDA) receptor, lack these sequence determinants. Instead, we identified conserved sites of phosphorylation that substitute for acidic residues and dramatically enhance SNX27 interactions. This newly identified mechanism suggests a likely regulatory switch for PDZ interaction and protein transport by the SNX27–retromer complex. Defining this SNX27 binding code allowed us to classify more than 400 potential SNX27 ligands with broad functional implications in signal transduction, neuronal plasticity and metabolite transport.

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Figure 1: High-affinity binding of PTHR to SNX27–retromer requires upstream acidic residues.
Figure 2: Electrostatic interactions established by a subset of PDZbms promote high-affinity binding to SNX27.
Figure 3: Crystal structure of the SNX27 PDZ domain in complex with the DGKζ PDZbm peptide.
Figure 4: Phosphorylation of upstream residues in the β2AR PDZbm promote specific association with SNX27.
Figure 5: Phosphorylation of the GluN1 and GluN2B subunits of NMDARs triggers interaction with SNX27.
Figure 6: Crystal structure of the SNX27 PDZ domain in complex with phosphorylated PDZbm peptides.
Figure 7: Classification of the SNX27 putative PDZ interactome, on the basis of C-terminal sequences.

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Acknowledgements

The authors acknowledge support from the staff and facilities of the University of Queensland Remote Operation Crystallization and X-ray (UQ ROCX) facility and the Australian Synchrotron. We thank B. Madio for assistance with sequence alignment and M. Mobli for assistance with NMR spectroscopy. This work was supported by funds from the Australian Research Council (ARC) (DP0985029), National Health and Medical Research Council (NHMRC) (APP1042082, APP1058734 and APP1078280), UWA-UQ Bilateral Research Collaboration Award (to N.J.P., R.D.T. and B.M.C.) and the John T. Reid Charitable Trusts (to V.A.). C.M. was supported by a University of Queensland Postdoctoral Fellowship; R.D.T. was supported by an NHMRC Senior Research Fellowship (APP1041929), and B.M.C. was supported by an NHMRC Career Development Fellowship (APP1061574) and a previously held ARC Future Fellowship (FT100100027). M.T.-L. was supported by an FPI fellowship from the Spanish Ministry of Economy and Competitiveness. I.M. received support from the Spanish Ministry of Economy and Competitiveness (BFU2013-47640-P) and the Madrid regional government (IMMUNOTHERCAM Consortium S2010/BMD-2326). We thank K. Roche (NIH) for providing GFP-GluN2B and R. Huganir (Johns Hopkins University) for providing antibodies.

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Authors and Affiliations

Authors

Contributions

All X-ray crystallography, bioinformatics and ITC studies were carried out by T.C., M.C. and B.M.C. with help from B.P. NMR experiments were performed by T.C. and C.M. Cell biology experiments were performed by M.T.-L., I.M., A.S.M.C., N.J.P., J.W., Z.Y., M.C.K. and V.A. B.M.C. and R.D.T. conceived the project, and B.M.C. coordinated the project and wrote the paper together with T.C., I.M., R.D.T., N.J.P. and V.A.

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Correspondence to Brett M Collins.

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Integrated supplementary information

Supplementary Figure 1 2D 1H-15N HSQC NMR spectrum of β2AR NMR titrations.

(related to Fig. 4)

(a) Overlays of the 2D 1H-15N HSQC spectra of 15N-13C-labeled SNX27 PDZ domain in its free form (blue) and titrated with between 0.5 to 4 molar equivalents of β2AR-pS-6 peptide. Resonances showing significant broadening and changes in chemical shift on β2AR-pS-6 binding are labelled with the residue number. Residues which chemical shift variation superior to Δδ=0.4 ppm are labelled on the histogram in the lower box. (b) Overlays of the 2D 1H-15N HSQC spectra of 15N-13C-labeled SNX27PDZ in its free form (black) and in the presence of 0.5 to 4 molar equivalents of β2AR or β2AR-pS-2. Resonances showing significant broadening and changes in chemical shift on VPS26A binding are labelled with the residue number.

Supplementary Figure 2 Proximity ligation assay (PLA) shows SNX27 interaction with GluN2B.

(related to Fig. 5)

Hippocampal neurons (DIV18) were transfected with GFP-SNX27 and stained with specific antibodies against GFP, GluN2B and MAP2 prior to PLA assay. The PLA signals between GFP-SNX27 and endogenous GluN2B are shown in the red channel, while GFP-SNX27 and MAP2 are labeled in green and blue channels, respectively. Scale bars = 50 μm (left), 20 μm (top right) and 5 μm (bottom right).

Supplementary Figure 3 SNX27 does not show significant binding to AMPARs, and binding is not enhanced by phosphorylation or VPS26 association.

(related to Fig. 5)

Binding of AMPARs to SNX27PDZ is not measurable by ITC suggesting weak binding or no interaction.

Supplementary Figure 4 NHERF1 PDZ domain 2 does not bind phosphorylated peptides.

(a) Selected sequence alignment of SNX27 PDZ domain with the first and second PDZ domains of NHERF1 and NHERF2. Highlights the three critical residues in SNX27 for binding phosphorylated residues at -3 and -5 (Asn56/Arg58/Ser82). (b) Overlay of the SNX27PDZ-DGKζ, NHERF1PDZ-CFTR and NHERF2PDZ-LPA co-crystal structures show the architecture of NHERF PDZ domains is optimal for exclusive and strong binding to the -3 residue. (C) ITC of CFTR and GluN2B peptides to SNX27 and NHERF1 PDZ2. CFTR control peptide binds strongly to CFTR as expected (green), as does SNX27 PDZ domain alone (purple) and SNX27 in complex with VPS26A (blue). Phosphorylated (green) and non-phosphorylated GluN2B (cyan) however do not bind significantly to the NHERF1 PDZ2 domain, compared to SNX27 PDZ domain alone (purple) or in complex with VPS26A (blue). A similar result is seen for both LRRC3B and GluN1 in their phosphorylated and non-phosphorylated states (data not shown).

Supplementary Figure 5 Classification of peptides on the basis of binding affinities from ITC and structural considerations.

(related to Fig. 7)

For PDZbm peptides, strong binding affinity correlates with the magnitude of the enthalpic contribution of binding to SNX27. Kd and ΔH values obtained from the present study and other work performed by our laboratory1 are plotted (errors show SD for 3 experiments). Detailed data can be found in Supplementary Table 1.

Supplementary Figure 6 Bioinformatic screening of the human proteome for SNX27 PDZbms.

(related to Fig. 7)

Potential SNX27 PDZbms identified bioinformatically after searching the human proteome were split into seven classes based on their sequences. Cargoes and soluble ligands for which experimental data indicating an interaction with SNX27 has previously been reported coloured in white and their proportion of each class (%) shown in boxes.

Supplementary Figure 7 Uncropped gel images.

(a) Related to Fig. 3d . GFP-DGKζ wild type and mutant constructs expressed in Jurkat T-cells were analysed for binding to SNX27 by GFP-trap immunoprecipitation. (b) Related to Fig. 5c . GST-pulldown experiments from HEK293 cells transfected with myc-SNX27 and GST-GluN1 or GST-GluN2B C-terminal tails, either WT, PDZ mutant, phosphodefective (AA) or phosphomimetic (DD) mutants. (c) Related to Fig. 7b . Steady state levels of putative SNX27 cargos were reduced following SNX27 knockout by CRISPR-Cas9 deletion. Whole cell lysates from control HeLa cells (stably expressing Cas9) and SNX27 knockout cells were probed for several PDZbm-containing cargos by western blotting. Reduction of PDGFRβ, ATP7A and SNX14 suggests a defect in endosomal transport leading to lysosomal degradation. EAAT1 total levels were apparently unaltered. Na+/K+-ATPase is a control plasma membrane protein.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Tables 1 and 2 (PDF 6930 kb)

Supplementary Table 3

Bioinformatic identification of putative SNX27 binding proteins (XLSX 261 kb)

Supplementary Table 4

Gene Ontology functional analysis of SNX27 putative PDZ binders (XLS 1172 kb)

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Clairfeuille, T., Mas, C., Chan, A. et al. A molecular code for endosomal recycling of phosphorylated cargos by the SNX27–retromer complex. Nat Struct Mol Biol 23, 921–932 (2016). https://doi.org/10.1038/nsmb.3290

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