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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Kennedy, M.B. Signal-processing machines at the postsynaptic density. Science 290, 750–754 (2000).
Zhang, M. & Wang, W. Organization of signaling complexes by PDZ-domain scaffold proteins. Acc. Chem. Res. 36, 530–538 (2003).
Kim, E. & Sheng, M. PDZ domain proteins of synapses. Nat. Rev. Neurosci. 5, 771–781 (2004).
Hung, A.Y. & Sheng, M. PDZ domains: structural modules for protein complex assembly. J. Biol. Chem. 277, 5699–5702 (2002).
Stiffler, M.A. et al. PDZ domain binding selectivity is optimized across the mouse proteome. Science 317, 364–369 (2007).
Hall, R.A. et al. The β2-adrenergic receptor interacts with the Na+/H+-exchanger regulatory factor to control Na+/H+ exchange. Nature 392, 626–630 (1998).
Ye, F. & Zhang, M. Structures and target recognition modes of PDZ domains: recurring themes and emerging pictures. Biochem. J. 455, 1–14 (2013).
Gallon, M. et al. A unique PDZ domain and arrestin-like fold interaction reveals mechanistic details of endocytic recycling by SNX27-retromer. Proc. Natl. Acad. Sci. USA 111, E3604–E3613 (2014).
Steinberg, F. et al. A global analysis of SNX27–retromer assembly and cargo specificity reveals a function in glucose and metal ion transport. Nat. Cell Biol. 15, 461–471 (2013).
Lauffer, B.E. et al. SNX27 mediates PDZ-directed sorting from endosomes to the plasma membrane. J. Cell Biol. 190, 565–574 (2010).
Temkin, P. et al. SNX27 mediates retromer tubule entry and endosome-to-plasma membrane trafficking of signalling receptors. Nat. Cell Biol. 13, 715–721 (2011).
Choy, R.W. et al. Retromer mediates a discrete route of local membrane delivery to dendrites. Neuron 82, 55–62 (2014).
Chan, A.S. et al. Sorting nexin 27 couples PTHR trafficking to retromer for signal regulation in osteoblasts during bone growth. Mol. Biol. Cell 27, 1367–1382 (2016).
Balana, B. et al. Mechanism underlying selective regulation of G protein-gated inwardly rectifying potassium channels by the psychostimulant-sensitive sorting nexin 27. Proc. Natl. Acad. Sci. USA 108, 5831–5836 (2011).
Lunn, M.L. et al. A unique sorting nexin regulates trafficking of potassium channels via a PDZ domain interaction. Nat. Neurosci. 10, 1249–1259 (2007).
Hussain, N.K., Diering, G.H., Sole, J., Anggono, V. & Huganir, R.L. Sorting Nexin 27 regulates basal and activity-dependent trafficking of AMPARs. Proc. Natl. Acad. Sci. USA 111, 11840–11845 (2014).
Loo, L.S., Tang, N., Al-Haddawi, M., Dawe, G.S. & Hong, W. A role for sorting nexin 27 in AMPA receptor trafficking. Nat. Commun. 5, 3176 (2014).
Wang, X. et al. Loss of sorting nexin 27 contributes to excitatory synaptic dysfunction by modulating glutamate receptor recycling in Down's syndrome. Nat. Med. 19, 473–480 (2013).
Joubert, L. et al. New sorting nexin (SNX27) and NHERF specifically interact with the 5-HT4a receptor splice variant: roles in receptor targeting. J. Cell Sci. 117, 5367–5379 (2004).
Lee, S., Chang, J. & Blackstone, C. FAM21 directs SNX27-retromer cargoes to the plasma membrane by preventing transport to the Golgi apparatus. Nat. Commun. 7, 10939 (2016).
Muhammad, A. et al. Retromer deficiency observed in Alzheimer's disease causes hippocampal dysfunction, neurodegeneration, and Aβ accumulation. Proc. Natl. Acad. Sci. USA 105, 7327–7332 (2008).
Vilariño-Güell, C. et al. VPS35 mutations in Parkinson disease. Am. J. Hum. Genet. 89, 162–167 (2011).
Wen, L. et al. VPS35 haploinsufficiency increases Alzheimer's disease neuropathology. J. Cell Biol. 195, 765–779 (2011).
Zimprich, A. et al. A mutation in VPS35, encoding a subunit of the retromer complex, causes late-onset Parkinson disease. Am. J. Hum. Genet. 89, 168–175 (2011).
Wang, X. et al. Sorting nexin 27 regulates Aβ production through modulating γ-secretase activity. Cell Rep. 9, 1023–1033 (2014).
Damseh, N. et al. A defect in the retromer accessory protein, SNX27, manifests by infantile myoclonic epilepsy and neurodegeneration. Neurogenetics 16, 215–221 (2015).
Tsika, E. et al. Parkinson's disease-linked mutations in VPS35 induce dopaminergic neurodegeneration. Hum. Mol. Genet. 23, 4621–4638 (2014).
Vardarajan, B.N. et al. Identification of Alzheimer disease-associated variants in genes that regulate retromer function. Neurobiol. Aging 33, 2231.e15–2231.e30 (2012).
Rincón, E. et al. Translocation dynamics of sorting nexin 27 in activated T cells. J. Cell Sci. 124, 776–788 (2011).
Rincón, E. et al. Proteomics identification of sorting nexin 27 as a diacylglycerol kinase zeta-associated protein: new diacylglycerol kinase roles in endocytic recycling. Mol. Cell. Proteomics 6, 1073–1087 (2007).
Sowa, M.E., Bennett, E.J., Gygi, S.P. & Harper, J.W. Defining the human deubiquitinating enzyme interaction landscape. Cell 138, 389–403 (2009).
Okabe, S. Molecular anatomy of the postsynaptic density. Mol. Cell. Neurosci. 34, 503–518 (2007).
Wang, J.Q. et al. Roles of subunit phosphorylation in regulating glutamate receptor function. Eur. J. Pharmacol. 728, 183–187 (2014).
Lussier, M.P., Sanz-Clemente, A. & Roche, K.W. Dynamic regulation of N-methyl-D-aspartate (nmda) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors by posttranslational modifications. J. Biol. Chem. 290, 28596–28603 (2015).
Ryan, T.J., Emes, R.D., Grant, S.G. & Komiyama, N.H. Evolution of NMDA receptor cytoplasmic interaction domains: implications for organisation of synaptic signalling complexes. BMC Neurosci. 9, 6 (2008).
Sakarya, O. et al. Evolutionary expansion and specialization of the PDZ domains. Mol. Biol. Evol. 27, 1058–1069 (2010).
Cushing, P.R., Fellows, A., Villone, D., Boisguérin, P. & Madden, D.R. The relative binding affinities of PDZ partners for CFTR: a biochemical basis for efficient endocytic recycling. Biochemistry 47, 10084–10098 (2008).
Pankow, S. et al. ΔF508 CFTR interactome remodelling promotes rescue of cystic fibrosis. Nature 528, 510–516 (2015).
Akizu, N. et al. Biallelic mutations in SNX14 cause a syndromic form of cerebellar atrophy and lysosome-autophagosome dysfunction. Nat. Genet. 47, 528–534 (2015).
Mas, C. et al. Structural basis for different phosphoinositide specificities of the PX domains of sorting nexins regulating G-protein signaling. J. Biol. Chem. 289, 28554–28568 (2014).
Ghai, R. et al. Phox homology band 4.1/ezrin/radixin/moesin-like proteins function as molecular scaffolds that interact with cargo receptors and Ras GTPases. Proc. Natl. Acad. Sci. USA 108, 7763–7768 (2011).
Ghai, R. et al. Phosphoinositide binding by the SNX27 FERM domain regulates its localization at the immune synapse of activated T-cells. J. Cell Sci. 128, 553–565 (2015).
Ghai, R. et al. Structural basis for endosomal trafficking of diverse transmembrane cargos by PX-FERM proteins. Proc. Natl. Acad. Sci. USA 110, E643–E652 (2013).
Cao, T.T., Deacon, H.W., Reczek, D., Bretscher, A. & von Zastrow, M. A kinase-regulated PDZ-domain interaction controls endocytic sorting of the β2-adrenergic receptor. Nature 401, 286–290 (1999).
Choi, J. et al. Phosphorylation of stargazin by protein kinase A regulates its interaction with PSD-95. J. Biol. Chem. 277, 12359–12363 (2002).
Chung, H.J., Huang, Y.H., Lau, L.F. & Huganir, R.L. Regulation of the NMDA receptor complex and trafficking by activity-dependent phosphorylation of the NR2B subunit PDZ ligand. J. Neurosci. 24, 10248–10259 (2004).
Chung, H.J., Xia, J., Scannevin, R.H., Zhang, X. & Huganir, R.L. Phosphorylation of the AMPA receptor subunit GluR2 differentially regulates its interaction with PDZ domain-containing proteins. J. Neurosci. 20, 7258–7267 (2000).
Cohen, N.A., Brenman, J.E., Snyder, S.H. & Bredt, D.S. Binding of the inward rectifier K+ channel Kir 2.3 to PSD-95 is regulated by protein kinase A phosphorylation. Neuron 17, 759–767 (1996).
Sanz-Clemente, A., Matta, J.A., Isaac, J.T. & Roche, K.W. Casein kinase 2 regulates the NR2 subunit composition of synaptic NMDA receptors. Neuron 67, 984–996 (2010).
Tanemoto, M., Fujita, A., Higashi, K. & Kurachi, Y. PSD-95 mediates formation of a functional homomeric Kir5.1 channel in the brain. Neuron 34, 387–397 (2002).
Tian, Q.B. et al. Interaction of LDL receptor-related protein 4 (LRP4) with postsynaptic scaffold proteins via its C-terminal PDZ domain-binding motif, and its regulation by Ca/calmodulin-dependent protein kinase II. Eur. J. Neurosci. 23, 2864–2876 (2006).
Lee, H.J. & Zheng, J.J. PDZ domains and their binding partners: structure, specificity, and modification. Cell Commun. Signal. 8, 8 (2010).
Liu, X., Shepherd, T.R., Murray, A.M., Xu, Z. & Fuentes, E.J. The structure of the Tiam1 PDZ domain/ phospho-syndecan1 complex reveals a ligand conformation that modulates protein dynamics. Structure 21, 342–354 (2013).
Nomme, J. et al. Structural basis of a key factor regulating the affinity between the zonula occludens first PDZ domain and claudins. J. Biol. Chem. 290, 16595–16606 (2015).
Zhang, N. et al. Phosphorylation of synaptic vesicle protein 2A at Thr84 by casein kinase 1 family kinases controls the specific retrieval of synaptotagmin-1. J. Neurosci. 35, 2492–2507 (2015).
Nobles, K.N. et al. Distinct phosphorylation sites on the β2-adrenergic receptor establish a barcode that encodes differential functions of β-arrestin. Sci. Signal. 4, ra51 (2011).
César-Razquin, A. et al. A call for systematic research on solute carriers. Cell 162, 478–487 (2015).
Belotti, E. et al. The human PDZome: a gateway to PSD95-Disc large-zonula occludens (PDZ)-mediated functions. Mol. Cell. Proteomics 12, 2587–2603 (2013).
Pim, D., Broniarczyk, J., Bergant, M., Playford, M.P. & Banks, L. A novel PDZ domain interaction mediates the binding between human papillomavirus 16 L2 and sorting nexin 27 and modulates virion trafficking. J. Virol. 89, 10145–10155 (2015).
Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).
McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).
Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).
Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).
de Castro, E. et al. ScanProsite: detection of PROSITE signature matches and ProRule-associated functional and structural residues in proteins. Nucleic Acids Res. 34, W362–W365 (2006).
Santos, T., Carrasco, S., Jones, D.R., Mérida, I. & Eguinoa, A. Dynamics of diacylglycerol kinase zeta translocation in living T-cells: study of the structural domain requirements for translocation and activity. J. Biol. Chem. 277, 30300–30309 (2002).
Anggono, V. et al. PICK1 interacts with PACSIN to regulate AMPA receptor internalization and cerebellar long-term depression. Proc. Natl. Acad. Sci. USA 110, 13976–13981 (2013).
Aubrey, B.J. et al. An inducible lentiviral guide RNA platform enables the identification of tumor-ess ential genes and tumor-promoting mutations in vivo. Cell Rep. 10, 1422–1432 (2015).
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.
Author information
Authors and Affiliations
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.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
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)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nsmb.3290
This article is cited by
-
RNA binding motif protein 45-mediated phosphorylation enhances protein stability of ASCT2 to promote hepatocellular carcinoma progression
Oncogene (2023)
-
Dynamic control of the dopamine transporter in neurotransmission and homeostasis
npj Parkinson's Disease (2021)
-
Phosphorylation-induced changes in the PDZ domain of Dishevelled 3
Scientific Reports (2021)
-
Kidins220 deficiency causes ventriculomegaly via SNX27-retromer-dependent AQP4 degradation
Molecular Psychiatry (2021)
-
SNXs take center stage in endosomal sorting
Nature Cell Biology (2019)