Phox homology band 4.1/ezrin/radixin/moesin-like proteins function as molecular scaffolds that interact with cargo receptors and Ras GTPases Rajesh Ghai, Mehdi Mobli, Suzanne J. Norwood, Andrea Bugarcic, Rohan D. Teasdale, Glenn F. King, and Brett M. Collins1 Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland 4072, Australia Edited by Frances M. Brodsky, University of California, San Francisco, CA, and accepted by the Editorial Board March 15, 2011 (received for review November 29, 2010) Following endocytosis, the fates of receptors, channels, and other the disease. The homology of SNX31 to SNX17 (approximately transmembrane proteins are decided via specific endosomal sort- 40% identity) suggests an involvement in similar endosomal ing pathways, including recycling to the cell surface for continued transport pathways. activity. Two distinct phox-homology (PX)-domain-containing pro- SNX27 is unique among the PX proteins, containing an teins, sorting nexin (SNX) 17 and SNX27, are critical regulators of N-terminal PDZ domain upstream of the PX domain. SNX27 recycling from endosomes to the cell surface. In this study we de- has also been annotated to possess a Ras-association domain monstrate that SNX17, SNX27, and SNX31 all possess a novel 4.1/ and extended C-terminal region (1, 2, 13). SNX27 was first iden- ezrin/radixin/moesin (FERM)-like domain. SNX17 has been shown tified as a binding partner for the 5-hydroxytryptamine type-4 to bind to Asn-Pro-Xaa-Tyr (NPxY) sequences in the cytoplasmic receptor (5-HT4R) (13), and overexpression of SNX27 directs tails of cargo such as LDL receptors and the amyloid precursor pro- localization of 5-HT4R and Kir3 potassium channels to early tein, and we find that both SNX17 and SNX27 display similar affi- endosomal autoantigen 1 (EEA1)-positive early endosomes nities for NPxY sorting motifs, suggesting conserved functions in (13, 14). There is accumulating evidence for a role for SNX27 endosomal recycling. Furthermore, we show for the first time that in coupling protein sorting to cell signaling. It can direct the all three proteins are able to bind the Ras GTPase through their endosome-to-cell surface recycling of the β2 adrenergic receptor FERM-like domains. These interactions place the PX-FERM-like (15), and SNX27 may also scaffold signaling and lipid modulating proteins at a hub of endosomal sorting and signaling processes. complexes by interacting with proteins such as diacylglycerolki- Studies of the SNX17 PX domain coupled with cellular localization nase ζ (16), NMDA receptors (17), and cytohesin associated experiments reveal the mechanistic basis for endosomal localiza- scaffolding protein (CASP) (18). All of these molecules bind tion of the PX-FERM-like proteins, and structures of SNX17 and to SNX27 via type-I PDZ-domain binding motifs. SNX27 determined by small angle X-ray scattering show that they Recent studies suggest that some PX proteins may play dual adopt non-self-assembling, modular structures in solution. In roles in membrane trafficking and cell signaling (19, 20), and summary, this work defines a novel family of proteins that parti- there is mounting evidence that endosomal sorting of receptors cipate in a network of interactions that will impact on both endo- is a key factor in determining differential signaling outcomes somal protein trafficking and compartment specific Ras signaling (21, 22). For example, signaling by the Ras oncogene has for cascades. many years been thought to occur primarily at the plasma mem- brane. More recent assessments of the spatiotemporal control of hox-homology (PX) domain-containing proteins are a diverse Ras signaling have demonstrated the existence of Ras-mediated Pfamily of proteins implicated in many protein trafficking pro- signaling events on intracellular membranes including Ras/ cesses, and there is emerging recognition of their importance in MAPK signaling on endosomes (23, 24). cell signaling (1, 2). The PX domain binds phosphatidylinositol Here we show that SNX17, SNX27, and SNX31 define a un- phospholipids (PIPs) to mediate localization to subcellular mem- ique subfamily of PX proteins possessing an unusual band 4.1/ branous compartments for regulation of cargo transport and ezrin/radixin/moesin (FERM)-like structure, which incorporates processing. Most PX proteins also contain a variety of other func- the previously annotated Ras-association domain of SNX27. We tional modules including Ras-association (RA) and PSD-95/discs find that the members of this family share both NPxY peptide- large/zona occludens (PDZ) domains. Thus PX proteins can binding properties and an ability to associate with H-Ras in a function as scaffolds that facilitate spatiotemporal assembly of GTP-dependent manner. Structural studies of the PX-FERM- BIOCHEMISTRY membrane trafficking and signaling complexes. like proteins reveal the molecular mechanisms for membrane The PX-protein sorting nexin 17 (SNX17) is important for endosomal sorting of transmembrane proteins from endosomes Author contributions: R.G., R.D.T., G.F.K., and B.M.C. designed research; R.G., M.M., S.J.N., to the cell surface. Identified cargo molecules include the low- A.B., and B.M.C. performed research; R.D.T. and G.F.K. contributed new reagents/analytic density lipoprotein receptor (LDLR), and other members of the tools; R.G., M.M., S.J.N., A.B., R.D.T., G.F.K., and B.M.C. analyzed data; and R.G., S.J.N., A.B., LDLR family including LDLR-related protein 1 (LRP1), suggest- and B.M.C. wrote the paper. ing an important role in lipid metabolism (3–5). SNX17 also The authors declare no conflict of interest. regulates the trafficking of P-selectin (6) and FEEL-1 (7) and as- This article is a PNAS Direct Submission. F.M.B. is a guest editor invited by the Editorial sociates with cytosolic factors Krit1 (8) and Kif1B (9). All of these Board. proteins have been found to bind SNX17 via a conserved Asn- Data deposition: Coordinates and structure factors for the SNX17 PX domain have been Pro-Xaa-Tyr (NPxY) sequence motif, but the molecular basis deposited in the RCSB Protein Data Bank, www.pdb.org/pdb/home/home.do (PDB ID of this interaction is unknown. Recent data indicate an important code 3LUI). Raw diffraction images are available on the Diffraction Image Experiment Repository (DIMER) at http://xr-diffraction.imb.uq.edu.au. NMR assignments have role for SNX17 in trafficking of the amyloid precursor protein been deposited in the Biological Magnetic Resonance Bank (http://www.bmrb.wisc.edu/) (APP) central to Alzheimer’s disease (AD) (10). As the LDLR (BMRB accession number 17497). family, in particular LRP1, have also been linked to AD and play 1To whom correspondence should be addressed. E-mail: [email protected]. direct roles in APP trafficking (11, 12), it appears SNX17 func- This article contains supporting information online at www.pnas.org/lookup/suppl/ tions at a nexus of endosomal trafficking pathways important for doi:10.1073/pnas.1017110108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1017110108 PNAS ∣ May 10, 2011 ∣ vol. 108 ∣ no. 19 ∣ 7763–7768 Downloaded by guest on September 26, 2021 recruitment and their overall domain architectures, highlighting a tein APP (Fig. 2A and Table S1). Significant binding was observed 32 Æ 5 μ structural scaffold primed for assembly of endosomal trafficking with a Kd of M. This low micromolar affinity is typical of and signaling complexes. Our work points to a role for PX- coat–cargo interactions in other systems such as clathrin-coated FERM-like proteins as interaction hubs that will have key func- vesicle formation. Titrations using a peptide in which the key tions in endosomal trafficking and Ras-mediated signaling and Tyr side chain was mutated to Ala showed no significant binding, provides a foundation for future studies of these processes. confirming the specificity of the interaction. Previous reports have indicated that the C terminus of SNX17 is required for Results NPxY binding (6, 8), but the mechanism underpinning this inter- Defining a Unique PX-FERM-Like Protein Family. FERM domains are action is unknown. As we have found that this region contains a found in numerous molecules where they regulate lipid and pro- FERM-like domain with a PTB-related F3 module, it is highly tein interactions. They are approximately 300 residues in length likely that this interaction occurs via binding to this structure, and contain three modules termed F1, F2, and F3 (25). F1 has a as observed in the complex formed between the FE65 PTB ubiquitin-related fold, F2 an α-helical structure, and F3 has struc- domain and APP (27). To investigate if other PX-FERM-like tural similarity to phosphotyrosine-binding (PTB) domains. Both proteins also interact with cargo receptors containing the NPxY SNX17 and SNX31 have been annotated to possess a C-terminal motif, SNX27 was titrated with the APP NPxY peptide (Fig. 2B). sequence similar to the N terminus of FERM domains (1, 2, 6, 8). SNX27 bound the APP peptide with similar affinity to SNX17 ¼ 27 6 Æ 0 1 μ We performed a secondary structure-based comparison of (Kd . M), and the SNX27 FERM-like domain binds SNX17 and SNX31 and found that the C-terminal region of both the APP peptide sequence with almost identical affinity to the ¼ 22 9 Æ 13 4 μ proteins, in fact, contains canonical F1 and F3 structures (Fig. 1). full-length protein (Kd . M). To date, SNX27 has However, unlike classical FERM domains, SNX17 and SNX31 been found to bind cargo only via its PDZ domain; hence we show possess an altered F2 module. Instead of four helices, the F2 that like SNX17, SNX27 is able to associate specifically with modules of SNX17 and SNX31 are predicted to have three and NPxY motif-containing cargo receptors.