Phox Homology Band 4.1/Ezrin/Radixin/Moesin-Like Proteins Function As Molecular Scaffolds That Interact with Cargo Receptors and Ras Gtpases

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 membrane. 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 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 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 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. are shorter in length than the canonical F2 structure (approximately 50 rather than approximately 100 residues). Thus SNX17 PX-FERM-Like Proteins Interact with H-Ras in Vitro via Their FERM-Like and SNX31 possess all of the expected features of the FERM Domain. As the F1 module of SNX27 was originally detected as a domain but with an altered F2 module, and we propose that this putative Ras-binding RA domain (1, 2), we examined whether the domain be referred to as a “FERM-like” domain to reflect this PX-FERM-like proteins have Ras-binding activity. The interac- difference. Similar analyses of other PX proteins unexpectedly tion of H-Ras with the PX-FERM-like proteins was investigated showed that SNX27 has a homologous FERM-like domain in vitro using GST pulldown assays and Western blotting. For (Fig. 1). SNX27 has been annotated to possess a C-terminal RA these experiments, soluble H-Ras truncated at residue 166 and domain (1, 2) and has an additional N-terminal PDZ domain. with a G12V activating mutation (26) was tested against GST- Δ A This bioinformatics-based classification likely results from the SNX17, GST-SNX27 PDZ, and GST-SNX31 (Fig. 3 ). The high degree of structural homology between the RA domain and PX-FERM-like proteins all showed binding to H-Ras(G12V) the FERM F1 module (26). In summary, SNX17, SNX27, and in these experiments. No binding to GST-SNX17-PX was ob- SNX31 form a distinct subfamily of PX proteins with a conserved served, and a GST-SNX17-FERM-like domain construct bound FERM-like structure. similarly to full-length SNX17, indicating that the binding of H-Ras occurs via the FERM-like domain as predicted. The lack A Interactions of PX-FERM-Like Proteins with NPxY-Containing Cargo of both stoichiometric amounts of H-Ras in pulldowns (Fig. 3 ) Molecules. SNX17 associates with transmembrane cargo proteins and strong binding signals by ITC indicate that the interactions 50–100 μ by recognizing an NPxY motif present in their cytosolic tails (4, 5, are of relatively low affinity (Kd > M). An essential 10). To examine the affinity of this interaction we used isothermal attribute of Ras effector proteins is that they bind active GTP- titration calorimetry (ITC) to monitor the interaction between loaded Ras but not inactive GDP-loaded Ras. To test this we used SNX17 and a 15-mer peptide derived from the model cargo pro- H-Ras loaded with either GTP or GDP and found that all three

Fig. 1. SNX17, SNX31, and SNX27 share a conserved C-terminal FERM-like Fig. 2. Binding of NPxY cargo motifs is conserved across the PX-FERM-like domain. Sequence alignment of SNX17, SNX27, and SNX31. Secondary struc- protein family. Binding of SNX17 (A) and SNX27 (B) to a peptide derived from ture predictions calculated with JPRED (41) are indicated for SNX17 (above) APP was measured using ITC. Experiments were performed at 10 °C with and SNX27 (below). Alignment was made with ESPript 2.2 (42). Identified 50 μM protein and 2,000 μM APP peptide. Also shown are the titrations subdomains are indicated by boxes. Red triangles indicate key residues in- of SNX17 and SNX27 with the APP Y A mutant peptide, which does not volved in PI(3)P binding. bind. (Top) Raw data; (Bottom) integrated normalized data.

7764 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1017110108 Ghai et al. Downloaded by guest on September 26, 2021 β-sheet (β1: Phe3-Arg11, β2: Tyr20-Val27, and β3: Val30-Val35) followed by a helical bundle (α1: Tyr37-Tyr51, α2: Pro69-Gln88, and α3: Gln96-Thr109) and a 310 helix. In addition, there is a long loop (Tyr52-Thr68) containing a polyproline region that connects helices α1 and α2. Each of the three chains of SNX17-PX in the crystallographic 2− asymmetric unit contain bound SO4 ions forming electrostatic 2− interactions with Arg36 (Fig. S3). The SO4 ion lies in a posi- tively charged cavity formed between α1, β3, and α2 and the adjacent polyproline loop. Overlay of SNX17 and SNX9 (rmsd 2− of 1.66 Å over 95 Cα atoms) shows that the bound SO4 ion is oriented identically to the 3 phosphate of the PI(3)P bound to SNX9 (Fig. 4 and Fig. S3) (28). Other critical side chains are also conserved in SNX17, including Tyr37, which in SNX9 forms a stacking interaction with the inositol ring of PI(3)P, Arg75, which contributes to the environment of the binding pocket, and Lys62, which forms an electrostatic contact with the 1-phosphate group. The electrostatic surface of SNX17 also shows a very similar elec- tropositive binding pocket to SNX9 (Fig. S3). We next used a combination of heteronuclear NMR experiments and molecular docking to determine the structure of the SNX17:PI(3)P complex. Complete 15N, 13C, and 1H resonance assignments were obtained for the SNX17 PX domain (Fig. S4), which enabled NMR chemical shift mapping to determine the identity of the diC8-PI(3)P binding site (Fig. 4D). These NMR 20 10 μ titration experiments yielded a Kd of M, which is very similar to that obtained by ITC. Those residues that incurred 15 the largest N and HN chemical shift changes upon addition of PI(3)P were mapped onto the crystal structure to identify the putative PI(3)P binding pocket (Fig. 4E and Fig. S5). These Fig. 3. PX-FERM-like proteins are effectors of the Ras GTPase. (A) PX-FERM- residues were used as restraints to dock PI(3)P onto the SNX17 like proteins were tested for H-Ras(G12V) binding in GST pulldown assays. crystal structure to obtain a model of the SNX17:PI(3)P complex (Top) Coomassie-stained gel of the pulldown samples; (Lower) Western blot (details in SI Text). The 3 phosphate of the docked PI(3)P over- with anti-Ras antibody. Note the SNX27 construct lacks the N-terminal PDZ 2− laps very well with the SO4 molecule bound to the SNX17 PX domain. A minimal SNX17-FERM-like domain associates with H-Ras similarly domain in our crystal structure (Fig. 4E). It also very closely to the full-length PX-FERM-like proteins. (B) The GTP dependence of the matches the previously solved structure of the SNX9:PI(3)P interaction was confirmed by loading H-Ras with either GDP or GTP and performing GST-pulldown assays as in A. complex (28). Overall then, we confidently predict that SNX17 (and SNX27 and SNX31) coordinates PI(3)P in a very similar PX-FERM-like proteins associate specifically with GTP-loaded manner to SNX9 and other members of the PX-protein family. H-Ras (Fig. 3B). This indicates they are likely bona fide effector The identity of the PI(3)P binding site was further confirmed molecules and suggests that apart from being involved in mem- by the finding that the binding of SNX17 to PI(3)P (Fig. 4A) brane trafficking, PX-FERM-like proteins could also play key and SNX17 localization in vivo (Fig. 4B) were both completely roles in Ras-mediated endosomal signal transduction. abrogated when the critical Arg36 was mutated to Gln.

PX Domains of PX-FERM-Like Proteins Bind Specifically to PI(3)P. Cri- Solution Structures of Full-Length PX-FERM-Like Proteins. The PX- tical to the function of PX-domain proteins is their ability to be FERM-like proteins represent a unique family with as yet poorly recruited to specific intracellular membranes via binding of the understood intradomain architectures and tertiary structures. As PX domain to PIPs. Liposome pelleting assays indicate a distinct many PX-domain proteins are known to self-associate, we first preference for PI(3)P lipids (Fig. S1). The PIP specificity of examined their oligomeric properties using multiangle laser light SNX17 and SNX27 was quantified by ITC using soluble short- scattering, confirming the monomeric nature of both SNX17 and A SNX27 (Table S3 and Fig. S6). We next used small angle X-ray

chain (di-C8) analogues (Fig. 4 and Table S1). The SNX17 and BIOCHEMISTRY SNX27 PX domains were found to interact only with PI(3)P, with scattering (SAXS) to examine the solution structures of full- almost identical thermodynamics and affinities (11.3 2.1 μM length SNX17 and SNX27. Scattering curves and Guinier plots and 12.7 4.5 μM, respectively). These data are consistent with are shown in Fig. 5 A and B, and structural parameters are sum- observations that SNX17 and SNX27 both localize to PI(3)P-rich marized in Table S3. Pair distance distribution functions, P(r), endosomal membranes (Fig. 4B and Fig. S2). Experiments using indicate that both SNX17 and SNX27 possess a compact globular inositolð1;3ÞP2 lacking the di-C8 hydrocarbon chain show that the conformation in solution (Fig. 5 C and D). The radius of gyration lipid acyl chain does not contribute to the interaction. Finally, (Rg) and maximum dimensions (Dmax) of SNX17 are significantly full-length SNX17 and SNX27 bound PI(3)P with the same affi- smaller than SNX27, likely due to the presence of the additional nity as the isolated PX domain, indicating that the interaction PDZ domain in SNX27. The ab initio structures of both SNX17 with PI(3)P lipids is solely driven by the PX domains. and SNX27 calculated with GASBOR display distinctive features, where a larger globular domain is apposed to a smaller globular Molecular Mechanism of Endosomal PI(3)P Binding. To assess the domain in the case of SNX17 or a more extended structure in the molecular determinants for PX-FERM-like protein membrane case of SNX27 (Fig. 5 E and F). A starting model composed of interaction, the structure of the SNX17-PX domain was deter- the SNX17 PX domain and radixin FERM domain was docked into mined by X-ray crystallography (Fig. 4C, Fig. S3, and Table S2). the SNX17 SAXS-derived envelope by treating the PX and FERM The SNX17 PX domain has a conventional PX-domain fold with domains as distinct rigid bodies. The derived structure shows a meander topology consisting of an N-terminal three-stranded excellent agreement with the ab initio model and confirms that

Ghai et al. PNAS ∣ May 10, 2011 ∣ vol. 108 ∣ no. 19 ∣ 7765 Downloaded by guest on September 26, 2021 Fig. 4. PX-FERM-like proteins bind specifically to endosomal PI(3)P phosphoinositides. (A) Binding of the SNX17 PX domain to PI(3)P measured by ITC at 25 °C. Data are shown for protein versus PI(3)P, and PIð3;5ÞP2, which does not bind. Also shown is the R36Q mutant, confirming it does not bind PI (3)P. (Top) Raw data; (Bottom) integrated normalized data. A full list of binding results is given in Table S1. (B) SNX27-GFP and GFP-SNX17 were examined for colocalization with early endosomal markers SNX1 and EEA1, respectively, using immunofluorescence microscopy. SNX17(R36Q)-GFP shows only cytosolic localization. Comparison with other cellular markers is shown in Fig. S5.(C) Ribbon representation of the superposition of SNX17 PX crystal structure (blue), SNX9-PX [green; (28)], and p40phox-PX [magenta; (43)]. (D) A superposition of the 1H-15N HSQC spectra of free (green) and PI(3)P-saturated SNX17 PX domain (red). (Bottom Right Inset) Chemical shift changes in the backbone amide resonances of Arg36, Lys62, and Lys69 with increasing PI(3)P concentration. The binding curves for these residues were plotted to show the chemical shift (Δδ)asa function of PI(3)P concentration (Top Left Inset). (E) Surface representation of the SNX17 PX domain highlighting residues showing significant chemical shift changes in blue (2× SD) and cyan (1× SD). The HADDOCK docked PI(3)P molecule (green) dis- 2− plays clear overlap with the bound SO4 ion in the crystal structure (yellow) and PI(3)P from the SNX9∶PIð3ÞP complex (white).

SNX17 has a compact architecture with closely associated PX and the N-terminal PDZ domain of SNX27 extends the protein signif- FERM-like domains (Fig. 5E). Similarly we determined the solu- icantly, but also appears to be closely associated with the PX and tion structure of SNX27, with an N-terminal PDZ domain added to FERM-like domains in a relatively rigid arrangement. the SNX17 structure, and modeling against the SNX27 SAXS data treating the PDZ, PX, and FERM-like domains as separate rigid Discussion entities. The refined PDZ, PX, and FERM-like domains form a A number of studies have confirmed the importance of the PX roughly linear overall structure, in excellent agreement with the ob- proteins SNX17 and SNX27 in distinct endosome-to-cell surface served scattering data (Fig. 5F). These results show that SNX17 and recycling processes. In contrast to previous reports we show that SNX27 possess analogous structures comprising the PX and the C-terminal regions of SNX17, SNX27, and the homologue FERM-like domains in close contact with each other, whereas SNX31 have a FERM-like domain analogous to the classical

Fig. 5. SAXS data and molecular models of the SNX17 and SNX27 proteins. (Top) Experimental SAXS scattering profiles for SNX17 (A) and SNX27 (B) after averaging and subtract- ing solvent scattering contribution. Theoretical scattering curves calculated from the ab initio models with lowest χ value (solid lines) overlaid on the experimental scattering data (black). (Insets) Guinier plots at the low-angle region × 1 3 (qmax Rg < . ). (Middle) P(r) functions for SNX17 (C) and SNX27 (D). (Lower) Ab initio models for SNX17 (E) and SNX27 (F) as transparent surfaces. Averaged and filtered en- velopes from GASBOR are shown in gray and blue, respectively. The domain models derived from BUNCH are overlaid with the ab intio structures, with PX and FERM domains colored blue and green, respectively, and the PDZ domain colored red.

7766 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1017110108 Ghai et al. Downloaded by guest on September 26, 2021 FERM domain, but with an altered F2 module, and share similar abilities to bind to NPxY cargo motifs. Canonical FERM domains recognize a variety of peptide signals including NPxY via the PTB domain-like F3 module (29); therefore, our determination that the PX-FERM-like proteins possess an F3 module as opposed to a truncated FERM domain provides a likely mechanism for NPxY signal binding by the proteins. SNX27 was previously shown to use its PDZ domain to bind transmembrane and cytosolic proteins with type-I PDZ binding motifs (13, 15, 16, 18); thus the demonstration of SNX27’s ability to also bind NPxY motifs significantly expands the range of potential cargo molecules for this protein. It will be important to determine if the three PX- FERM-like proteins have overlapping roles in endosomal sorting of NPxY-containing proteins or whether they have different functions mediated via regulatory interactions, alternate expression patterns, or tissue distributions. Fig. 6. A model for the network of interactions driven by the PX-FERM-like Several studies have demonstrated that endosomal localization proteins. H-Ras (purple ribbons), NPxY peptide (gold cylinders), and Type-I of SNX17 and SNX27 is essential for regulating trafficking of PDZ-binding peptides (gold cylinders) were modeled onto the SNX27 SAXS- cargo molecules (4, 5, 8, 13, 14, 16). Our ITC and NMR titration derived structure following structural alignment of respective complexes data demonstrate the exclusive binding of the PX domains of with PLC RA domain, FE65 PTB domain, and syntenin PDZ domain. The bound SNX17 and SNX27 to PI(3)P, and structural modeling confirms PI(3)P (spheres) is shown from our docking studies (Fig. 4E). The PX domain SNX17 binds PI(3)P via a conserved mechanism relying on spe- binds to PI(3)P, and the similarity of the F3 module of the FERM domain sug- cific recognition of the 3 phosphate and exclusion of potential 4- gests it will be responsible for binding membrane and cytoplasmic NPxY- containing proteins. The similarity of the F1 module to RA domains suggests and 5-phosphate moieties. The association of SNX17 and SNX27 this region governs interaction with Ras. SNX27 possesses an additional with PI(3)P explains their interaction with PI(3)P-enriched endo- N-terminal PDZ domain that binds cytosolic and transmembrane proteins somal membranes, and this depends on a functional PX domain. via C-terminal type-I PDZ binding motifs, E½S∕T × ½F∕V. Note that the An intriguing finding of this study is the binding of the PX- FERM-like F2 module is expected to differ from the canonical F2 module FERM-like proteins to the GTPase Ras. Central to the relay depicted. of a cellular signal is the spatiotemporal organization of suitable signaling components, and the interplay between cellular locali- and effectors, it is clear that many other proteins with similar zation and signaling, and in particular the role of endosomes in motifs will be potential interacting partners. In particular, a large differential signaling mechanisms, has become a field of intense number of receptor tyrosine kinases contain NPxY sorting signals interest (21, 22). Furthermore, it is increasingly recognized that and are therefore interesting candidates as PX-FERM-like cargo the cellular localization of Ras proteins at different organelles molecules (33). such as endosomes is critical for differential Ras-mediated signal- In summary, we have shown that SNX17, SNX27, and SNX31 ing (23). Here we identified a trio of PX proteins that are endo- belong to a subfamily of PX proteins, and our biochemical and some-associated and bind to H-Ras(GTP) via a unique FERM- structural studies present important details on the overall struc- like domain interaction. As H-Ras is highly similar to other ture of PX-FERM-like proteins and the mechanisms by which Ras-family members, it is not yet possible to link PX-FERM-like they engage biological ligands. This provides the basis for future proteins directly to a specific Ras protein functionally (30). It is studies aimed at dissecting the role of these proteins in endoso- interesting to speculate that other FERM domain proteins mal sorting, and the potential coupling of transport to down- outside of the PX family will have similar binding activity. This is stream signaling outcomes. supported by the identification of Krit1 as an effector of Rap1, to stabilize junctional integrity in epithelial cells (31). Individually Materials and Methods the Ras GTPase family and FERM domain-containing protein Detailed methods are given in SI Text. families play critical roles in many signaling cascades, so the demonstration of a direct association between these protein families Molecular Biology and Protein Expression Expression. cDNAs encoding human would have important implications for our understanding of these SNX17, SNX17-PX(1-110), mouse SNX27b (herein referred to as SNX27), processes. SNX27ΔPDZ(156-526), and mouse SNX31 were expressed in Escherichia coli The precise mechanism of action of the PX-FERM-like with N-terminal GST tags. SNX27, SNX27-PX(156-265), SNX17-PX, human proteins in endosomal trafficking and signaling still remains H-Ras(1-166), and H-Ras(G12V)(1-166) were expressed with N-terminal His tags. A synthetic gene encoding an R36Q mutant of the SNX17-PX domain BIOCHEMISTRY unknown. What is clear, though, is that these proteins are primed was synthesized by Genesearch and expressed with an N-terminal GST tag. to act as membrane and receptor interaction scaffolds and can Human SNX17 was cloned into pEGFP-N1 for expression in mammalian cells direct numerous molecular associations important for these pro- with a C-terminal GFP tag. SNX17(R36Q)-GFP was generated using the Quick cesses. Our results, in combination with previous studies, allow us change site-directed mutagenesis kit (Stratagene). Mouse SNX27 was cloned to propose a model for PX-FERM-like protein interactions at the into pEGFP-C1 with an N-terminal GFP tag for mammalian cell expression. endosomal membrane (Fig. 6). The proteins are directly coupled to endosomes via their PX domain, and it is likely that membrane Isothermal Titration Calorimetry. All ITC experiments were performed using a association is further stabilized by other interactions through co- Microcal iTC200 instrument. APP peptides at 3 mM were titrated into incidence detection (32). The proteins once localized will then 0.05 mM SNX17 and SNX27 proteins in 13 × 3.1 μL aliquots at 10 °C. Similarly, 13 × 3 1 μ engage with receptors containing NPxY sorting motifs for endo- PIP head groups were titrated at 0.5 mM into 0.02 mM proteins in . L somal to cell surface recycling, via binding to the PTB-related F3 aliquots at 25 °C. module. SNX27 will in addition engage a separate subset of trans- Δ ½ ∕ × ½ ∕ H-Ras-Binding Assay. GST-tagged SNX17, SNX17 PX domain, SNX27 PDZ, and membrane cargo containing type-I PDZ motifs (E S T F V ) SNX31 proteins were mixed with H-Ras(G12V) or H-Ras, bound to glutathione via the PDZ domain. Finally, the proteins can also associate with sepharose, washed, and then eluted in SDS-PAGE sample buffer. Western blot and recruit other molecules and cytosolic effectors, including Ras analysis was performed using mouse anti-Ras. For nucleotide dependency via the F1 module, and other PDZ motif and NPxY-containing experiments, H-Ras was loaded with GTP or GDP by adding a 5-fold molar soluble factors. Although Fig. 6 shows all known binding partners excess of EDTA and a 20-fold molar excess of GTP/GDP. Samples were incu-

Ghai et al. PNAS ∣ May 10, 2011 ∣ vol. 108 ∣ no. 19 ∣ 7767 Downloaded by guest on September 26, 2021 bated for 30 min at room temperature, reactions stopped with 100 mM Indirect Immunoflourescence. HeLa cells were transiently transfected with MgCl2, and proteins purified by gel filtration. SNX17-GFP or GFP-SNX27 constructs using LipofectAMINE (Invitrogen). Cells were fixed and permeabilized before staining with appropriate primary and SNX17 PX-Domain Crystal Structure Determination. SNX17 PX domain was secondary antibodies for 1–2 h at room temperature. Monolayers were cleaved from GST using tobacco etch virus (TEV) protease, purified further examined using a Zeiss LSM 510 META confocal fluorescent microscope. by gel filtration (in 20 mM Hepes (pH 7.5), 100 mM NaCl, 1 mM DTT), then 15 ∕ concentrated to mg mL. Crystals were grown by vapor diffusion in 0.1 M SAXS Data Collection and Modeling. SAXS data were collected at the Austra- Tris (pH 8.0), 1.8 mM LiSO4. Data were collected at the University of Queens- lian Synchrotron using four protein concentrations between 1.25 and land Remote Op Crystallisation and X-ray (UQ ROCX) diffraction facility and 8.0 mg∕mL. Scattering data were background-corrected, averaged, and integrated and scaled with d*TREK (34). The structure was solved by mole- scaled using SAXS15ID software. All further processing was carried out using cular replacement using PHASER (35) and automated model building with the ATSAS programme suite (version 2.3) (http://www.embl-hamburg.de/ ARP/wARP (36). Refinement was done with PHENIX (37). biosaxs/software.html). Ab initio models were calculated using GASBOR, NMR Spectroscopy. Samples of 15N∕13C-labeled or 15N-labeled SNX17 PX do- with a total of 20 independent simulations carried out for each protein, main were produced as described (38). The His tag was removed with TEV and averaged with DAMAVER. Rigid body modeling was performed using protease, then the protein was gel filtered into 10 mM Hepes (pH 7.0), BUNCH. 100 mM NaCl, 2 mM DTT, and 10 mM Li2SO4 as required, and concentrated to 0.8 mM. All spectra were collected at 298 K on a Bruker 900-MHz spectro- ACKNOWLEDGMENTS. We acknowledge support from the UQ ROCX facility meter with cryoprobe and Z-axis gradients. Triple resonance experiments and the Queensland NMR Network. Thanks to Kevin Jack and staff of the were performed in 10% D2O. Sequential backbone assignments were ob- Australian Synchrotron for assistance with SAXS data collection, Jasmine tained by recording 3D HNCACB, CBCA(CO)NH, HNCO, and HN(CA)CO spec- Davis for protein expression, Natalie Castro for cellular localization studies, tra. (H)CC(CO)NH-TOCSY and H(CC)(CO)NH-TOCSY experiments were used for Rodrigo Morales for peptide purification, and Kirill Alexandrov for the H-Ras side-chain assignments. All data were acquired using nonuniform sampling plasmid. We are grateful to David Owen, Andrew Whitten, and Kirill and processed using maximum entropy reconstruction (39, 40). For lipid bind- Alexandrov for critical reading. This work was supported by funds from ing experiments 15N HSQC spectra of SNX17 PX domain were recorded with the Australian Research Council (ARC) (DP0878608 and DP1095728) and increasing amounts of diC8-PI(3)P to a final molar ratio of 8∶1. Docking of the National Health and Medical Research Council (NHMRC) (511072). R.D.T. is PI(3)P into the SNX17 PX-domain structure was done with HADDOCK using supported by an NHMRC Senior Research Fellowship (511042). B.M.C. is restraints derived from NMR experiments, as described in SI Text. supported by an ARC Future Fellowship (FT100100027).

1. Cullen PJ (2008) Endosomal sorting and signalling: An emerging role for sorting nex- 20. Zheng B, et al. (2001) RGS-PX1, a GAP for GalphaS and sorting nexin in vesicular ins. Nat Rev Mol Cell Biol 9:574–582. trafficking. Science 294:1939–1942. 2. Seet LF, Hong W (2006) The Phox (PX) domain proteins and membrane traffic. Biochim 21. Scita G, Di Fiore PP (2010) The endocytic matrix. Nature 463:464–473. Biophys Acta 1761:878–896. 22. Sorkin A, von Zastrow M (2009) Endocytosis and signalling: Intertwining molecular 3. Burden JJ, Sun XM, Garcia AB, Soutar AK (2004) Sorting motifs in the intracellular do- networks. Nat Rev Mol Cell Biol 10:609–622. main of the low density lipoprotein receptor interact with a novel domain of sorting 23. Fehrenbacher N, Bar-Sagi D, Philips M (2009) Ras/MAPK signaling from endomem- nexin-17. J Biol Chem 279:16237–16245. branes. Mol Oncol 3:297–307. 4. Stockinger W, et al. (2002) The PX-domain protein SNX17 interacts with members of 24. Lu A, et al. (2009) A clathrin-dependent pathway leads to KRas signaling on late the LDL receptor family and modulates endocytosis of the LDL receptor. EMBO J endosomes en route to lysosomes. J Cell Biol 184:863–879. 21:4259–4267. 25. Chishti AH, et al. (1998) The FERM domain: A unique module involved in the linkage of – 5. van Kerkhof P, et al. (2005) Sorting nexin 17 facilitates LRP recycling in the early cytoplasmic proteins to the membrane. Trends Biochem Sci 23:281 282. endosome. EMBO J 24:2851–2861. 26. Bunney TD, et al. (2006) Structural and mechanistic insights into ras association – 6. Knauth P, et al. (2005) Functions of sorting nexin 17 domains and recognition motif for domains of phospholipase C epsilon. Mol Cell 21:495 507. P-selectin trafficking. J Mol Biol 347:813–825. 27. Radzimanowski J, et al. (2008) Structure of the intracellular domain of the amyloid – 7. Adachi H, Tsujimoto M (2010) Adaptor protein sorting nexin 17 interacts with the precursor protein in complex with Fe65-PTB2. EMBO Rep 9:1134 1140. scavenger receptor FEEL-1/stabilin-1 and modulates its expression on the cell surface. 28. Pylypenko O, Lundmark R, Rasmuson E, Carlsson SR, Rak A (2007) The PX-BAR – Biochim Biophys Acta 1803:553–563. membrane-remodeling unit of sorting nexin 9. EMBO J 26:4788 4800. 8. Czubayko M, Knauth P, Schluter T, Florian V, Bohnensack R (2006) Sorting nexin 17, a 29. Garcia-Alvarez B, et al. (2003) Structural determinants of integrin recognition by talin. Mol Cell 11:49–58. non-self-assembling and a PtdIns(3)P high class affinity protein, interacts with the cer- 30. Colicelli J (2004) Human RAS superfamily proteins and related GTPases. Sci STKE ebral cavernous malformation related protein KRIT1. Biochem Biophys Res Commun 2004:RE13. 345:1264–1272. 31. Francalanci F, et al. (2009) Structural and functional differences between KRIT1A and 9. Seog DH, Han J (2008) Sorting nexin 17 interacts directly with kinesin superfamily KRIT1B isoforms: a framework for understanding CCM pathogenesis. Exp Cell Res KIF1Bbeta protein. Korean J Physiol Pha 12:199–204. 315:285–303. 10. Lee J, et al. (2008) Adaptor protein sorting nexin 17 regulates amyloid precursor pro- 32. Di Paolo G, De Camilli P (2006) Phosphoinositides in cell regulation and membrane tein trafficking and processing in the early endosomes. J Biol Chem 283:11501–11508. dynamics. Nature 443:651–657. 11. Andersen OM, Willnow TE (2006) Lipoprotein receptors in Alzheimer’s disease. Trends 33. Smith MJ, Hardy WR, Murphy JM, Jones N, Pawson T (2006) Screening for PTB domain Neurosci 29:687–694. binding partners and ligand specificity using proteome-derived NPXY peptide arrays. 12. Bu G (2009) Apolipoprotein E and its receptors in Alzheimer’s disease: Pathways, Mol Cell Biol 26:8461–8474. pathogenesis and therapy. Nat Rev Neurosci 10:333–344. 34. Pflugrath JW (1999) The finer things in X-ray diffraction data collection. Acta Crystal- 13. Joubert L, et al. (2004) New sorting nexin (SNX27) and NHERF specifically interact logr D 55:1718–1725. with the 5-HT4a receptor splice variant: Roles in receptor targeting. J Cell Sci 35. McCoy AJ, et al. (2007) Phaser crystallographic software. J Appl Crystallogr 40:658–674. – 117:5367 5379. 36. Langer G, Cohen SX, Lamzin VS, Perrakis A (2008) Automated macromolecular model 14. Lunn ML, et al. (2007) A unique sorting nexin regulates trafficking of potassium building for X-ray crystallography using ARP/wARP version 7. Nat Protoc 3:1171–1179. – channels via a PDZ domain interaction. Nat Neurosci 10:1249 1259. 37. Adams PD, et al. (2010) PHENIX: A comprehensive Python-based system for macromo- 15. Lauffer BE, et al. (2010) SNX27 mediates PDZ-directed sorting from endosomes to the lecular structure solution. Acta Crystallogr D 66:213–221. – plasma membrane. J Cell Biol 190:565 574. 38. Marley J, Lu M, Bracken C (2001) A method for efficient isotopic labeling of recombi- 16. Rincon E, et al. (2007) Proteomics identification of sorting nexin 27 as a diacylglycerol nant proteins. J Biomol NMR 20:71–75. kinase zeta-associated protein: New diacylglycerol kinase roles in endocytic recycling. 39. Mobli M, Maciejewski MW, Gryk MR, Hoch JC (2007) Automatic maximum entropy Mol Cell Proteomics 6:1073–1087. spectral reconstruction in NMR. J Biomol NMR 39:133–139. 17. Cai L, Loo LS, Atlashkin V, Hanson BJ, Hong W (2011) Deficiency of sorting nexin 27 40. Mobli M, Maciejewski MW, Gryk MR, Hoch JC (2007) An automated tool for maximum (SNX27) leads to growth retardation and elevated levels of N-methyl-D-aspartate entropy reconstruction of biomolecular NMR spectra. Nat Methods 4:467–468. (NMDA) receptor 2C (NR2C). Mol Cell Biol 31:1734–1747. 41. Cole C, Barber JD, Barton GJ (2008) The Jpred 3 secondary structure prediction server. 18. MacNeil AJ, Mansour M, Pohajdak B (2007) Sorting nexin 27 interacts with the Nucleic Acids Res 36:W197–201. cytohesin associated scaffolding protein (CASP) in lymphocytes. Biochem Biophys 42. Gouet P, Courcelle E, Stuart DI, Metoz F (1999) ESPript: Analysis of multiple sequence Res Commun 359:848–853. alignments in PostScript. Bioinformatics 15:305–308. 19. Zheng B, et al. (2004) Regulation of epidermal growth factor receptor degradation by 43. Bravo J, et al. (2001) The crystal structure of the PX domain from p40(phox) bound to heterotrimeric Galphas protein. Mol Biol Cell 15:5538–5550. phosphatidylinositol 3-phosphate. Mol Cell 8:829–839.

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