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. 2011 May 10;108(19):7763-8.
doi: 10.1073/pnas.1017110108. Epub 2011 Apr 21.

Phox homology band 4.1/ezrin/radixin/moesin-like proteins function as molecular scaffolds that interact with cargo receptors and Ras GTPases

Rajesh Ghai  1 , Mehdi Mobli, Suzanne J Norwood, Andrea Bugarcic, Rohan D Teasdale, Glenn F King, Brett M Collins
Affiliations

Phox homology band 4.1/ezrin/radixin/moesin-like proteins function as molecular scaffolds that interact with cargo receptors and Ras GTPases

Rajesh Ghai et al. Proc Natl Acad Sci U S A. .

Abstract

Following endocytosis, the fates of receptors, channels, and other transmembrane proteins are decided via specific endosomal sorting pathways, including recycling to the cell surface for continued activity. Two distinct phox-homology (PX)-domain-containing proteins, sorting nexin (SNX) 17 and SNX27, are critical regulators of recycling from endosomes to the cell surface. In this study we demonstrate that SNX17, SNX27, and SNX31 all possess a novel 4.1/ezrin/radixin/moesin (FERM)-like domain. SNX17 has been shown to bind to Asn-Pro-Xaa-Tyr (NPxY) sequences in the cytoplasmic tails of cargo such as LDL receptors and the amyloid precursor protein, and we find that both SNX17 and SNX27 display similar affinities for NPxY sorting motifs, suggesting conserved functions in endosomal recycling. Furthermore, we show for the first time that all three proteins are able to bind the Ras GTPase through their FERM-like domains. These interactions place the PX-FERM-like proteins at a hub of endosomal sorting and signaling processes. Studies of the SNX17 PX domain coupled with cellular localization experiments reveal the mechanistic basis for endosomal localization of the PX-FERM-like proteins, and structures of SNX17 and SNX27 determined by small angle X-ray scattering show that they adopt non-self-assembling, modular structures in solution. In summary, this work defines a novel family of proteins that participate in a network of interactions that will impact on both endosomal protein trafficking and compartment specific Ras signaling cascades.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
SNX17, SNX31, and SNX27 share a conserved C-terminal FERM-like domain. Sequence alignment of SNX17, SNX27, and SNX31. Secondary structure predictions calculated with JPRED (41) are indicated for SNX17 (above) and SNX27 (below). Alignment was made with ESPript 2.2 (42). Identified subdomains are indicated by boxes. Red triangles indicate key residues involved in PI(3)P binding.
Fig. 2.
Fig. 2.
Binding of NPxY cargo motifs is conserved across the PX-FERM-like protein family. Binding of SNX17 (A) and SNX27 (B) to a peptide derived from APP was measured using ITC. Experiments were performed at 10 °C with 50 μM protein and 2,000 μM APP peptide. Also shown are the titrations of SNX17 and SNX27 with the APP Y*A mutant peptide, which does not bind. (Top) Raw data; (Bottom) integrated normalized data.
Fig. 3.
Fig. 3.
PX-FERM-like proteins are effectors of the Ras GTPase. (A) PX-FERM-like proteins were tested for H-Ras(G12V) binding in GST pulldown assays. (Top) Coomassie-stained gel of the pulldown samples; (Lower) Western blot with anti-Ras antibody. Note the SNX27 construct lacks the N-terminal PDZ domain. A minimal SNX17-FERM-like domain associates with H-Ras similarly to the full-length PX-FERM-like proteins. (B) The GTP dependence of the interaction was confirmed by loading H-Ras with either GDP or GTP and performing GST-pulldown assays as in A.
Fig. 4.
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 PI(3,5)P2, 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) as a 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) displays clear overlap with the bound formula image ion in the crystal structure (yellow) and PI(3)P from the SNX9∶PI(3)P complex (white).
Fig. 5.
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 subtracting 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 (qmax × Rg < 1.3). (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 envelopes 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.
Fig. 6.
Fig. 6.
A model for the network of interactions driven by the PX-FERM-like proteins. H-Ras (purple ribbons), NPxY peptide (gold cylinders), and Type-I PDZ-binding peptides (gold cylinders) were modeled onto the SNX27 SAXS-derived structure following structural alignment of respective complexes with PLC RA domain, FE65 PTB domain, and syntenin PDZ domain. The bound PI(3)P (spheres) is shown from our docking studies (Fig. 4E). The PX domain binds to PI(3)P, and the similarity of the F3 module of the FERM domain suggests it will be responsible for binding membrane and cytoplasmic NPxY-containing proteins. The similarity of the F1 module to RA domains suggests this region governs interaction with Ras. SNX27 possesses an additional N-terminal PDZ domain that binds cytosolic and transmembrane proteins via C-terminal type-I PDZ binding motifs, E[S/T] × [F/V]. Note that the FERM-like F2 module is expected to differ from the canonical F2 module depicted.
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