Phox Homology Research Articles

Cyclophilin A Is Required for Angiotensin II–Induced p47phox Translocation to Caveolae in Vascular Smooth Muscle Cells

Angiotensin II (AngII) signal transduction in vascular smooth muscle cells (VSMC) is mediated by reactive oxygen species (ROS). Cyclophilin A (CyPA) is a ubiquitously expressed cytosolic protein that possesses peptidyl-prolyl cis-trans isomerase activity, scaffold function, and significantly enhances AngII-induced ROS production in VSMC. We hypothesized that CyPA regulates AngII-induced ROS generation by promoting translocation of NADPH oxidase cytosolic subunit p47phox to caveolae of the plasma membrane. Overexpression of CyPA in CyPA-deficient VSMC (CyPA(-/-)VSMC) significantly increased AngII-stimulated ROS production. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors (VAS2870 or diphenylene iodonium) significantly attenuated AngII-induced ROS production in CyPA and p47phox-overexpressing CyPA(-/-)VSMC. Cell fractionation and sucrose gradient analyses showed that AngII-induced p47phox plasma membrane translocation, specifically to the caveolae, was reduced in CyPA(-/-)VSMC compared with wild-type-VSMC. Immunofluorescence studies demonstrated that AngII increased p47phox and CyPA colocalization and translocation to the plasma membrane. In addition, immunoprecipitation of CyPA followed by immunoblotting of p47phox and actin showed that AngII increased CyPA and p47phox interaction. AngII-induced p47phox and actin cell cytoskeleton association was attenuated in CyPA(-/-)VSMC. Mechanistically, inhibition of p47phox phosphorylation and phox homology domain deletion attenuated CyPA and p47phox interaction. Finally, cyclosporine A and CyPA-peptidyl-prolyl cis-trans isomerase mutant, R55A, inhibited AngII-stimulated CyPA and p47phox association in VSMC, suggesting that peptidyl-prolyl cis-trans isomerase activity was required for their interaction. These findings provide the mechanism by which CyPA is an important regulator for AngII-induced ROS generation in VSMC through interaction with p47phox and cell cytoskeleton, which enhances the translocation of p47phox to caveolae.

Structure of Sorting Nexin 11 (SNX11) Reveals a Novel Extended Phox Homology (PX) Domain Critical for Inhibition of SNX10-induced Vacuolation

Sorting nexins are phox homology (PX) domain-containing proteins involved in diverse intracellular endosomal trafficking pathways. The PX domain binds to certain phosphatidylinositols and is recruited to vesicles rich in these lipids. The structure of the PX domain is highly conserved, containing a three-stranded β-sheet, followed by three α-helices. Here, we report the crystal structures of truncated human SNX11 (sorting nexin 11). The structures reveal that SNX11 contains a novel PX domain, hereby named the extended PX (PXe) domain, with two additional α-helices at the C terminus. We demonstrate that these α-helices are indispensible for the in vitro functions of SNX11. We propose that this PXe domain is present in SNX10 and is responsible for the vacuolation activity of SNX10. Thus, this novel PXe domain constitutes a structurally and functionally important PX domain subfamily.

SNX3 recruits to phagosomes and negatively regulates phagocytosis in dendritic cells

Phagocytes such as dendritic cells (DC) and macrophages employ phagocytosis to take up pathogenic bacteria into phagosomes, digest the bacteria and present the bacteria-derived peptide antigens to the adaptive immunity. Hence, efficient antigen presentation depends greatly on a well-regulated phagocytosis process. Lipids, particularly phosphoinositides, are critical components of the phagosomes. Phosphatidylinositol-3,4,5-triphosphate [PI(3,4,5)P3 ] is formed at the phagocytic cup, and as the phagosome seals off from the plasma membrane, rapid disappearance of PI(3,4,5)P3 is accompanied by high levels of phosphatidylinositol-3-phosphate (PI3P) formation. The sorting nexin (SNX) family consists of a diverse group of Phox-homology (PX) domain-containing cytoplasmic and membrane-associated proteins that are potential effectors of phosphoinositides. We hypothesized that SNX3, a small sorting nexin that contains a single PI3P lipid-binding PX domain as its only protein domain, localizes to phagosomes and regulates phagocytosis in DC. Our results show that SNX3 recruits to nascent phagosomes and silencing of SNX3 enhances phagocytic uptake of bacteria by DC. Furthermore, SNX3 competes with PI3P lipid-binding protein, early endosome antigen-1 (EEA1) recruiting to membranes. Our results indicate that SNX3 negatively regulates phagocytosis in DC possibly by modulating recruitment of essential PI3P lipid-binding proteins of the phagocytic pathways, such as EEA1, to phagosomal membranes.

Structural basis for endosomal trafficking of diverse transmembrane cargos by PX-FERM proteins.

Transit of proteins through the endosomal organelle following endocytosis is critical for regulating the homeostasis of cell-surface proteins and controlling signal transduction pathways. However, the mechanisms that control these membrane-transport processes are poorly understood. The Phox-homology (PX) domain-containing proteins sorting nexin (SNX) 17, SNX27, and SNX31 have emerged recently as key regulators of endosomal recycling and bind conserved Asn-Pro-Xaa-Tyr-sorting signals in transmembrane cargos via an atypical band, 4.1/ezrin/radixin/moesin (FERM) domain. Here we present the crystal structure of the SNX17 FERM domain bound to the sorting motif of the P-selectin adhesion protein, revealing both the architecture of the atypical FERM domain and the molecular basis for recognition of these essential sorting sequences. We further show that the PX-FERM proteins share a promiscuous ability to bind a wide array of putative cargo molecules, including receptor tyrosine kinases, and propose a model for their coordinated molecular interactions with membrane, cargo, and regulatory proteins.

Phosphatidylinositol 4-Phosphate 5-Kinase α Facilitates Toll-like Receptor 4-mediated Microglial Inflammation through Regulation of the Toll/Interleukin-1 Receptor Domain-containing Adaptor Protein (TIRAP) Location*

Phosphatidylinositol (PI) 4,5-bisphosphate (PIP(2)), generated by PI 4-phosphate 5-kinase (PIP5K), regulates many critical cellular events. PIP(2) is also known to mediate plasma membrane localization of the Toll/IL-1 receptor domain-containing adaptor protein (TIRAP), required for the MyD88-dependent Toll-like receptor (TLR) 4 signaling pathway. Microglia are the primary immune competent cells in brain tissue, and TLR4 is important for microglial activation. However, a functional role for PIP5K and PIP(2) in TLR4-dependent microglial activation remains unclear. Here, we knocked down PIP5Kα, a PIP5K isoform, in a BV2 microglial cell line using stable expression of lentiviral shRNA constructs or siRNA transfection. PIP5Kα knockdown significantly suppressed induction of inflammatory mediators, including IL-6, IL-1β, and nitric oxide, by lipopolysaccharide. PIP5Kα knockdown also attenuated signaling events downstream of TLR4 activation, including p38 MAPK and JNK phosphorylation, NF-κB p65 nuclear translocation, and IκB-α degradation. Complementation of the PIP5Kα knockdown cells with wild type but not kinase-dead PIP5Kα effectively restored the LPS-mediated inflammatory response. We found that PIP5Kα and TIRAP colocalized at the cell surface and interacted with each other, whereas kinase-dead PIP5Kα rendered TIRAP soluble. Furthermore, in LPS-stimulated control cells, plasma membrane PIP(2) increased and subsequently declined, and TIRAP underwent bi-directional translocation between the membrane and cytosol, which temporally correlated with the changes in PIP(2). In contrast, PIP5Kα knockdown that reduced PIP(2) levels disrupted TIRAP membrane targeting by LPS. Together, our results suggest that PIP5Kα promotes TLR4-associated microglial inflammation by mediating PIP(2)-dependent recruitment of TIRAP to the plasma membrane.

N-terminal domain of vacuolar SNARE Vam7p promotes trans -SNARE complex assembly

SNARE-dependent membrane fusion in eukaryotic cells requires that the heptad-repeat SNARE domains from R- and Q-SNAREs, anchored to apposed membranes, assemble into four-helix coiled-coil bundles. In addition to their SNARE and transmembrane domains, most SNAREs have N-terminal domains (N-domains), although their functions are unclear. The N-domain of the yeast vacuolar Qc-SNARE Vam7p is a binding partner for the homotypic fusion and vacuole protein sorting complex (a master regulator of vacuole fusion) and has Phox homology, providing a phosphatidylinositol 3-phosphate (PI3P)-specific membrane anchor. We now report that this Vam7p N-domain has yet another role, one that does not depend on its physical connection to the Vam7p SNARE domain. By attaching a transmembrane anchor to the C terminus of Vam7p to create Vam7tm, we bypass the requirement for the N-domain to anchor Vam7tm to reconstituted proteoliposomes. The N-domain of Vam7tm is indispensible for trans-SNARE complex assembly in SNARE-only reactions. Introducing Vam7(1-125)p as a separate recombinant protein suppresses the defect caused by N-domain deletion from Vam7tm, demonstrating that the function of this N-domain is not constrained to covalent attachment to Vam7p. The Vam7p N-domain catalyzes the docking of apposed membranes by promoting transinteractions between R- and Q-SNAREs. This function of the Vam7p N-domain depends on the presence of PI3P and its affinity for PI3P. Added N-domain can even promote SNARE complex assembly when Vam7 still bears its own N-domain.

Frank-ter Haar Syndrome Protein Tks4 Regulates Epidermal Growth Factor-dependent Cell Migration

Mutations in the SH3PXD2B gene coding for the Tks4 protein are responsible for the autosomal recessive Frank-ter Haar syndrome. Tks4, a substrate of Src tyrosine kinase, is implicated in the regulation of podosome formation. Here, we report a novel role for Tks4 in the EGF signaling pathway. In EGF-treated cells, Tks4 is tyrosine-phosphorylated and associated with the activated EGF receptor. This association is not direct but requires the presence of Src tyrosine kinase. In addition, treatment of cells with LY294002, an inhibitor of PI 3-kinase, or mutations of the PX domain reduces tyrosine phosphorylation and membrane translocation of Tks4. Furthermore, a PX domain mutant (R43W) Tks4 carrying a reported point mutation in a Frank-ter Haar syndrome patient showed aberrant intracellular expression and reduced phosphoinositide binding. Finally, silencing of Tks4 was shown to markedly inhibit HeLa cell migration in a Boyden chamber assay in response to EGF or serum. Our results therefore reveal a new function for Tks4 in the regulation of growth factor-dependent cell migration.

Associations between PXK and TYK2 polymorphisms and systemic lupus erythematosus: a meta-analysis

The aim of this study was to determine whether phox homology domain containing serine/threonine kinase (PXK) and tyrosine kinase 2 (TYK2) confer susceptibility to systemic lupus erythematosus (SLE). The authors conducted meta-analyses on associations between SLE susceptibility and the rs6445975 polymorphism of PXK and the rs2304256, rs12720270, rs280519, and rs1272036 polymorphisms of TYK2. A total of 13 separate comparisons studies were included in this meta-analysis. Meta-analysis identified an association between SLE and the 2 allele of the rs6445975 polymorphism in the overall population [odds ratio (OR)=1.151, 95% confidence interval (CI)=1.086-1.291, P=1.8E-06]. Stratification by ethnicity identified a significant association between this polymorphism and SLE in Europeans (OR=1.198, 95% CI=1.118-1.285, P=3.4E-07), but not in Asians. Meta-analysis identified a significant negative association between SLE and the 2 allele of the rs2304256 polymorphism in the overall population (OR=0.808, 95% CI=0.659-0.990, P=0.040), and a significant negative association was found in Europeans, but not in Asians. This meta-analysis shows that the rs6445975 polymorphism of PXK and the rs2304256 polymorphism of TYK2 are associated with the development of SLE in Europeans.

Atypical Membrane-embedded Phosphatidylinositol 3,4-Bisphosphate (PI(3,4)P2)-binding Site on p47phox Phox Homology (PX) Domain Revealed by NMR

The Phox homology (PX) domain is a functional module that targets membranes through specific interactions with phosphoinositides. The p47(phox) PX domain preferably binds phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)) and plays a pivotal role in the assembly of phagocyte NADPH oxidase. We describe the PI(3,4)P(2) binding mode of the p47(phox) PX domain as identified by a transferred cross-saturation experiment. The identified PI(3,4)P(2)-binding site, which includes the residues of helices α1 and α1' and the following loop up to the distorted left-handed PP(II) helix, is located at a unique position, as compared with the phosphoinositide-binding sites of all other PX domains characterized thus far. Mutational analyses corroborated the results of the transferred cross-saturation experiments. Moreover, experiments with intact cells demonstrated the importance of this unique binding site for the function of the NADPH oxidase. The low affinity and selectivity of the atypical phosphoinositide-binding site on the p47(phox) PX domain suggest that different types of phosphoinositides sequentially bind to the p47(phox) PX domain, allowing the regulation of the multiple events that characterize the assembly and activation of phagocyte NADPH oxidase.

Sorting Nexin 27 mediates PDZ‐directed trafficking of Zona‐Occludens (ZO)‐2 to the Tight Junction

Proteins of the sorting nexin (SNX) superfamily are characterized by the presence of a phox‐homology (PX) domain. SNX27 is unique in being the only sorting nexin to contain a PDZ domain. We used a proteomic approach to identify a novel interaction between SNX27 and Zona Occludens‐2 (ZO‐2, TJP2), a component of the epithelial tight junction. The SNX27‐ZO‐2 interaction requires the PDZ domain of SNX27 and the C‐terminal PDZ‐binding motif of ZO‐2. When tight junctions were perturbed by chelation of intracellular Ca2+, we observed a transient co‐localization of ZO‐1 and ZO‐2 on SNX27‐positive early endosomes. Decreasing SNX27 expression in mpkCCD monolayers caused a decrease in the rate of ZO‐2 mobility, an enhancement of junctional permeability to large solutes, and an increase in the transepithelial resistance of monolayers. In addition to identifying an important new binding partner of SNX27, these findings suggest a role for endocytic pathways in the intracellular trafficking of ZO‐2 and possibly other tight junction proteins. Our results also indicate the involvement of the SNX27‐ZO‐2 interaction in tight junction maintenance and function.

Tks5-dependent formation of circumferential podosomes mediates cell-cell fusion

Multinucleation of osteoclasts during osteoclastogenesis requires dynamic rearrangement of the plasma membrane and cytoskeleton, and this process involves numerous previously characterized factors. However, the mechanism underlying osteoclast fusion remains obscure. Live-imaging analysis of osteoclastogenesis revealed that the products of PI3-kinase are enriched at the sites of osteoclast fusion. Among the downstream molecules whose expression was screened, the expression of Tks5, an adaptor protein with the phox homology (PX) domain with multiple Src homology 3 domains, was induced during osteoclastogenesis. Tks5 was localized in the podosomes and fusing membranes of osteoclasts, and reducing its expression impaired both formation of circumferential podosomes and osteoclast fusion without altering osteoclast differentiation. In addition, the expression of a deletion mutant of the PX domain abrogated circumferential podosome formation as well as osteoclast fusion, suggesting that Tks5-dependent circumferential podosomes function as fusion machinery during osteoclastogenesis. As Tks5 is known to promote the formation of podosomes/invadopodia in transformed/cancer cells, we tested if these cells also have the potential to fuse with osteoclasts. Among the cells tested, B16F0 melanoma cells formed circumferential podosomes with Tks5 accumulation in the presence of RANKL, TGFβ and TNFα. Co-culture of B16F0 melanoma cells with osteoclasts in an inflammatory milieu promoted increased formation of melanoma-osteoclast hybrid cells. Our results revealed a previously unknown mechanism of regulation of both circumferential podosome formation and cell-cell fusion by Tks5.

The NOXO1β PX Domain Preferentially Targets PtdIns(4,5)P2 and PtdIns(3,4,5)P3

NOXO1β [NOXO1 (Nox organizer 1) β] is a cytosolic protein that, in conjunction with NOXA1 (Nox activator 1), regulates generation of reactive oxygen species by the NADPH oxidase 1 (Nox1) enzyme complex. NOXO1β is targeted to membranes through an N-terminal PX (phox homology) domain. We have used NMR spectroscopy to solve the structure of the NOXO1β PX domain and surface plasmon resonance (SPR) to assess phospholipid specificity. The solution structure of the NOXO1β PX domain shows greatest similarity to that of the phosphatidylinositol 3-kinase-C2α PX domain with regard to the positions and types of residues that are predicted to interact with phosphatidylinositol phosphate (PtdInsP) head groups. SPR experiments identify PtdIns(4,5)P2 and PtdIns(3,4,5)P3 as preferred targets of NOXO1β PX. These findings contrast with previous lipid overlay experiments showing strongest binding to monophosphorylated PtdInsP and phosphatidylserine. Our data suggest that localized membrane accumulation of PtdIns(4,5)P2 or PtdIns(3,4,5)P2 may serve to recruit NOXO1β and activate Nox1.

Insights into the PX (phox-homology) domain and SNX (sorting nexin) protein families: structures, functions and roles in disease

The mammalian genome encodes 49 proteins that possess a PX (phox-homology) domain, responsible for membrane attachment to organelles of the secretory and endocytic system via binding of phosphoinositide lipids. The PX domain proteins, most of which are classified as SNXs (sorting nexins), constitute an extremely diverse family of molecules that play varied roles in membrane trafficking, cell signalling, membrane remodelling and organelle motility. In the present review, we present an overview of the family, incorporating recent functional and structural insights, and propose an updated classification of the proteins into distinct subfamilies on the basis of these insights. Almost all PX domain proteins bind PtdIns3P and are recruited to early endosomal membranes. Although other specificities and localizations have been reported for a select few family members, the molecular basis for binding to other lipids is still not clear. The PX domain is also emerging as an important protein-protein interaction domain, binding endocytic and exocytic machinery, transmembrane proteins and many other molecules. A comprehensive survey of the molecular interactions governed by PX proteins highlights the functional diversity of the family as trafficking cargo adaptors and membrane-associated scaffolds regulating cell signalling. Finally, we examine the mounting evidence linking PX proteins to different disorders, in particular focusing on their emerging importance in both pathogen invasion and amyloid production in Alzheimer's disease.

Class III PI-3-kinase activates phospholipase D in an amino acid–sensing mTORC1 pathway

The rapamycin-sensitive mammalian target of rapamycin (mTOR) complex, mTORC1, regulates cell growth in response to mitogenic signals and amino acid availability. Phospholipase D (PLD) and its product, phosphatidic acid, have been established as mediators of mitogenic activation of mTORC1. In this study, we identify a novel role for PLD1 in an amino acid-sensing pathway. We find that amino acids activate PLD1 and that PLD1 is indispensable for amino acid activation of mTORC1. Activation of PLD1 by amino acids requires the class III phosphatidylinositol 3-kinase hVps34, which stimulates PLD1 activity through a functional interaction between phosphatidylinositol 3-phosphate and the Phox homology (PX) domain of PLD1. Furthermore, amino acids stimulate PLD1 translocation to the lysosomal region where mTORC1 activation occurs in an hVps34-dependent manner, and this translocation is necessary for mTORC1 activation. The PX domain is required for PLD1 translocation, mTORC1 activation, and cell size regulation. Finally, we show that the hVps34-PLD1 pathway acts independently of, and in parallel to, the Rag pathway in regulating amino acid activation of mTORC1.

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

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.

Expression of Sorting Nexin 18 (SNX18) Is Dynamically Regulated in Developing Spinal Motor Neurons

The sorting nexin (SNX) family proteins, which contain a Phox homology (PX) domain, play crucial roles in regulating the intracellular membrane trafficking of the endocytic pathway. The proper coordination of this pathway is important for axonal elongation; however, little is known about the expression and intracellular dynamics of the SNX members during the formation of the nervous system. Here the authors found that SNX18, which belongs to the Src-homology-3-PX-Bin/Amphiphysin/Rvs domain-containing SNX subfamily, was specifically expressed in differentiating motor neurons in the chick and mouse embryonic spinal cord. The expression of SNX18 in embryonic spinal motor neurons was transient and was downregulated as the neurons matured. The authors further demonstrated that the localization of EGFP-SNX18 in growth cones was dynamically regulated and accumulated especially at areas in contact with permissive substrates. These findings collectively suggest that SNX18 may play an active role in axonal elongation.

Backbone 1H, 15N, and 13C resonance assignments for the NOXO1β PX domain

NOXO1 (Nox Organizer 1) is a homolog of the NAPDH oxidase protein p47(phox). NADPH oxidases transfer electrons from NADPH to molecular oxygen, generating the superoxide anion. NOXO1 contains an N-terminal PX (phox homology) domain and is one of several PX domain-containing proteins found in the cytosolic subunits of the NADPH oxidase complex. These PX domains bind to membrane lipids and target the protein to membranes, recruiting other cytosolic components to the membrane bound components and aiding formation of a active enzyme complex. This recruitment represents a level of regulation of these oxidases. Here we report the backbone assignments of NOXO1β PX.

P47phox Phox Homology Domain Regulates Plasma Membrane but Not Phagosome Neutrophil NADPH Oxidase Activation

The assembly of cytosolic subunits p47(phox), p67(phox), and p40(phox) with flavocytochrome b(558) at the membrane is required for activating the neutrophil NADPH oxidase that generates superoxide for microbial killing. The p47(phox) subunit plays a critical role in oxidase assembly. Recent studies showed that the p47(phox) Phox homology (PX) domain mediates phosphoinositide binding in vitro and regulates phorbol ester-induced NADPH oxidase activity in a K562 myeloid cell model. Because the importance of the p47(phox) PX domain in neutrophils is unclear, we investigated its role using p47(phox) knock-out (KO) mouse neutrophils to express human p47(phox) and derivatives harboring R90A mutations in the PX domain that result in loss of phosphoinositide binding. Human p47(phox) proteins were expressed at levels similar to endogenous murine p47(phox), with the exception of a chronic granulomatous disease-associated R42Q mutant that was poorly expressed, and wild type human p47(phox) rescued p47(phox) KO mouse neutrophil NADPH oxidase activity. Plasma membrane NAPDH oxidase activity was reduced in neutrophils expressing p47(phox) with Arg(90) substitutions, with substantial effects on responses to either phorbol ester or formyl-Met-Leu-Phe and more modest effects to particulate stimuli. In contrast, p47(phox) Arg(90) mutants supported normal levels of intracellular NADPH oxidase activity during phagocytosis of a variety of particles and were recruited to phagosome membranes. This study defines a differential and agonist-dependent role of the p47(phox) PX domain for neutrophil NADPH oxidase activation.

NHE3 Activity Is Dependent on Direct Phosphoinositide Binding at the N Terminus of Its Intracellular Cytosolic Region

The small intestinal BB Na(+)/H(+) antiporter NHE3 accounts for the majority of intestinal sodium and water absorption. It is highly regulated with both postprandial inhibition and stimulation sequentially occurring. Phosphatidylinositide 4,5-bisphosphate (PI(4,5)P(2)) and phosphatidylinositide 3,4,5-trisphosphate (PI(3,4,5)P(3)) binding is involved with regulation of multiple transporters. We tested the hypothesis that phosphoinositides bind NHE3 under basal conditions and are necessary for its acute regulation. His(6) proteins were made from the NHE3 C-terminal region divided into four parts as follows: F1 (amino acids 475-589), F2 (amino acids 590-667), F3 (amino acids 668-747), and F4 (amino acids 748-832) and purified by a nickel column. Mutations were made in the F1 region of NHE3 and cloned in pet30a and pcDNA3.1 vectors. PI(4,5)P(2) and PI(3,4,5)P(3) bound only to the NHE3 F1 fusion protein (amino acids 475-589) on liposomal pulldown assays. Mutations were made in the putative lipid binding region of the F1 domain and studied for alterations in lipid binding and Na(+)/H(+) exchange as follows: Y501A/R503A/K505A; F509A/R511A/R512A; R511L/R512L; R520/FR527F; and R551L/R552L. Our results indicate the following. 1) The F1 domain of the NHE3 C terminus has phosphoinositide binding regions. 2) Mutations of these regions alter PI(4,5)P(2) and PI(3,4,5)P(3) binding and basal NHE3 activity. 3) The magnitude of serum stimulation of NHE3 correlates with PI(4,5)P(2) and PI(3,4,5)P(3) binding of NHE3. 4) Wortmannin inhibition of PI3K did not correlate with PI(4,5)P(2) or PI(3,4,5)P(3) binding of NHE3. Two functionally distinct phosphoinositide binding regions (Tyr(501)-Arg(512) and Arg(520)-Arg(552)) are present in the NHE3 F1 domain; both regions are important for serum stimulation, but they display differences in phosphoinositide binding, and the latter but not the former alters NHE3 surface expression.

SNX27 mediates PDZ-directed sorting from endosomes to the plasma membrane

Postsynaptic density 95/discs large/zonus occludens-1 (PDZ) domain-interacting motifs, in addition to their well-established roles in protein scaffolding at the cell surface, are proposed to act as cis-acting determinants directing the molecular sorting of transmembrane cargo from endosomes to the plasma membrane. This hypothesis requires the existence of a specific trans-acting PDZ protein that mediates the proposed sorting operation in the endosome membrane. Here, we show that sorting nexin 27 (SNX27) is required for efficient PDZ-directed recycling of the beta(2)-adrenoreceptor (beta(2)AR) from early endosomes. SNX27 mediates this sorting function when expressed at endogenous levels, and its recycling activity requires both PDZ domain-dependent recognition of the beta(2)AR cytoplasmic tail and Phox homology (PX) domain-dependent association with the endosome membrane. These results identify a discrete role of SNX27 in PDZ-directed recycling of a physiologically important signaling receptor, and extend the concept of cargo-specific molecular sorting in the recycling pathway.