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Activated Chloride Channel Stoichiometrically Interacts with an Ezrin–Radixin–Moesin Network

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Activated Chloride Channel Stoichiometrically Interacts with an Ezrin–Radixin–Moesin Network Anoctamin 1 (Tmem16A) Ca2+-activated chloride channel stoichiometrically interacts with an ezrin–radixin–moesin network Patricia Perez-Cornejoa,1, Avanti Gokhaleb,1, Charity Duranb,1, Yuanyuan Cuib, Qinghuan Xiaob, H. Criss Hartzellb,2, and Victor Faundezb,2 aPhysiology Department, School of Medicine, Universidad Autónoma de San Luis Potosí, San Luis Potosí, SLP 78210, Mexico; and bDepartment of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322 Edited by David E. Clapham, Howard Hughes Medical Institute, Children’s Hospital Boston, Boston, MA, and approved May 9, 2012 (received for review January 4, 2012) The newly discovered Ca2+-activated Cl− channel (CaCC), Anocta- approach to identify Ano1-interacting proteins. We find that min 1 (Ano1 or TMEM16A), has been implicated in vital physiolog- Ano1 forms a complex with two high stochiometry interactomes. ical functions including epithelial fluid secretion, gut motility, and One protein network is centered on the signaling/scaffolding smooth muscle tone. Overexpression of Ano1 in HEK cells or Xen- actin-binding regulatory proteins ezrin, radixin, moesin, and opus oocytes is sufficient to generate Ca2+-activated Cl− currents, RhoA. The ezrin–radixin–moesin (ERM) proteins organize the but the details of channel composition and the regulatory factors cortical cytoskeleton by linking actin to the plasma membrane that control channel biology are incompletely understood. We and coordinate cell signaling events by scaffolding signaling used a highly sensitive quantitative SILAC proteomics approach molecules (19). The other major interactome is centered on the to obtain insights into stoichiometric protein networks associated SNARE and SM proteins VAMP3, syntaxins 2 and -4, and the with the Ano1 channel. These studies provide a comprehensive syntaxin-binding proteins munc18b and munc18c. This complex footprint of putative Ano1 regulatory networks. We find that is involved in docking and translocation of vesicles to the plasma Ano1 associates with the signaling/scaffolding proteins ezrin, rad- membrane (20). These studies provide a comprehensive foot- ixin, moesin, and RhoA, which link the plasma membrane to the print of putative Ano1 regulatory networks encompassing 2+ cytoskeleton with very high stoichiometry. Ano1, ezrin, and moe- a spectrum from Ca sensors to actin cytokeleton scaffolding sin/radixin colocalize apically in salivary gland epithelial cells, and networks and suggest mechanisms for the polarized localization overexpression of moesin and Ano1 in HEK cells alters the sub- of Ano1 in epithelial cells. cellular localization of both proteins. Moreover, interfering RNA Results for moesin modifies Ano1 current without affecting its surface expression level. Another network associated with Ano1 includes To characterize the Ano1 interactome, we developed a method the SNARE and SM proteins VAMP3, syntaxins 2 and -4, and syn- for high-level purification of Ano1 with specifically interacting taxin-binding proteins munc18b and munc18c, which are integral proteins while minimizing nonspecific protein associations. This to translocation of vesicles to the plasma membrane. A number of was accomplished using immunoaffinity chromatography of the other regulatory proteins, including GTPases, Ca2+-binding pro- Ano1 complex, which was stabilized with the cross-linker DSP teins, kinases, and lipid-interacting proteins are enriched in the [dithiobis(succinimidyl propionate)]. We have previously vali- Ano1 complex. These data provide stoichiometrically prioritized dated the robustness and specificity of this method using genetic information about mechanisms regulating Ano1 function and tools (21). We constructed a HEK-293 cell line stably expressing trafficking to polarized domains of the plasma membrane. Ano1 tagged on its C terminus with three FLAG epitopes – × 2+ (Ano1 FLAG3 ). The cell line− exhibited robust Ca -de- calcium | interactome | cross-linker | apical targeting pendent, outwardly rectifying Cl selective Ano1 currents (>200 pA/pF at +100 mV with 1 μM intracellular Ca2+). The cells were − a2+-activated Cl channels (CaCCs) play critical roles in treated with the cell-permeant, amino-reactive, homobifunc- tional cross-linker DSP to stabilize low-affinity protein–protein Cepithelial secretion, sensory transduction and adaptation, fi regulation of smooth muscle contraction, control of neuronal interactions. DSP has a 12-Å spacer arm and a disul de bond, and cardiac excitability, and nociception (1, 2). In 2008, after which is cleaved with reducing agents to release cross-linked many years of controversy about the molecular identity of proteins (22, 23). Effective cross-linking was demonstrated by – CaCCs, Ano1 and Ano2 (also called Tmem16A and Tmem16B) a moderate increase in the sedimentation velocity of Ano1 of the Anoctamin superfamily were identified as essential sub- FLAG3× protein complexes in sucrose gradients (Fig. S1). units of CaCCs (3–5). However, the regulatory networks that Cross-linking was limited to closely neighboring proteins and did control Ano1 function remain largely unexplored. not result in large protein aggregates, because the sedimentation The physiological significance of Ano1 cannot be understated. profile of total protein visualized by silver stain was not signifi- Ano1 is widely expressed in epithelia including salivary gland, cantly altered by the DSP treatment (Fig. S1) (22, 24). pancreas, gut, mammary gland, and airway. Disruption of the − Ano1 gene in mice eliminates Ca2+-dependent Cl secretion in several epithelial tissues including salivary gland (3, 5–9). Ano1 Author contributions: P.P.-C., A.G., C.D., Y.C., H.C.H., and V.F. designed research; P.P.-C., also performs important functions in nonepithelial tissues in- A.G., C.D., Y.C., Q.X., and V.F. performed research; P.P.-C., A.G., C.D., Y.C., Q.X., H.C.H., cluding pacemaker activity in the gut and regulation of vascular and V.F. analyzed data; and P.P.-C., A.G., C.D., H.C.H., and V.F. wrote the paper. and airway smooth muscle tone. Ano1 has also attracted the The authors declare no conflict of interest. interest of cancer biologists because the gene is amplified in oral, This article is a PNAS Direct Submission. head, and neck squamous cell carcinomas, and its expression 1P.P-C., A.G., and C.D. contributed equally to this work. level may correlate with cell proliferation (see recent reviews, 2To whom correspondence may be addressed. E-mail: [email protected] or refs. 10–18). [email protected]. To identify potential accessory subunits and/or regulatory pro- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tein networks, we used a highly sensitive quantitative proteomic 1073/pnas.1200174109/-/DCSupplemental. 10376–10381 | PNAS | June 26, 2012 | vol. 109 | no. 26 www.pnas.org/cgi/doi/10.1073/pnas.1200174109 Downloaded by guest on September 27, 2021 The cross-linked Ano1 protein complex was captured by in- cubating the cell lysate with anti-FLAG antibody-coated magnetic beads. The material that bound to the anti-FLAG beads consisted of a spectrum of bands on silver-stained gels (Fig. 1, lane 3). The prominent 130-kDa and 260-kDa bands correspond to Ano1 monomers and dimers as shown by Western blot (lane 3′). The predicted molecular mass of Ano1–FLAG3× is 113 kDa, but the glycosylated monomers and dimers migrate as diffuse bands at 130 kDa and 260 kDa (25). Many of the other bound proteins were nonspecific, because they bound to magnetic beads lacking anti- FLAG (lane 1), to beads coated with an irrelevant antibody (lane 2), or to beads coated with anti-FLAG in the presence of excess competing FLAG3× peptide (lane 4). Under these control con- ditions, Ano1–FLAG3× did not bind (lanes 1′,2′,and4′). The bound Ano1–FLAG3× complex was separated from non- specifically bound proteins by elution from the anti-FLAG beads with excess FLAG3× peptide. The eluted material was excep- tionally clean on silver-stained gels, consisting predominantly of the 130-kDa and 260-kDa Ano1 bands (lanes 6 and 6′). To analyze Ano1 interacting proteins quantitatively, we cou- pled the immunoaffinity purification just described with stable isotope labeling with amino acids in culture (SILAC) (Fig. 2). Untransfected HEK cells were grown in medium containing ar- 12 14 ginine and lysine with “light” C and N, (R0K0), whereas the Ano1–FLAG3× stable cells were grown in medium containing 13 15 “heavy” C- and N-labeled arginine and lysine (R10K8) for more than six passages to ensure equilibrium labeling of the proteome (97.5% R10K8 saturation). The cross-linked Ano1 Fig. 2. Summary of SILAC experiment. Untransfected HEK cells were in- “ ” complex purified by immunoaffinity chromatography (Fig. S2B) cubated in isotopically light (R0K0) DMEM. HEK cells stably transfected – × “ ” was analyzed by nano-LC MS/MS. A total of 509 proteins were with Ano1 FLAG3 were incubated in isotopically heavy (R10K8) medium. identified at a 1% false discovery rate. Of these, there was suf- Cells were substoichiometrically cross-linked using DSP. Lysates were ficient signal to quantify SILAC enrichment for 392 (Fig. 2). The immunoprecipitated using magnetic beads decorated with FLAG antibody. list was refined to 209 by curation against a database of poly- Ano1 supramolecular complexes were eluted with FLAG3× peptide. Samples peptides that nonselectively bind to anti-FLAG beads from were combined at a 1:1 ratio and analyzed by nano-LS MS/MS. Peptides – enriched more than twofold with the R10K8 amino acids were considered as untransfected DSP cross-linked cell extracts (26 28). A total of fi 93 of these proteins were enriched more than twofold and 73 potential Ano1 interactors. The list of proteins was re ned by curation fi against a list of peptides that nonspecifically bind to the immunomagnetic were enriched more than vefold in the Ano1-expressing cells beads. The Venn diagram shows the number of peptides identified in the CELL BIOLOGY (Fig. 2 and Dataset S1). A twofold SILAC enrichment is experiment. A total of 509 proteins were identified, 93 of which were a stringent cutoff criterion to reliably detect differences between enriched more than twofold in R10K8 and did not bind nonspecifically to two samples (26, 28, 29).
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