Identification and biochemical characterization of the SLC9A7 interactome.
Organellar and cytosolic pH homeostasis is central to most cellular processes, including vesicular trafficking, post-translational modification/processing of proteins, and receptor-ligand interactions. SLC9A7 (NHE7) was identified as a unique (Na+, K+)/H+ exchanger that dynamically cycles between the trans-Golgi network (TGN), endosomes and the plasma membrane. Here we have used mass spectrometry to explore the affinity-captured interactome of NHE7, leading to the identification of cytoskeletal proteins, cell adhesion molecules, membrane transporters, and signaling molecules. Among these binding proteins, calcium-calmodulin, but not apo-calmodulin, binds to NHE7 and regulates the organellar transporter activity. Vimentin was co-immunoprecipitated with endogenous NHE7 protein in human breast cancer MDA-MB-231 cells. A sizable population of NHE7 relocalized to focal complexes in migrating cells and showed colocalization with vimentin and actin in focal complexes. Among the NHE7-binding proteins identified, CD44, a cell surface glycoprotein receptor for hyaluronate and other ligands, showed regulated interaction with NHE7. Pretreatment of the cells with phorbol ester facilitated the NHE7-CD44 interaction and the lipid raft association of CD44. When lipid rafts were chemically disrupted, the NHE7-CD44 interaction was markedly reduced. These results suggest potential dual roles of NHE7 in intracellular compartments and subdomains of cell-surface membranes.
Kagami
Takashi
T
Department of Biochemistry and Molecular Biology, The University of British Columbia, Life Sciences Centre, Vancouver, British Columbia, Canada.
Chen
Sam
S
Memar
Pouya
P
Choi
Matthew
M
Foster
Leonard J
LJ
Numata
Masayuki
M
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism; D011992:Endosomes / Q000235:genetics / Q000378:metabolism; D006056:Golgi Apparatus / Q000235:genetics / Q000378:metabolism; D006706:Homeostasis / Q000502:physiology*; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism*; D006820:Hyaluronic Acid / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D021962:Membrane Microdomains / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*
C497483:CD44 protein, human; D002147:Calmodulin; D003598:Cytoskeletal Proteins; D018960:Hyaluronan Receptors; D008024:Ligands; C429538:SLC9A7 protein, human; D017923:Sodium-Hydrogen Exchangers; D006820:Hyaluronic Acid; D002118:Calcium