The PDZ-interacting domain of TRPC4 controls its localization and surface expression in HEK293 cells.
Mammalian homologs of the Drosophila TRP protein have been shown to form cation-permeable channels in the plasma membrane but very little is known about the mechanisms that control their cell surface localization. Recently it has been demonstrated that the last three C-terminal amino acids (TRL) of TRPC4 comprise a PDZ-interacting domain that binds to the scaffold protein EBP50 [ezrin/moesin/radixin-binding phosphoprotein 50]. In this report, we have examined the influence of the TRL motif on the subcellular distribution of TRPC4 in human embryonic kidney (HEK) 293 cells. We have also analyzed the consequences of the interaction between EBP50 and the membrane-cytoskeletal adaptors of the ezrin/radixin/moesin (ERM) family for the cell surface expression of TRPC4. Using immunofluorescence microscopy, we found that the mutant lacking the TRL motif accumulated into cell outgrowths and exhibited a punctate distribution pattern whereas the wild-type channel was evenly distributed on the cell surface. Deletion of the PDZ-interacting domain also decreased the expression of TRPC4 in the plasma membrane by 2.4-fold, as assessed by cell surface biotinylation experiments. Finally, in a large percentage of cells co-expressing TRPC4 and an EBP50 mutant lacking the ERM-binding site, TRPC4 was not present in the plasma membrane but co-localized with the truncated scaffold in a perinuclear compartment (most probably representing the Golgi apparatus) and in vesicles associated with actin filaments. Our data demonstrate that the PDZ-interacting domain of TRPC4 controls its localization and surface expression in transfected HEK293 cells. They also point to a yet unexplored role of the EBP50-ERM complex in the regulation of protein insertion into the plasma membrane.
Mery
Laurence
L
Department of Physiology, University of Saarland, D-66421 Homburg, Germany.
Strauss
Bettina
B
Dufour
Jean F
JF
Krause
Karl H
KH
Hoth
Markus
M
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers; D050052:TRPC Cation Channels
D015220:Calcium Channels; D002352:Carrier Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D017923:Sodium-Hydrogen Exchangers; D050052:TRPC Cation Channels; C093376:sodium-hydrogen exchanger regulatory factor