RNF24, a new TRPC interacting protein, causes the intracellular retention of TRPC.
TRPCs function as cation channels in non-excitable cells. The N-terminal tails of all TRPCs contain an ankyrin-like repeat domain, one of the most common protein-protein interaction motifs. Using a yeast two-hybrid screening approach, we found that RNF24, a new membrane RING-H2 protein, interacted with the ankyrin-like repeat domain of TRPC6. GST pull-down and co-immunoprecipitation assays showed that RNF24 interacted with all TRPCs. Cell surface-labelling assays showed that the expression of TRPC6 at the surface of HEK 293T cells was greatly reduced when it was transiently co-transfected with RNF24. Confocal microscopy showed that TRPC3 and TRPC6 co-localized with RNF24 in a perinuclear compartment and that RNF24 co-localized with mannosidase II, a marker of the Golgi cisternae. Using a pulse-chase approach, we showed that RNF24 did not alter the maturation process of TRPC6. Moreover, in HEK 293T cells, RNF24 did not alter carbachol-induced Ca(2+) entry via endogenous channels or TRPC6. These results indicate that RNF24 interacts with TRPCs in the Golgi apparatus and affects TRPC intracellular trafficking without affecting their activity.
Lussier
Marc P
MP
Department of Pharmacology, Université de Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4.
Lepage
Pascale K
PK
Bousquet
Simon M
SM
Boulay
Guylain
G
D000595:Amino Acid Sequence; D017089:Ankyrin Repeat; D002217:Carbachol / Q000494:pharmacology; D002352:Carrier Proteins / Q000032:analysis / Q000737:chemistry / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006056:Golgi Apparatus / Q000737:chemistry; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry / Q000502:physiology*; D008969:Molecular Sequence Data; D050052:TRPC Cation Channels / Q000737:chemistry / Q000378:metabolism*; D000074287:TRPC6 Cation Channel; D044767:Ubiquitin-Protein Ligases
D002352:Carrier Proteins; D008565:Membrane Proteins; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; D002217:Carbachol; C530470:RNF24 protein, human; D044767:Ubiquitin-Protein Ligases