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.

The self-association of two N-terminal interaction domains plays an important role in the tetramerization of TRPC4.

Transient receptor potential canonical (TRPC) channels function as cation channels. In a previous study, we identified the molecular determinants involved in promoting TRPC subunit assembly. In the present study, we used size-exclusion chromatography assays to show that the N-terminus of TRPC4 can self-associate and form a tetramer in cellulo. We further showed that the N-terminus of TRPC4 self-associates via the ankyrin repeat domain and the region downstream from the coiled-coil domain. GST pull-down, yeast two-hybrid, and circular dichroism approaches demonstrated that both domains can self-associate. These findings indicated that the self-association of two distinct domains in the N-terminus of TRPC4 is involved in the assembly of the tetrameric channel.

Identification of two domains involved in the assembly of transient receptor potential canonical channels.

Transient receptor potential canonical (TRPC) channels are associated with calcium entry activity in nonexcitable cells. TRPCs can form homo- or heterotetrameric channels, in which case they can assemble together within a subfamily groups. TRPC1, 4, and 5 represent one group, and TRPC3, 6, and 7 represent the other. The molecular determinants involved in promoting subunit tetramerization are not known. To identify them, we generated chimeras by swapping the different domains of TRPC4 with the same regions in TRPC6. We showed that TRPC4 coimmunoprecipitated with the chimeras containing the ankyrin repeats and coiled-coil domains of TRPC4 into TRPC6. However, chimeras containing only the ankyrin repeats or only the coiled-coil domain of TRPC4 did not coimmunoprecipitate with TRPC4. We also showed that a second domain of interaction composed of the pore region and the C-terminal tail is involved in the oligomerization of TRPC4. However, chimeras containing only the pore region or only the C-terminal tail of TRPC4 did not coimmunoprecipitate with TRPC4. Furthermore, we showed that the N terminus of TRPC6 coimmunoprecipitated with the C terminus of TRPC6. Overexpression in HEK293T cells of chimeras that contained an N terminus and a C terminus from different subfamily groups increased intracellular calcium entry subsequent to stimulation of G(q) protein-coupled receptors. These results suggest that two types of interactions are involved in the assembly of the four subunits of the TRPC channel. The first interaction occurs between the N termini and involves two regions. The second interaction occurs between the N terminus and the C terminus and does not appear to be necessary for the activity of TRPCs.

MxA, a member of the dynamin superfamily, interacts with the ankyrin-like repeat domain of TRPC.

Mammalian transient receptor potential canonical channels have been proposed as the molecular entities associated with calcium entry activity in nonexcitable cells. Amino acid sequence analyses of TRPCs revealed the presence of ankyrin-like repeat domains, one of the most common protein-protein interaction motifs. Using a yeast two-hybrid interaction assay, we found that the second ankyrin-like repeat domain of TRPC6 interacted with MxA, a member of the dynamin superfamily. Using a GST pull-down and co-immunoprecipitation assay, we showed that MxA interacted with TRPC1, -3, -4, -5, -6, and -7. Overexpression of MxA in HEK293T cells slightly increased endogenous calcium entry subsequent to stimulation of G(q) protein-coupled receptors or store depletion by thapsigargin. Co-expression of MxA with TRPC6 enhanced agonist-induced or OAG-induced calcium entry activity. GTP binding-defective MxA mutants had only a minor potentiating effect on OAG-induced TRPC6 activity. However, a MxA mutant that could bind GTP but that lacked GTPase activity produced the same effect as MxA on OAG-induced TRPC6 activity. These results indicated that MxA interacted specifically with the second ankyrin-like repeat domain of TRPCs and suggested that monomeric MxA regulated the activity of TRPC6 by a mechanism requiring GTP binding. Additional results showed that an increase in the endogenous expression of MxA, induced by a treatment with interferon alpha, regulated the activity of TRPC6. The study clearly identified MxA as a new regulatory protein involved in Ca2+ signaling.