MxA, a Member of the Dynamin Superfamily, Interacts with the Ankyrin-like Repeat Domain of TRPC

Abstract

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 ␣, regulated the activity of TRPC6. The study clearly identified MxA as a new regulatory protein involved in Ca 2؉ signaling.[ Mammalian members of the TRPC (transient receptor potential canonical) subfamily (TRPC1 to -7) are calcium-permeable cation channels involved in the mechanism of [Ca 2ϩ ] i elevation in cells stimulated with G q -coupled receptors or tyrosine kinase receptor agonists (3, 4). Functional TRPC complexes are presumed to be homo-as well as heterotetramers, and each TRPC subunit contains typical domains that probably interact with other proteins. There are growing efforts to identify proteins that interact with TRPCs and to clarify the role of these interactions in the regulation of the Ca 2ϩ entry. The IP 3 receptor, calmodulin, HOMER, INAD, NHERF, the a-isoform of the inhibitor of the myogenic family (I-mfa), and stathmin 2 (5-17) all interact with TRPCs. A typical TRPC subunit is composed of six membrane-spanning domains and a hydrophobic segment between the fifth and sixth transmembrane domains that forms the putative pore region loop. Cytosolic N terminus and C terminus tails flank the six transmembrane domains. Each TRPC subunit possesses two coiled-coil regions (one in the N terminus and the other in the C terminus) and a hyperconserved region composed of three to four ankyrin-like repeat domains (ARDs) in the N terminus (18).ARDs are found in more than 4,000 different proteins, making one of the most common motifs involved in protein-protein interactions. The 33 amino acids of ARDs form two ␣-helices linked by a ␤-hairpin/loop (19,20). When ARDs are involved in prote...

A New Role for PTEN in Regulating Transient Receptor Potential Canonical Channel 6-mediated Ca2+ Entry, Endothelial Permeability, and Angiogenesis*

“…Exocytosis of the TRPC6 channel to the cell surface is a known requirement for channel activity (65). We showed that knockdown of PTEN impaired TRPC6 localization at the cell membrane.…”

Organization and function of TRPC channelosomes

“…MxA [54] MxA [54] MxA [54] MxA [54] MxA [54] MxA [54] MxA [54] RhoA [57] RhoA RhoA SNARES [89] SNARES [95] SNARES [8,26] Only proteins common to 2 or more TRPCs have been listed. Components involved in Ca 2+ signaling are shown in the top half of the PMCA [38,39,50] Caveolin [1,13,51] STIM1 [32,52,72] SERCA [50,38,39] proteins involved in vesicle trafficking, cytoskeletal interactions, and scaffolding (see Table 2).…”

“…MxA, a member of the dynamin superfamily [54], and this interaction is most likely involved in trafficking of the channel. However, the exact components of the TRPC3 channelosome that are involved in constitutive and regulated trafficking of TRPC3, as well as its ability to generate SOC channels, have not yet been resolved.…”

Sipl1 and Rbck1 Are Novel Eya1-Binding Proteins with a Role in Craniofacial Development

“…Colonies were analyzed by quantitative ␤-galactosidase (␤-Gal) liquid assay, according to the Clontech protocol. Expression of the constructs used for the ␤-Gal liquid assay was confirmed by yeast protein extraction as described previously (27).…”