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Abstract
Filamins are important actin cross-linking proteins implicated in scaffolding, membrane stabilization and signal transduction, through interaction with ion channels, receptors and signaling proteins. Here we report the physical and functional interaction between filamins and polycystin-2, a TRP-type cation channel mutated in 10–15% patients with autosomal dominant polycystic kidney disease. Yeast two-hybrid and GST pull-down experiments demonstrated that the C-termini of filamin isoforms A, B and C directly bind to both the intracellular N- and C-termini of polycystin-2. Reciprocal co-immunoprecipitation experiments showed that endogenous polycystin-2 and filamins are in the same complexes in renal epithelial cells and human melanoma A7 cells. We then examined the effect of filamin on polycystin-2 channel function by electrophysiology studies with a lipid bilayer reconstitution system and found that filamin-A substantially inhibits polycystin-2 channel activity. Our study indicates that filamins are important regulators of polycystin-2 channel function, and further links actin cytoskeletal dynamics to the regulation of this channel protein.
Highlights
Mammalian filamin (FLN) was first isolated from rabbit macrophages in 1975 as an actin-binding protein [1]
Given that the three mammalian FLN isoforms share high sequence similarities, we further explored whether FLNA and FLNB, which are more abundantly and universally expressed than FLNC, bound PC2
In this study we have demonstrated, by various protein-protein interaction methods including yeast two-hybrid screen, GST pulldown and co-IP, that PC2 physically interacts with the actin crosslinking protein filamin
Summary
Mammalian filamin (FLN) was first isolated from rabbit macrophages in 1975 as an actin-binding protein [1]. The mammalian FLN family consists of three ,280-kDa isoforms, filamin-A (FLNA), -B (FLNB) and -C (FLNC), sharing 60–80% whole sequence homology, of which FLNA is the most abundant and widely distributed [2]. FLNs contain an N-terminal actinbinding domain (ABD) that shares sequence similarity with other actin-binding proteins, and a rod domain consisting of 24 repeated anti-parallel b-sheets with one or two short ‘hinges’ inserted before repeats 16 and 24 (Fig. 1A). By cross-linking actin filaments at wide angles, FLNs act as important actin cytoskeleton organizers implicated in sol-gel transformations and membrane stabilization as anchors of many transmembrane proteins, and as scaffolding proteins for various signaling molecules [4]. Mutations in the FLNA and FLNB genes are known to cause a variety of developmental disorders in humans, including bone anomalies, periventricular heterotopia, aortic dissection and aneurysm [5,6,7]
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