CLIC5A binds to and stabilizes the open and active conformation of ezrin

Ezrin, radixin, and moesin (ERM) proteins regulate assembly of actin-based structures, link membrane-spanning proteins to cortical actin, and are part of cell signaling hubs. The chloride intracellular channel (CLIC) 5A protein is very abundant in radixin-dependent inner ear hair cell stereocilia and in ezrin-dependent kidney glomerular podocyte foot processes and is essential for the structural integrity of these actin-based cellular projections. The functional relationship between ERM proteins and CLIC5A is incompletely understood and whether CLIC5A functions as a chloride channel is controversial. We determined whether CLIC5A is membrane-spanning protein and sought direct CLIC5A binding partners. While CLIC5A localized predominantly to the dorsal plasma membrane domain, we found CLIC5A to be a soluble, intracellular protein, without characteristics expected of a membrane-spanning channel. In the yeast two-hybrid assay, CLIC5A interacted directly with the C-terminal domains of ERM with a hierarchy of ezrin > radixin = moesin. The last 16 amino acids of ezrin were essential but not sufficient for CLIC5A binding, and phosphorylation of ezrin at T567 enhanced the interaction. The affinity of purified CLIC5A for a phosphomimetic ezrin_482-586 (T567E) C-terminal fragment was in the 30 μM range. Silencing of ERM dislodged CLIC5A from the peripheral location, and the CLIC5A-ezrin interaction augmented Rho guanine nucleotide dissociation inhibitor sequestration by ezrin and Rac1 activity. Thus, CLIC5A functions as a direct binding partner of ezrin, stabilizing its open/active conformation and resulting in localized small GTPase activation.