Ezrin, radixin and moesin regulate assembly of actin-based structures, link membrane-spanning proteins to cortical actin and are part of cell signalling hubs. The CLIC5A 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 ezrin, radixin and moesin 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 ezrin482-586 (T567E) C-terminal fragment was in the 30 μM range. Silencing of ezrin, radixin and moesin dislodged CLIC5A from the peripheral location, and the CLIC5A-ezrin interaction augmented Rho-GDI 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.
Rahman et al. (Fri,) studied this question.