The secretion-stimulated 80K phosphoprotein of parietal cells is ezrin, and has properties of a membrane cytoskeletal linker in the induced apical microvilli.

Abstract

Stimulation of gastric acid secretion in parietal cells involves the translocation of the proton pump (H,K‐ATPase) from cytoplasmic tubulovesicles to the apical membrane to form long, F‐actin‐containing, microvilli. Following secretion, the pump is endocytosed back into tubulovesicles. The parietal cell therefore offers a system for the study of regulated membrane recycling, with temporally separated endocytic and exocytic steps. During cAMP‐mediated stimulation, an 80 kDa peripheral membrane protein becomes phosphorylated on serine residues. This protein is a major component, together with actin and the pump, of the isolated apical membrane from stimulated cells, but not the resting tubulovesicular membrane. Here we show that the gastric 80 kDa phosphoprotein is closely related or identical to ezrin, a protein whose phosphorylation on serine and tyrosine residues was recently implicated in the induction by growth factors of cell surface structures on cultured cells [Bretscher, A. (1989) J. Cell Biol., 108, 921–930]. Light and electron microscopy reveal that ezrin is associated with the actin filaments of the microvilli of stimulated cells, but not with the filaments in the terminal web. In addition, a significant amount of ezrin is present in the basolateral membrane infoldings of both resting and stimulated cells. Extraction studies show that ezrin is a cytoskeletal protein in unstimulated and stimulated cells, and its association with the cytoskeleton is more stable in stimulated cells. These studies indicate that ezrin is a membrane cytoskeletal linker that may play a key role in the control of the assembly of secretory apical microvilli in parietal cells and ultimately in the regulation of acid secretion. Taken together with the earlier studies, we suggest that ezrin might be a general substrate for kinases involved in the regulation of actin‐containing cell surface structures.

Polarized distribution of actin isoforms in gastric parietal cells.

“…Because the immuofluorescence using anti-actin antibodies gives the total signal from both monomeric actin (G-actin) and F-actin, we carried out two kinds of immunofluorescence: one recognizing both G-and F-actin in which the cells were fixed then carried through immunostaining; the other in which cells were extracted with a mild nonionic detergent, to remove G-actin and other soluble proteins, before fixation and immunostaining. For the latter case, we first extracted glands with extraction buffer (0.1% NP-40, 100 mM PIPES, pH 6.9, 0.18 g/ml glycerol, 1.2 mM MgCl2, 2 mM EGTA, 5 jiM calpain inhibitor I, 1 mM phenylmethylsulfonyl fluoride, 1 mM pepstatin A), then fixed the glands with 4% formaldehyde, and finally settled the glands onto polylysine-coated coverslips as described by Hanzel et al (1991). The extraction caused no change in the distribution of F-actin (phalloidin) or ezrin, but did tend to improve clarity of stain.…”

“…Differential Distribution of Actin Isoforms in Gastric Glandular Cells Our previous studies have characterized the distribution of filamentous actin in gastric glandular cells (Wolosin et al, 1983;Hanzel et al, 1991). Because actin isoforms were clearly represented in the gastric extracts, it was of interest to determine if an isoformspecific distribution of (3-and y-actin could be detected within the cells using immunofluorescence microscopy.…”

“…In parietal cells the distribution of filamentous actin (F-actin) is polarized toward the apical plasma membrane where actin-based microvillar structures are present. When the cells are stimulated to secrete acid, a dramatic extension of apical plasma membrane occurs after the recruitment of H,K-ATPase-containing cytoplasmic membranes (Hanzel et al, 1991). Ezrin, a putative actinbinding protein, co-localizes with filamentous actin in the apical membrane of parietal cells, and has been implicated in stimulation-mediated membrane-cytoskeletal interaction Hanzel et al, 1991).…”

Identification of a phosphatidylinositol‐4,5‐bisphosphate‐binding domain in the N‐terminal region of ezrin

“…Ezrin is a member of the protein 4.1-superfamily which is involved in the interaction of the cell cytoskeleton with the plasma membrane [1,2,3]. Sequence analysis shows, that ezrin contains an N-terminal domain with homology to protein 4.1 and talin, followed by a long c~-helical region and a charged C-terminal domain [4].…”

“…A putative membrane anchor of ezrin and the closely related proteins moesin and radixin may be the cell surface glycoprotein CD44 [10]. The membrane association of the tyrosine kinase substrate ezrin may be regulated by phosphorylation [3]. *Corresponding author.…”