Identification of clathrin and clathrin adaptors on tubulovesicles of gastric acid secretory (oxyntic) cells

Abstract

γ-Adaptin and clathrin heavy chain were identified on tubulovesicles of gastric oxyntic cells with the anti-γ-adaptin monoclonal antibody (MAb) 100/3 and an anti-clathrin heavy chain MAb (MAb 23), respectively. In Western blots, crude gastric microsomes from rabbit and rat and density gradient-purified, H-K-ATPase-rich microsomes from these same species were immunoreactive for γ-adaptin and clathrin. In immunofluorescent labeling of isolated rabbit gastric glands, anti-γ-adaptin and anti-clathrin heavy chain immunoreactivity appeared to be concentrated in oxyntic cells. In primary cultures of rabbit oxyntic cells, the immunocytochemical distribution of γ-adaptin immunoreactivity was similar to that of the tubulovesicular membrane marker in oxyntic cells, the H-K-ATPase. Further biochemical characterization of the tubulovesicular γ-adaptin-containing complex suggested that it has a subunit composition that is typical of that for a clathrin adaptor: in addition to the γ-adaptin subunit, it contains a β-adaptin subunit and other subunits of apparent molecular masses of 50 kDa and 19 kDa. From solubilized gastric microsomes from rabbit, γ-adaptin could be copurified with the major cargo protein of tubulovesicles, the H-K-ATPase. Thus this tubulovesicular coat may bind directly to the H-K-ATPase and may thereby mediate the regulated trafficking of the H-K-ATPase at the apical membrane of the oxyntic cell during the gastric acid secretory cycle. Given the similarities of the regulated trafficking of the H-K-ATPase with recycling of cargo through the apical recycling endosome of many epithelial cells, we propose that tubulovesicular clathrin and adaptors may regulate some part of an apical recycling pathway in other epithelial cells.

Hip1r is expressed in gastric parietal cells and is required for tubulovesicle formation and cell survival in mice

“…Once the proton pump is localized at the apical membrane, acid secretion into the lumen of the stomach takes place. Upon return to the resting state, proton pumps are sequestered back into tubulovesicles, a process thought to involve clathrin-coated vesicle formation similar to endocytosis (13,21). Previous gene expression microarray studies of purified parietal cells showed abundant expression of Hip1r but little or no Hip1 (14), suggesting that Hip1r may serve a unique function in parietal cells (5,8).…”

“…Upon stimulation of the parietal cell, tubulovesicles fuse with the apical plasma membrane to form an expanded surface membrane termed the canaliculus, which is capable of acid secretion into the lumen of the stomach. To return the parietal cell to the basal state, tubulovesicles containing the proton pump are reformed through a process thought to involve clathrin-coated vesicle formation similar to endocytosis (13). Thus, parietal cells rely on vesicular fusion and reformation to regulate acid secretion.…”

Isolation, Cloning, and Characterization of a New Mammalian Coronin Family Member, Coroninse, Which Is Regulated within the Protein Kinase C Signaling Pathway

“…Another potentially relevant finding is that there is a fairly well conserved clathrin adaptor motif in coronin se . Since clathrin and clathrin adaptors have recently been localized to tubulovesicular membranes in parietal cells (36), it is possible that there is a regulated interaction between coronin se and clathrin or a related protein that serves to direct, for example, the endocytic retrieval of the H ϩ ,K ϩ -ATPase from the apical membrane to subapical tubulovesicular membranes.…”

Molecular Cloning and Characterization of a Novel Chloride Intracellular Channel-related Protein, Parchorin, Expressed in Water-secreting Cells

“…Upon stimulation, tubulovesicles containing H ϩ ,K ϩ -ATPase are incorporated into the apical membrane, the latter acquiring K ϩ and Cl Ϫ permeability, and thus H ϩ ,K ϩ -ATPase can exchange K ϩ for protons, creating HCl together with water secretion (1). Recent studies on mechanisms of membrane fusion in parietal cells have made rapid progress, and several proteins involved in the recruitment/recycling process have been identified (2)(3)(4). In contrast, little is known of how the apical membrane acquires K ϩ and Cl Ϫ ion permeability, which is thought to be the direct trigger for activation of the H ϩ ,K ϩ -ATPase.…”