A Tyrosine-Based Signal Targets H/K-ATPase to a Regulated Compartment and Is Required for the Cessation of Gastric Acid Secretion

In order to understand the regulatory role of protein kinase C (PKC) in secretory epithelia, it is necessary to identify and characterize specific downstream targets. We previously identified one such protein in studies of gastric parietal cells. This protein was referred to as pp66 because it migrated with an apparent molecular mass of 66 kDa on SDS-polyacrylamide gels. The phosphorylation of pp66 is increased by the cholinergic agonist, carbachol, and by the PKC activator, phorbol-12-myristate-13-acetate, in a calcium-independent manner. In this study, we have purified pp66 to homogeneity and cloned the complete open reading frame. GenBank TM searches revealed a 45% homology with the Dictyostelium actin-binding protein, coronin, and ϳ67% homology with the previously cloned human and bovine coronin-like homologue, p57. pp66 appears to be most highly expressed in the gastrointestinal mucosa and in kidney and lung. Confocal microscopic studies of an enhanced green fluorescent protein fusion construct of pp66 in cultured parietal cells and in Madin-Darby canine kidney cells indicate that pp66 preferentially localizes in F-actin-rich regions. On the basis of our findings, we propose that pp66 may play an important, PKC-dependent role in regulating membrane/cytoskeletal rearrangements in epithelial cells. We have tentatively named this protein coronin se , because it appears to be highly expressed in secretory epithelia.

Section: Discussionmentioning

confidence: 99%

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

Parente

Chen

Zhou

et al. 1999

“…Briefly, HA-Y543-expressing cells grown to confluency on Transwell filters were incubated at 37°C for 40 min in Met-Cys-free medium and labeled for 30 min with 35 S-Met and Cys (ICN Radiochemicals, Irving, CA). Cells were chased with complete medium for 2 h at 37°C.…”

Section: Internalization Assaymentioning

confidence: 99%

Tyrosine-based Membrane Protein Sorting Signals Are Differentially Interpreted by Polarized Madin-Darby Canine Kidney and LLC-PK1 Epithelial Cells

Roush¹,

Gottardi²,

Naim³

et al. 1998

114

Tyrosine-dependent sequence motifs are implicated in sorting membrane proteins to the basolateral domain of Madin-Darby canine kidney (MDCK) cells. We find that these motifs are interpreted differentially in various polarized epithelial cell types. The H,K-ATPase ␤ subunit, which contains a tyrosine-based motif in its cytoplasmic tail, was expressed in MDCK and LLC-PK 1 cells. This protein was restricted to the basolateral membrane in MDCK cells, but was localized to the apical membrane in LLC-PK 1 cells. Similarly, HA-Y543, a construct in which a tyrosine-based motif was introduced into the cytoplasmic tail of influenza hemagglutinin, was sorted to the basolateral membrane of MDCK cells and retained at the apical membrane of LLC-PK 1 cells. A chimera in which the cytoplasmic tail of the H,K-ATPase ␤ subunit protein was replaced with the analogous region of the Na,K-ATPase ␤ subunit polypeptide was localized to both surface domains of MDCK cells. Mutation of tyrosine-20 of the H,K-ATPase ␤ subunit cytoplasmic sequence to an alanine was sufficient to disrupt basolateral localization of this polypeptide. In contrast, these constructs all remain localized to the apical membrane in LLC-PK 1 cells. The FcRII-B2 protein bears a dileucine motif and is found at the basolateral membrane of both MDCK and LLC-PK 1 cells. These results demonstrate that polarized epithelia are able to discriminate between different classes of specifically defined membrane protein sorting signals.

“…The ␣-subunit contains ATP and cation binding sites and carries out the catalytic and transport functions of the enzyme (1), and it also contains sequences responsible for apical membrane localization (4). The heavily glycosylated ␤-subunit is required for endocytic retrieval of the H,K-ATPase from the canilicular membranes as the cell passes from the stimulated to the resting state (5) and may also contribute to proper folding and membrane localization of the enzyme (1).…”

mentioning

confidence: 99%

Stomachs of Mice Lacking the Gastric H,K-ATPase α-Subunit Have Achlorhydria, Abnormal Parietal Cells, and Ciliated Metaplasia

Spicer¹,

Miller²,

Andringa³

et al. 2000

162

135

The H,K-ATPase of the gastric parietal cell is the most critical component of the ion transport system mediating acid secretion in the stomach. To study the requirement of this enzyme in the development, maintenance, and function of the gastric mucosa, we used gene targeting to prepare mice lacking the ␣-subunit. Homozygous mutant (Atp4a ؊/؊ ) mice appeared healthy and exhibited normal systemic electrolyte and acid-base status but were achlorhydric and hypergastrinemic. Immunocytochemical, histological, and ultrastructural analyses of Atp4a ؊/؊ stomachs revealed the presence of chief cells, demonstrating that the lack of acid secretion does not interfere with their differentiation. Parietal cells were also present in normal numbers, and despite the absence of ␣-subunit mRNA and protein, the ␤-subunit was expressed. However, Atp4a ؊/؊ parietal cells had dilated canaliculi and lacked typical canalicular microvilli and tubulovesicles, and subsets of these cells contained abnormal mitochondria and/or massive glycogen stores. Stomachs of adult Atp4a؊/؊ mice exhibited metaplasia, which included the presence of ciliated cells. We conclude that ablation of the H,K-ATPase ␣-subunit causes achlorhydria and hypergastrinemia, severe perturbations in the secretory membranes of the parietal cell, and metaplasia of the gastric mucosa; however, the absence of the pump appears not to perturb parietal cell viability or chief cell differentiation.