IQGAPs are differentially expressed and regulated in polarized gastric epithelial cells

Abstract

. IQGAPs are differentially expressed and regulated in polarized gastric epithelial cells. Am J Physiol Gastrointest Liver Physiol 288: G376 -G387, 2005. First published September 30, 2004; doi:10.1152/ajpgi.00290.2004.-IQGAPs, GTPase-activating proteins with an IQ motif, are thought to regulate many actin cytoskeleton-based activities through interactions with Cdc42 and Rac. Recently, Cdc42 was implicated in regulation of gastric parietal cell HCl secretion, and IQGAP2 was immunolocalized with Cdc42 to F-actin-rich intracellular canalicular membranes of isolated gastric parietal cells in primary culture. Here we sought to define distribution and localization of IQGAP1 and IQGAP2 in major oxyntic (acid-secreting) gastric mucosal cell types and to determine whether secretory agonists modulate these proteins. Differential staining protocols were used to identify different cell populations (parietal, chief, surface/pit, and mucous neck cells) in semi-intact glands isolated from rabbit gastric mucosae and to characterize these same cells after dispersion and fractionation on isopycnic density gradients with simultaneous staining for F-actin, H ϩ -K ϩ -ATPase, and GSII lectinbinding sites. There was a pronounced increase in intracellular F-actin staining in dispersed chief cells, apparently from internalization of F-actin-rich apical membranes that normally abut the gland lumen. Therefore, other membrane-associated proteins might also be redistributed by disruption of cell-cell contacts. Western blot analyses were used to quantitate relative concentrations of IQGAPs in defined mucosal cell fractions, and gastric glands were used for in situ localizations. We detected uniform levels of IQGAP2 expression in oxyntic mucosal cells with predominant targeting to regions of cellcell contact and nuclei of all cell types. IQGAP2 was not detected in parietal cell intracellular canaliculi. IQGAP1 expression was variable and targeted predominantly to the cortex of chief and mucous neck cells. Parietal cells expressed little or no IQGAP1 vs. other mucosal cell types. Phosphoprotein affinity chromatography, isoelectric focusing, and phosphorylation site analyses indicated that both IQGAP1 and IQGAP2 are phosphoproteins potentially regulated by [Ca 2ϩ ]i/ PKC and cAMP signaling pathways, respectively. Stimulation of glands with carbachol, which elevates [Ca 2ϩ ]i and activates PKC, induced apparent translocation of IQGAP1, but not IQGAP2, to apical poles of chief (zymogen) and mucous neck cells. This response was mimicked by PMA but not by ionomycin or by elevation of [cAMP] i with forskolin. Our observations support a novel, PKC-dependent role for IQGAP1 in regulated exocytosis and suggest that IQGAP2 may play a more general role in regulating cell-cell interactions and possibly migration within the gastric mucosa. parietal cell; chief cell; protein kinase C; carbachol; actin THE RHO GTPASES Cdc42 and Rac1 regulate actin cytoskeletal dynamics by interacting with a number of effectors, including the IQGAPs (5, 15, 43), which are s...

Use of Fluorescence-activated Vesicle Sorting for Isolation of Naked2-associated, Basolaterally Targeted Exocytic Vesicles for Proteomics Analysis

“…The same phenomenon was observed in the live polarized MDCK cells (data not shown). As mentioned above, IQGAP1 has been linked to basolateral trafficking (29,30). Endogenous IQGAP1 partially co-localized on Naked2-associated vesicles in MDCK cells stably expressing wild-type Naked2-EGFP (Fig.…”

“…Forty-seven unique IQGAP1 peptides from 65 spectra were identified in Pool 2 G2A Naked2 flow-sorted vesicles. IQGAP1 is a widely expressed protein that has been linked to basolateral trafficking (29,30). Of potential interest, we also identified RACK1, which has been shown to interact with IQGAP1 (31).…”

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

“…Gastric gland immunostaining and fractionation. Gastric mucosal cells were dispersed from the oxyntic mucosa of New Zealand White rabbits by sequential pronase and collagenase digestion, and cell fractions were separated by Optiprep density gradient centrifugation as described previously (50). The percentage of parietal cells in each fraction was calculated by immunostaining for H + , K + -ATPase (α subunit, clone HK 12.18; Calbiochem).…”

“…Reactions were terminated by addition of 50 ml DMEM/F12 containing 0.2% BSA and 1 mM DTT. PFA fixation and immunostaining were performed as previously described (50,51). The Hip1r mouse mAb (1:100; BD Biosciences) was diluted in tris-buffered saline containing 1% nonfat milk (BioRad).…”

“…Donkey anti-mouse secondary antibody conjugated to Cy5 (1:100; Jackson ImmunoResearch Laboratories), SYTOX green (0.5 μM; Invitrogen), and Oregon green- or Alexa Fluor 568-conjugated phalloidin (1:40; Invitrogen) were diluted in tris-buffered saline with 0.1% nonfat milk. Stained glands were mounted with Prolong Gold antifade reagent (Invitrogen) and visualized with a Zeiss LSM 510 META confocal microscope using Meta software (version 3.2; Zeiss) (50). Controls for nonspecific binding and fluorescent emission crossover were included in each experiment as described previously (52).…”