Vesicular trafficking machinery, the actin cytoskeleton, and H+-K+-ATPase recycling in the gastric parietal cell

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Vesicular trafficking machinery, the actin cytoskeleton, and H⁺-K⁺-ATPase recycling in the gastric parietal cell

Curtis T. Okamoto and John G. Forte*

Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089-9121 and *Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA

MS 11947 Received 20 November 2000; accepted after revision 12 February 2001

ABSTRACT

Gastric HCl secretion by the parietal cell involves the secretagogue-regulated re-cycling of the H⁺-K⁺-ATPase at the apical membrane. The trafficking of the H⁺-K⁺-ATPase and the remodelling of the apical membrane during this process are likely to involve the co-ordination of the function of vesicular trafficking machinery and the cytoskeleton. This review summarizes the progress made in the identification and characterization of components of the vesicular trafficking machinery that are associated with the H⁺-K⁺-ATPase and of components of the actin-based cytoskeleton that are associated with the apical membrane of the parietal cell. Since many of these proteins are also expressed at the apical pole of other epithelial cells, the parietal cell may represent a model system to characterize the protein- protein interactions that regulate apical membrane trafficking in many other epithelial cells.

Historical background

Recruitment and recycling of specific membrane transport proteins is now recognized as a major means for up- and downregulation of transport activity in many cells. The gastric acid secreting cell (referred to as the parietal cell) was the first system in which the recruitment and recycling of a transport protein, the H⁺-K⁺-ATPase, was proposed as the principal means for regulating secretion (Forte et al. 1977). Electron microscopy provided evidence that physiological stimulation of parietal cells led to a large expansion of the apical plasma membrane, with the increased membrane surface presumably coming from a compartment of cytoplasmic membranes known as tubulovesicles (Sedar & Friedman, 1961; Helander & Hirschowitz, 1974; Ito & Schofield, 1974; T. M. Forte et al. 1975; Schofield et al. 1979; Gibert & Hersey, 1982). The putative interconversion of tubulovesicles and apical plasma membrane during the secretory cycle formed the basis for the membrane recycling hypothesis of HCl secretion (Forte et al. 1977), as shown in schematic form in Fig. 1. With the identification of H⁺-K⁺-ATPase as the primary gastric proton pump (J. G. Forte et al. 1967, 1975; Ganser & Forte, 1973), it was proposed that activation of H⁺ secretion occurred by incorporation of H⁺-K⁺-ATPase-rich tubulovesicles into the apical plasma membrane, and that the pumps were re-sequestered back into the cytoplasmic compartment on return to the resting state (Forte & Lee, 1977). The hypothesis was later modified to include the recruitment of K⁺ and Cl^- conductance channels to the apical surface (Wolosin & Forte, 1981a,b, 1983, 1984; Hirst & Forte, 1985).

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Figure 1. Schematic diagram of regulated membrane recycling in parietal cells during the gastric HCl secretory cycle

Stimulation of the parietal cell results in the exocytotic insertion of H⁺-K⁺-ATPase-rich tubulovesicles with the apical (canalicular) membrane. Morphological changes occurring as a consequence of this exocytotic event are depletion of intracellular tubulovesicles, elongated apical microvilli and expanded canaliculi. As the stimulus is withdrawn, an endocytotic retrieval of the H⁺-K⁺-ATPase and membrane from the apical (canalicular) membrane results in the re-establishment of the tubulovesicular system, poised for another round of secretion. The illustration is not drawn to scale.

There has been controversy over the mechanism by which the apical surface transformation occurs. An alternative hypothesis holds that the H⁺-K⁺-ATPase-rich membranes are not a separate vesicular compartment, but that the membranes are really in physical contiguity with the apical plasma membrane (Berglindh et al. 1980; Pettitt et al. 1995). This so-called osmotic expansion hypothesis requires no fusion; rather, the elaboration of apical membrane surface in stimulated cells would supposedly result from expansion of a highly involuted membrane system using osmotic work or cytoskeletal forces.

Lessons from primary cultures of parietal cells

Recently, the use of isolated parietal cells in primary culture has provided a clear distinction between the fusion-based recycling hypothesis and the osmotic expansion hypothesis of HCl secretion (Agnew et al. 1999), as well as a very useful model to study regulated recruitment and recycling of membranes and marker proteins. In culture, parietal cells lose or re-orient their polarity: the apical canalicular membrane is engulfed as a series of vacuolar inclusions (so-called VACs), and the basolateral membrane becomes the surrounding plasma membrane (Chew et al. 1989; Mangeat et al. 1990; Soroka et al. 1993; Agnew et al. 1999). In non-secreting parietal cells F-actin is distinctly localized to the VACs and to the basolateral membrane, while H⁺-K⁺-ATPase is distributed throughout the cytoplasm in the compartment of tubulovesicles. After stimulation, H⁺-K⁺-ATPase co-localizes with F-actin in the VACs, which ordinarily have become enormously swollen due to the large volume of acidic fluid transport. However, even when the osmotic swelling forces are eliminated by pump inhibitors or protonophores, parietal cell stimulation still results in the clearing of H⁺-K⁺-ATPase from the cytoplasm and its translocation to the VACs (Agnew et al. 1999). Thus, these data clearly favour the idea that H⁺-K⁺-ATPase-rich tubulovesicle membranes migrate to, and fuse with, the apical membrane rather than an expansion of the apical membrane back into the cytoplasmic locale (Berglindh et al. 1980; Pettitt et al. 1996).

Vesicular trafficking machinery in parietal cells

As described above, a major function of the fully differentiated parietal cell is to regulate HCl secretion by secretagogue-dependent trafficking of the proton pump to and from the apical membrane. Thus, the H⁺-K⁺-ATPase is the major vesicular cargo in this particular trafficking pathway, and it provides a good biochemical handle for the isolation of accessory proteins involved in regulating the trafficking of H⁺-K⁺-ATPase-rich vesicles. Moreover, the parietal cell should express a relative abundance of machinery involved in the regulation of H⁺-K⁺-ATPase trafficking, thereby facilitating the biochemical and molecular identification of these interacting proteins. We report here on the progress in identifying and characterizing proteins ostensibly involved in the regulation of vesicular transport and H⁺-K⁺-ATPase traffic in parietal cells.

Rab11 and rab25. The first molecular evidence that the vesicular trafficking machinery in parietal cells was similar to that found in other cells was the cloning and biochemical characterization of two members of the rab family of GTPases, rab11 and rab25, from parietal cell cDNA libraries (Goldenring et al. 1993, 1994). Rab proteins belong to the family of ras-like small GTPases that now include over 50 members (Schimmöller et al. 1998). They display distinctive intracellular membranous localizations and tissue- or organ-specific expression. In many cases, they have been shown to regulate protein sorting and vesicular trafficking between two membrane compartments by recruiting other effector proteins (Schimmöller et al. 1998; Brennwald, 2000). In the parietal cell, rab11a, rab11b, and rab25 have been found on H⁺-K⁺-ATPase-rich membranes (Goldenring et al. 1993, 1994; Calhoun & Goldenring, 1997; Calhoun et al. 1998; Duman et al. 1999). Moreover, rab11 and rab25 are found in membranes immuno-isolated with an antibody against the H⁺-K⁺-ATPase, suggesting a physically close association between these rab proteins and the H⁺-K⁺-ATPase (Calhoun & Goldenring, 1997). A stoichiometry of six H⁺-K⁺-ATPase molecules per one rab11 molecule has been estimated (Duman et al. 1999), implying that rab11 is a relatively abundant component of H⁺-K⁺-ATPase-rich membranes and may be a key candidate for regulating vesicular traffic.

One function of rab proteins is to provide intracellular spatial cues for the localized recruitment and/or activation of effector proteins (Schimmöller et al. 1998; Brennwald, 2000). In parietal cells, rab11, like the H⁺-K⁺-ATPase, was found to translocate from an intracellular membranous pool to the apical membrane, suggesting that rab11 accompanies the H⁺-K⁺-ATPase to the target membrane (Calhoun et al. 1998). The kinesis of rab11 to the apical membrane suggests that it may act at the target membrane as well as at the donor membrane. Such a mode of action would have some parallel to that reported for the yeast rab homologue Ypt1, which appears to regulate membrane fusion at the target membrane (Cao & Barlowe, 2000).

In another study, expression of a dominant-negative form of rab11a (N124I) in primary cultures of parietal cells inhibited secretory activity (Duman et al. 1999). Moreover, this mutant construct specifically inhibited the stimulatory recruitment of H⁺-K⁺-ATPase to the apical membrane, as determined by immunofluorescence assays. These results are consistent with a major regulatory function for rab11 on donor membranes (i.e. tubulovesicles) in the secretagogue-stimulated membrane transformations of parietal cells. An interesting finding of this study is that the generation of HCl appears to be obligatorily linked to the membrane transformations; that is, acid secretion cannot be stimulated independently of membrane transformations.

Illustrating the value of the parietal cell as a model to study regulated membrane trafficking, since the discovery of rab11, numerous studies now offer evidence to support a role for rab11 in the regulation of membrane recycling in many cell types, epithelial and non-epithelial (Goldenring et al. 1996; Ullrich et al. 1996; Ren et al. 1998; Casanova et al. 1999; Cox et al. 2000), as well as in the regulation of exocytotic traffic from the Golgi in some cells (Urbé et al. 1993; Chen et al. 1998).

Although the functional role of rab25 has not been assessed in parietal cells, several features deserve mention. In cultured Madin-Darby canine kidney cell lines rab25 is associated with subapical membranes, and overexpression of wild-type rab25 inhibits transcytotic and apical recycling traffic through this compartment (Casanova et al. 1999). In addition, rab25 is apparently unique in that it contains a natural amino acid substitution that would be predicted to reduce its GTPase activity, thus rendering it as a constitutively active rab. However, despite this amino acid substitution, rab25 has been shown to be an active GTPase (Casanova et al. 1999).

Receptors for soluble N-ethylmaleimide-sensitive factor attachment protein (SNARE) proteins. SNARE proteins are a family of membrane proteins that have been implicated in mediating homotypic and heterotypic vesicle-vesicle or vesicle-organelle fusion (reviewed in Jahn & Südhof, 1999). Indeed, evidence from in vitro reconstituted systems suggests that SNARE proteins comprise the minimal machinery for vesicular fusion (Weber et al. 1998). Thus, SNARE proteins would be predicted to regulate tubulovesicle trafficking and fusion during the secretory cycle of the parietal cell. Data supporting vesicle-dependent trafficking of the H⁺-K⁺-ATPase was provided by the identification of two SNARE proteins in H⁺-K⁺-ATPase-rich membranes, syntaxin 3 and vesicle-associated membrane protein (VAMP) 2 (Calhoun & Goldenring, 1997; Peng et al. 1997; Calhoun et al. 1998). Syntaxin 3 and VAMP 2 have been shown to be associated with the apical membrane trafficking pathway in other epithelial cells (Braun et al. 1994; Gaisano et al. 1994; Low et al. 1996; Delgrossi et al. 1997; Riento et al. 2000).

Clathrin and clathrin adaptors. Clathrin and clathrin adaptors are the best-characterized family of vesicular coat proteins. The structure and function of clathrin and clathrin adaptors have been the subject of many excellent recent reviews (Kirchhausen, 1999; Marsh & McMahon, 1999) and will not be reviewed here. Vesicular coat proteins such as clathrin and clathrin adaptors are thought to mediate transport vesicle formation by: (1) providing the means to select and to concentrate cargo into nascent vesicles; and (2) recruiting other proteins that will deform the membrane into a transport vesicle or tubule. The presence and function of such proteins would be predicted in the retrieval of the H⁺-K⁺-ATPase from the apical membrane and in the biogenesis and continual re-formation of tubulovesicles.

Clathrin and the AP-1 and AP-2 clathrin adaptors have been identified in parietal cells (Okamoto et al. 1998, 2000). Evidence from biochemical and morphological studies show that clathrin and AP-1 clathrin adaptors are associated with a compartment of cytoplasmic tubulovesicles at steady state and are distinct from their association with the Golgi apparatus (Okamoto et al. 1998, 2000). The AP-1 clathrin adaptor appears to interact with, and can be co-purified with, the H⁺-K⁺-ATPase from tubulovesicles (Okamoto et al. 1998). Clathrin on tubulovesicles appears to be comprised of conventional clathrin subunits (Okamoto et al. 2000), but the AP-1 clathrin adaptor on tubulovesicles appears to possess an immunologically distinct $beta$ -adaptin subunit (Okamoto & Jeng, 1998). The nature of this immunological distinction has not been characterized, but could represent isoform differences or post-translational modification, such as phosphorylation (Wilde & Brodsky, 1996). Tubulovesicles thus appear to be a novel clathrin- and AP-1 adaptor-coated organelle in epithelial cells.

In addition to its association with tubulovesicles, clathrin is also associated with the apical membrane of resting parietal cells, and has been specifically localized to endocytic inclusions at the apical surface by immunoelectron microscopy (Okamoto et al. 2000). The AP-2 clathrin adaptor is also localized to the apical membrane at steady state (Okamoto et al. 2000). These results suggest that, even in resting cells, active membrane trafficking occurs between the apical membrane and the tubulovesicular compartment. Thus, the secretory cycle may be regulated with respect to the relative rates of exocytosis (stimulation) and endocytosis (recycling), rather than a binary 'on or off' process. Alternatively, the active vesicular trafficking observed in resting cells may reflect an extended recovery phase from stimulation. In this scenario, once the secretagogue has been withdrawn, there is a massive, relatively non-specific re-uptake of apical membrane, analogous to a 'bulk flow' process. In the time between the next stimulation, the parietal cell performs a 'proofreading' function, returning improperly endocytosed proteins to the apical membrane (e.g. t-SNAREs) and re-internalizing tubulovesicular proteins improperly left at the apical membrane (e.g. v-SNAREs and H⁺-K⁺-ATPase). Thus, in this case, the ratio of intracellular to apical membrane H⁺-K⁺-ATPase and SNAREs should change with time, while in the first scenario, the ratio would not be predicted to change.

The dynamic behaviour of clathrin and clathrin adaptors during the stimulatory recruitment of the H⁺-K⁺-ATPase to the apical membrane has been studied in primary cultures of parietal cells (Okamoto et al. 2000). Perhaps not surprisingly, clathrin and the AP-1 clathrin adaptor did not translocate with the H⁺-K⁺-ATPase to the apical membrane upon stimulation. Thus, the major role of clathrin and clathrin adaptors may be during the massive re-uptake of membrane and protein and re-formation of the tubulovesicular compartment after withdrawal of the stimulus. In fact, in cells in the re-uptake phase, coated profiles have been reported in an early ultrastructural study of the parietal cell secretory cycle (Schofield et al. 1979). The challenge will be to develop a recycling assay with the parietal cell to test the role of clathrin and clathrin adaptors and to characterize the mechanism by which their assembly is regulated temporally and spatially.

Sorting signals in the H⁺-K⁺-ATPase for the interaction with clathrin adaptors. Clathrin adaptors mediate the sorting of many membrane proteins by direct interaction of adaptor µ or $beta$ -subunits with Tyr- or di-Leu-based motifs in the cytoplasmic domains of the membrane protein cargo (Bonifacino & Dell'Angelica, 1999; Kirchhausen, 1999). The H⁺-K⁺-ATPase appears to interact with AP-1 clathrin adaptors in tubulovesicles (Okamoto et al. 1998), and the $beta$ -subunit of the H⁺-K⁺-ATPase (HK $beta$ ) has both a Tyr- and a Met-Leu motif in its N-terminal cytoplasmic domain. The Tyr-based motif bears a strong similarity to the internalization motif of the transferrin receptor that mediates its interaction with AP-2 adaptors (Collawn et al. 1990). The role of the Tyr-based motif was tested directly in transgenic mice in which the critical Tyr residue in HK $beta$ was replaced with Ala (Courtois-Coutry et al. 1997). These mice developed gastric mucosal pathologies resembling those in idiopathic hypersecretion. Immunoelectron microscopic evidence suggested that HK $beta$ , and presumably HK $alpha$ , in these transgenic mice were constitutively expressed at the apical membrane. Thus, it was concluded that the mutated HK $beta$ , and therefore the holoenzyme, could not interact with the endocytotic machinery to allow its re-sequestration, resulting in the constitutive secretion of HCl and consequent pathologies.

The recruitment of vesicular coat proteins has been shown to depend upon both the presence of membrane protein cargo and the lipid composition of the target membranes (Kirchhausen, 1999). Thus, cargo can regulate the formation of vesicular carriers. Results from knockout mice lacking either HK $alpha$ (Spicer et al. 2000) or HK $beta$ (Scarff et al. 1999) are relevant to the genesis of the tubulovesicular compartment. Predictably, both knockout mice are achlorhydric, but histologically identifiable parietal cells are clearly present. On the other hand, both knockout mice exhibit a dramatic diminution of membranes of the tubulovesicular compartment, suggesting that the H⁺-K⁺-ATPase is required as cargo for the development of the tubulovesicular compartment. Interestingly, the residual amounts of HK $beta$ expressed in the HK $alpha$ knockout mice were not sufficient to produce a tubulovesicular network (Spicer et al. 2000). It would be of interest to determine whether the expressed HK $beta$ in these mice resides in the remnant tubulovesicular membranes; such data would provide insight into the sorting of HK $beta$ .

Dynamin. Dynamins are members of a family of large (~100 kDa), multidomain GTPases that regulate vesicular fission (reviewed in Schmid et al. 1998). The mechanism by which dynamins regulate vesicular fission is still under debate (Sever et al. 2000), with evidence for: (1) active severing of membrane tubules via a GTPase-dependent conformational change; or (2) acting as a classical GTPase that recruits fission machinery to the neck of a nascent budding vesicle. Notwithstanding the mechanism, dynamins are localized to sites of active vesicle production, with splice variants often exhibiting markedly different intracellular localizations (Cao et al. 1998). Thus far, 25 splice variants distributed among three isoforms (dynamins I, II and III) have been identified (Cao et al. 1998).

From the distribution of clathrin and clathrin adaptors in the parietal cell, one prediction would be that a dynamin-like molecule should be associated with the apical membrane and tubulovesicles. Indeed, a member of the dynamin family, most probably dynamin II, has been immunolocalized to the apical membrane of parietal cells (Calhoun et al. 1998; Okamoto et al. 2000). Interestingly, among the several cell types in gastric glands, dynamin appears to be expressed predominantly, if not exclusively, in the parietal cell (Okamoto et al. 2000). In addition, preliminary data suggest that dynamin II is associated with isolated tubulovesicles (C. T. Okamoto, unpublished observations).

Many dynamin-interactive proteins have been characterized, particularly in neuronal tissue, that link dynamin to the clathrin vesicular machinery and to the cytoskeleton (Schmid et al. 1998; Simpson et al. 1999; Jarousse & Kelly, 2000; Ochoa et al. 2000). Many of these proteins contain src homology 3 (SH3) domains that bind to the C-terminal proline-arginine-rich domain of dynamin and stimulate its GTPase activity in vitro (Gout et al. 1993; Herskovits et al. 1993; Okamoto et al. 1997). In neurons (Shupliakov et al. 1997), fibroblasts (Simpson et al. 1999), and osteoclasts (Ochoa et al. 2000), SH3 domain-containing proteins have been shown to modulate dynamin function. The presence of dynamin in parietal cells suggests that this system may provide a testing ground to identify and characterize binding proteins that regulate dynamin function in epithelial cells.

Other GTPases. In a permeabilized parietal cell model, the non-hydrolysable GTP analogue, GTP $gamma$ S, blocked the generation of HCl, suggesting a key role for GTPases in the stimulatory process (Miller & Hersey, 1996; Akagi et al. 1999). Based upon the assumption that the target of GTP $gamma$ S was a small GTPase, several potential inhibitory peptides were tested for their ability to block the secretory response in this system (Akagi et al. 1999). The peptides were chosen from within sequences of rab proteins (rab3, rab11 and rab25) and ADP-ribosylation factor 1 (ARF1); however, only the ARF1 peptide was found to function in an inhibitory manner (Akagi et al. 1999). The inhibition by ARF1 peptide is intriguing, given that ARF1 regulates the association of AP-1 and AP-3 clathrin adaptors with target membranes (Stamnes & Rothman, 1993; Ooi et al. 1998).

Incubation of permeabilized cultured fibroblast cells with GTP $gamma$ S and cytosol results in the recruitment of AP-1 clathrin adaptor complexes to target membranes (Robinson & Kreis, 1992; Traub et al. 1993). Thus, the inhibitory effect of GTP $gamma$ S in permeabilized parietal cells may be a consequence of the recruitment of AP-1 adaptors to tubulovesicles, which might inhibit docking and/or fusion due to the steric hindrance of SNARE-SNARE interactions in coated membranes.

In other systems, certain aspects of dynamin function are inhibited by GTP $gamma$ S. Treatment of perforated cells with GTP $gamma$ S blocks endocytosis (Carter et al. 1993), and in permeabilized synaptosomes, incubation with GTP $gamma$ S results in the formation of dynamin spirals on endocytic membranes (Takei et al. 1995). However, since dynamin is thought to regulate vesicle fission, it is difficult to reconcile how GTP $gamma$ S, through dynamin, might result in a block of HCl secretion, which appears to be primarily a fusion event. In summary, it will be important to determine the relative contributions of all these GTPases in vesicular trafficking in the parietal cell.

Secretory carrier membrane proteins (SCAMPs). SCAMPs were identified on vesicles immuno-isolated with an anti-H⁺-K⁺-ATPase antibody (Calhoun & Goldenring, 1997). SCAMPs are ubiquitous, transmembrane proteins found in vesicles involved in membrane and protein recycling between the plasma membrane, endosomes and the trans-Golgi network (TGN) (Singleton et al. 1997). They possess four highly conserved transmembrane domains, and they contain multiple Asn-Pro-Phe (NPF) motifs in their amino terminus, which have the potential to interact with proteins containing eps15 homology (EH) domains (Di Fiore et al. 1997; Chen et al. 1998; Page et al. 1999; Fernández-Chac�n et al. 2000). Eps15 is a protein first identified as a substrate for the epidermal growth factor receptor (EGFR) tyrosine kinase (Wong et al. 1995), and subsequently, eps15 was shown to be linked to AP-2 clathrin adaptor-based endocytotic machinery at the plasma membrane (Benmerah et al. 1998). Thus, SCAMPs may mediate the interaction of endocytic membranes with other members of the endocytic machinery.

Biochemical, molecular and functional characterization of actin-based cytoskeletal proteins

There has been an exponential increase in the number of different cellular systems, from yeast to mammalian epithelial cells, in which physical and functional links between membrane trafficking and the cytoskeleton have been characterized (Vallee & Sheetz, 1996; Finger & Novick, 1998; Mermall et al. 1998; Brown, 1999; Gaidarov et al. 1999; Nielsen et al. 1999; Kreitzer et al. 2000; Ochoa et al. 2000; Qualmann et al. 2000; Tuxworth & Titus, 2000). These data are beginning to confirm a long-held suspicion that the regulation of membrane trafficking must be co-ordinated with regulated remodelling of the submembranous cytoskeleton or the recruitment of mechanochemical enzymes such as myosin, dynein and kinesins. It is in this spirit that the role of components of the actin-based cytoskeleton in the regulation of the secretory cycle in the parietal cell (for which there are significant data) is reviewed below.

Actin microfilament dynamics. As in many other epithelial cells, actin microfilaments line the microvilli at the canalicular (apical) membrane of the parietal cell (Forte et al. 1977; Vial et al. 1979; Black et al. 1982; Wolosin et al. 1983; Mercier et al. 1989). Further characterization of actin at the canalicular membrane has revealed that it consists predominantly of the $beta$ -actin isoform (Yao et al. 1995). During the secretory cycle, ultrastructural changes in the microfilaments can be clearly observed, particularly during the membrane re-uptake phase (Black et al. 1982). As the stimulus is withdrawn, apical microvilli collapse upon themselves and microfilaments become fragmented as a prelude to membrane recovery. This fragmentation is suggestive of a regulated activity of a microfilament-severing protein, but whose identity in this case remains unknown.

As mentioned above, upon the fusion of tubulovesicular membrane, the length of the microvilli increases severalfold. This elongation might be predicted to be accompanied by an increase in the polymerization of actin. Biochemical studies have shown that parietal cell actin is predominantly in the polymerized form (F-actin), and interestingly, no change was observed in the ratio of F- to G-actin when the cells were stimulated (Forte et al. 1998), although a qualitative impression from ultrastructural images or immunofluorescence suggests more polymerized actin at the apical surface of stimulated parietal cells (Forte et al. 1977; Black et al. 1982; Mercier et al. 1989; Soroka et al. 1993; Chew et al. 2000). Moreover, several inhibitor studies have clearly shown that polymerized actin is required for membrane transformation and HCl secretion (Rosenfeld et al. 1981; Black et al. 1982; Forte et al. 1998). Thus, the resting-to-stimulated transition may not depend upon a simple G-actin-to-F-actin transition, but may require a rearrangement among F-actin pools, e.g. from a more labile cortical pool to the highly stable pool of microvillar microfilaments. Alternatively, there may be a more subtle remodelling of the cytoskeleton by actin-binding proteins or small GTPases such as rac, rho or ARF6 (Schmidt & Hall, 1998; Chavrier & Goud, 1999), that is not amenable to detection by current assays. As probes become more readily available for the study of actin dynamics, this area of investigation should yield interesting results.

Actin-associated proteins

Ezrin. Ezrin is an 80 kDa actin-binding protein belonging to the family which partially bears its name, the ezrin-radixin-moesin (ERM) family of proteins (Tsukita et al. 1997; Mangeat et al. 1999). These proteins are involved in regulating the interaction of the plasma membrane with microfilaments. When ezrin was discovered in parietal cells, it was first identified as an 80 kDa protein whose phosphorylation increased upon cAMP-dependent stimulation of acid secretion (Urushidani et al. 1987, 1989) and was localized to regions rich in F-actin, especially the apical microvilli (Hanzel et al. 1989, 1991). Further studies demonstrated that parietal cell ezrin co-localizes with the $beta$ -actin isoform (Yao et al. 1995), presumably due to its direct binding to $beta$ -actin (Yao et al. 1995, 1996).

The phosphorylated form of ezrin biochemically co-purified with the apical membrane of stimulated cells (Urushidani et al. 1987, 1989), suggesting that ezrin may be involved in secretagogue-stimulated, phosphorylation-dependent modulation of the actin cytoskeleton at the apical membrane. Also, ezrin appears to be a low affinity protein kinase A anchoring protein (AKAP) (Dransfield et al. 1997). Thus, in the parietal cell, ezrin has the capacity to integrate signalling pathways to the remodelling of the actin cytoskeleton.

Coronin. Coronins are a family of proteins expressed from yeast to man that are involved in the remodelling of the cortical actin cytoskeleton, particularly during phagocytosis and macropinocytosis (de Hostos, 1999). They also promote actin polymerization and interact with microtubules. Structurally, they are predicted to form a $beta$ -propeller near the N-terminus and to dimerize via the C-terminal $alpha$ -helical coiled-coil domain (de Hostos, 1999). Coronin was first discovered in parietal cells as a 66 kDa substrate of cholinergic- and phorbol ester-stimulated kinases (Parente et al. 1999). Thus, phosphorylation of coronin is via a different pathway than that of ezrin. Coronin is localized predominantly to the canalicular membrane, where it may co-ordinate the remodelling of the apical cytoskeleton in response to other secretagogues (Parente et al. 1999). Intriguingly, the coronin isoform expressed in parietal cells has several putative motifs that are not found in other coronin family members; these motifs are similar to motifs found in other signalling, cytoskeletal, and vesicular trafficking proteins (Parente et al. 1999).

Lasp-1. Lasp-1 was identified as a 40 kDa protein phosphorylated by protein kinase A upon cAMP-dependent stimulation of parietal cells (Chew et al. 1998), which translocates from a more basal membrane distribution to the canalicular membrane upon cAMP-dependent stimulation (Chew et al. 2000). It is a multidomain-containing protein, featuring an N-terminal Cys-rich, Zn²⁺ finger LIM domain, an actin-binding nebulin-like repeat, a C-terminal SH3 domain, putative SH2-binding sites, and several putative phosphorylation sites for protein kinase A, protein kinase C, casein kinase II, and tyrosine kinases (Chew et al. 1998). Lasp-1 also appears to be expressed in a wide variety of, but not exclusively in, epithelial tissues and appears to be expressed in F-actin-rich secretory cells of these tissues (Chew et al. 2000). With the abundance of sites for protein-protein interaction and stimulation-dependent phosphorylation, lasp-1, like ezrin, may serve to integrate signalling pathways to the actin cytoskeleton and perhaps proteins involved in the regulation of membrane trafficking.

Myosin. Myosins are a family of proteins presently categorized with 12 isoforms (Mermall et al. 1998). As its diversity implies, myosins play a role in a plethora of cellular processes, from muscle contraction to visual signalling (Mermall et al. 1998). There exist abundant data supporting the role of various isoforms of myosin in the regulation of vesicular traffic (Vallee & Sheetz, 1996; Mermall et al. 1998). In permeabilized parietal cells, the addition of a pseudosubstrate for myosin light chain kinase blocks the production of HCl by cAMP (Akagi et al. 1999). While these results do not demonstrate directly the role of myosin in the regulation of vesicular traffic, they suggest the involvement of an upstream regulator of myosin activity in this process. Clearly, the characterization of the role of myosins in the regulation of vesicular trafficking in the parietal cell deserves further attention.

Conclusions

A summary of the vesicular machinery and cytoskeletal proteins identified and characterized in the parietal cell, in their respective locations, is shown in Fig. 2. The parietal cell has been exploited as a rich source of the gastric H⁺-K⁺-ATPase, resulting in extensive biochemical and structural characterization of this membrane transporter. The parietal cell is now proving to be an excellent source with which to study vesicular trafficking and cytoskeletal machinery that regulate HCl secretion and H⁺-K⁺-ATPase function and apical recycling. The characterization of the regulation of apical recycling in parietal cells may provide insight into this trafficking pathway in many other epithelial cells.

Corresponding author

C. T. Okamoto: Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089-9121, USA.

Email: cokamoto{at}hsc.usc.edu

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The calcium-sensing receptor acts as a modulator of gastric acid secretion in freshly isolated human gastric glands
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[Abstract]

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				D. Fu and B. D. Roufogalis Actin disruption inhibits endosomal traffic of P-glycoprotein-EGFP and resistance to daunorubicin accumulation Am J Physiol Cell Physiol, April 1, 2007; 292(4): C1543 - C1552. [Abstract] [Full Text] [PDF]

AGNEW B. I., DUMAN, J. G., WATSON, C. L., COLING, D. E. & FORTE, J. G. (1999). Cytological transformations associated with parietal cell stimulation: critical steps in the activation cascade. Journal of Cell Science 112, 2639-2646	[Abstract]
AKAGI K., NAGAO, T. & URUSHIDANI, T. (1999). Responsiveness of $beta$ -escin-permeabilized rabbit gastric gland model: effects of functional peptide fragments. American Journal of Physiology 277, G736-744	[Medline]
BENMERAH A., LAMAZE, C., B�GUE, B., SCHMID, S. L., DAUTRY-VARSAT, A. & CERF-BENSUSSAN, N. (1998). AP-2/eps15 interaction is required for receptor-mediated endocytosis. Journal of Cell Biology 140, 1055-1062	[Abstract/Full Text]
BERGLINDH T., DIBONA, D. R., ITO, S. & SACHS, G. (1980). Probes of parietal cell function. American Journal of Physiology 238, G165-176	[Medline]
BLACK J. A., FORTE, T. M. & FORTE, J. G. (1982). The effects of microfilament disrupting agents on HCl secretion and ultrastructure of piglet gastric oxyntic cells. Gastroenterology 83, 595-604
BONIFACINO J. S. & DELL'ANGELICA, E. C. (1999). Molecular bases for the recognition of tyrosine-based sorting signals. Journal of Cell Biology 145, 923-926	[Full Text]
BRAUN J. E., FRITZ, B. A., WONG, S. M. & LOWE, A. W. (1994). Identification of a vesicle-associated membrane protein (VAMP)-like membrane protein in zymogen granules of the rat exocrine pancreas. Journal of Biological Chemistry 269, 5328-5335	[Abstract]
BRENNWALD P. (2000). Reversal of fortune: do rab GTPases act on the target membrane? Journal of Cell Biology 149, 1-3	[Abstract/Full Text]
BROWN S. S. (1999). Cooperation between microtubule- and actin-based motor proteins. Annual Review of Cell and Developmental Biology 15, 63-80	[Abstract/Full Text]
CALHOUN B. C. & GOLDENRING, J. R. (1997). Two rab proteins, VAMP-2, and SCAMPS are present on immunoisolated parietal cell tubulovesicles. Biochemical Journal 325, 559-564	[Medline]
CALHOUN B. C., LAPIERRE, L. A., CHEW, C. S. & GOLDENRING, J. R. (1998). Rab11a redistributes to apical secretory canaliculus during stimulation of gastric parietal cells. American Journal of Physiology 275, C163-170	[Medline]
CAO H., GARCIA, F. & MCNIVEN, M. A. (1998). Differential distribution of dynamin isoforms in mammalian cells. Molecular Biology of the Cell 9, 2595-2609	[Abstract/Full Text]
CAO X. & BARLOWE, C. (2000). Asymmetric requirements for a rab GTPase and SNARE proteins in fusion of COPII-vesicles with acceptor membranes. Journal of Cell Biology 149, 55-65	[Abstract/Full Text]
CARTER L. L., REDELMEIER, T. E., WOOLLENWEBER, L. A. & SCHMID, S. L. (1993). Multiple GTP-binding proteins participate in clathrin-coated vesicle-mediated endocytosis. Journal of Cell Biology 120, 37-45	[Abstract]
CASANOVA J. E., WANG, X., KUMAR, R., BHARTUR, S. G., NAVARRE, J., WOODRUM, J. E., ALTSCHULER, Y., RAY, G. S. & GOLDENRING, J. R. (1999). Association of Rab25 and Rab11a with the apical recycling system of polarized Madin-Darby canine kidney cells. Molecular Biology of the Cell 10, 47-61	[Abstract/Full Text]
CHAVRIER P. & GOUD, B. (1999). The role of ARF and rab GTPases in membrane transport. Current Opinion in Cell Biology 11, 466-475	[Medline]
CHEN H., FRE, S., SLEPNEV, V. I., CAPUA, M. R., TAKEI, K., BUTLER, M. H., DI FIORE, P. P. & DE CAMILLI, P. (1998). Epsin is an EH-domain-binding protein implicated in clathrin-mediated endocytosis. Nature 394, 793-797	[Medline]
CHEN W., FENG, Y., CHEN, D. & WANDINGER-NESS, A. (1998). Rab11 is required for trans-Golgi network-to-plasma membrane transport and a preferential target for GDP dissociation inhibitor. Molecular Biology of the Cell 9, 3241-3257	[Abstract/Full Text]
CHEW C. S., LJUNGSTROM, M., SMOLKA, A. & BROWN, M. R. (1989). Primary culture of secretagogue-responsive parietal cells from rabbit gastric mucosa. American Journal of Physiology 256, G254-263	[Medline]
CHEW C. S., PARENTE, J. A. J., CHEN, X., CHAPONNIER, C. & CAMERON, R. S. (2000). The LIM and SH3 domain-containing protein, lasp-1, may link the cAMP signaling pathway with dynamic membrane restructuring activities in ion transporting epithelia. Journal of Cell Science 113, 2035-2045	[Abstract]
CHEW C. S., PARENTE, J. A. J., ZHOU, C.-J., BARANCO, E. & CHEN, X. (1998). Lasp-1 is a regulated phosphoprotein within the cAMP signaling pathway in the gastric parietal cell. American Journal of Physiology 275, C56-67	[Medline]
COLLAWN J. F., STANGEL, M., KUHN, L. A., ESEKOGWU, V., JING, S., TROWBRIDGE, I. S. & TAINER, J. A. (1990). Transferrin receptor internalization sequence YXRF implicates a tight turn as the structural recognition motif for endocytosis. Cell 63, 1061-1072	[Medline]
COURTOIS-COUTRY N., ROUSH, D., RAJENDRAN, V., MCCARTHY, J. B., GEIBEL, J., KASHGARIAN, M. & CAPLAN, M. J. (1997). A tyrosine-based signal targets H/K-ATPase to a regulated compartment and is required for the cessation of gastric acid secretion. Cell 90, 501-510	[Medline]
COX D., LEE, D. J., DALE, B. M., CALAFAT, J. & GREENBERG, S. (2000). A Rab11-containing rapidly recycling compartment in macrophages that promotes phagocytosis. Proceedings of the National Academy of Sciences of the USA 97, 680-685	[Abstract/Full Text]
DE HOSTOS E. L. (1999). The coronin family of actin-associated proteins. Trends in Cell Biology 9, 345-350	[Medline]
DELGROSSI M. H., BREUZA, L., MIRRE, C., CHAVRIER, P. & LE BIVIC, A. (1997). Human syntaxin 3 is localized apically in human intestinal cells. Journal of Cell Science 110, 2207-2214	[Abstract]
DI FIORE P. P., PELICCI, P. G. & SORKIN, A. (1997). EH: a novel protein-protein interaction domain potentially involved in intracellular sorting. Trends in Biochemical Sciences 22, 411-413	[Medline]
DRANSFIELD D. T., BRADFORD, A. J., SMITH, J., MARTIN, M., ROY, C., MANGEAT, P. & GOLDENRING, J. R. (1997). Ezrin is a cyclic AMP-dependent protein kinase anchoring protein. EMBO Journal 16, 35-43	[Abstract/Full Text]
DUMAN J. G., TYAGARAJAN, K., KOLSI, M. S., MOORE, H.-P. H. & FORTE, J. G. (1999). Expression of rab11a N124I in gastric parietal cells inhibits stimulatory recruitment of the H⁺-K⁺-ATPase. American Journal of Physiology 277, C361-372	[Medline]
FERNÁNDEZ-CHAC�N R., ACHIRILOAIE, M., JANZ, R., ALBANESI, J. P. & SÜDHOF, T. C. (2000). SCAMP1 function in endocytosis. Journal of Biological Chemistry 275, 12752-12756	[Abstract/Full Text]
FINGER F. P. & NOVICK, P. (1998). Spatial regulation of exocytosis: lessons from yeast. Journal of Cell Biology 142, 609-612	[Full Text]
FORTE J. G., FORTE, T. M. & SALTMAN, P. (1967). K⁺-stimulated phosphatase of microsomes from gastric mucosa. Journal of Cellular Physiology 69, 293-304	[Medline]
FORTE J. G., GANSER, A. L., BEESLEY, R. & FORTE, T. M. (1975). Unique enzymes of purified microsomes from pig fundic mucosa. Gastroenterology 69, 175-189	[Abstract]
FORTE J. G. & LEE, H. C. (1977). Gastric adenosinetriphosphatases: a review of their possible role in HCl secretion. Gastroenterology 73, 921-926	[Abstract]
FORTE J. G., LY, B., RONG, Q., OGIHARA, S., RAMILO, M., AGNEW, B. & YAO, X. (1998). State of actin in gastric parietal cells. American Journal of Physiology 274, C97-104	[Medline]
FORTE T. M., MACHEN, T. E. & FORTE, J. G. (1975). Ultrastructural and physiological changes in piglet oxyntic cells during histamine stimulation and metabolic inhibition. Gastroenterology 69, 1208-1222	[Abstract]
FORTE T. M., MACHEN, T. E. & FORTE, J. G. (1977). Ultrastructural changes in oxyntic cells associated with secretory function: a membrane recycling hypothesis. Gastroenterology 73, 941-955	[Medline]
GAIDAROV I., SANTINI, F., WARREN, R. A. & KEEN, J. H. (1999). Spatial control of coated-pit dynamics in living cells. Nature Cell Biology 1, 1-7	[Medline]
GAISANO H. Y., SHEU, L., FOSKETT, J. K. & TRIMBLE, W. S. (1994). Tetanus toxin light chain cleaves a vesicle-associated membrane protein (VAMP) isoform 2 in rat pancreatic zymogen granules and inhibits enzyme secretion. Journal of Biological Chemistry 269, 17062-17077	[Abstract]
GANSER A. L. & FORTE, J. G. (1973). K⁺-stimulated ATPase in purified microsomes of bullfrog oxyntic cells. Biochimica et Biophysica Acta 307, 169-180	[Medline]