Cyclic AMP-mediated endocytosis of intestinal epithelial NHE3 requires binding to synaptotagmin 1

Musch, Mark W.; Arvans, Donna L.; Wang, Yunwei; Nakagawa, Yasushi; Solomaha, Elena; Chang, Eugene B.

doi:10.1152/ajpgi.00379.2009

Cited by 22 publications

(16 citation statements)

References 48 publications

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“…Interestingly, and similar to our results, Chang and colleagues (36,37) recently demonstrated that synaptotagmin 1 is present in intestinal epithelial cells by RT-PCR, immunoblotting and direct sequencing, and further showed synaptotagmin 1 localizes to the apical membrane by immunofluorescence. We also found that synaptotagmin 1 localized to the apical aspects of acini but surprisingly showed incomplete overlap with VAMP-2 on the plasma membrane.…”

Section: Discussionsupporting

confidence: 91%

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Expression, localization, and functional role for synaptotagmins in pancreatic acinar cells

Falkowski¹,

Thomas²,

Messenger³

et al. 2011

American Journal of Physiology-Gastrointestinal and Liver Physi

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induced changes in intracellular Ca 2ϩ play a pivotal role in secretion in pancreatic acini yet the molecules that respond to Ca 2ϩ are uncertain. Zymogen granule (ZG) exocytosis is regulated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. In nerve and endocrine cells, Ca 2ϩ -stimulated exocytosis is regulated by the SNARE-associated family of proteins termed synaptotagmins. This study examined a potential role for synaptotagmins in acinar secretion. RT-PCR revealed that synaptotagmin isoforms 1, 3, 6, and 7 are present in isolated acini. Immunoblotting and immunofluorescence using three different antibodies demonstrated synaptotagmin 1 immunoreactivity in apical cytoplasm and ZG fractions of acini, where it colocalized with vesicle-associated membrane protein 2. Synaptotagmin 3 immunoreactivity was detected in membrane fractions and colocalized with an endolysosomal marker. A potential functional role for synaptotagmin 1 in secretion was indicated by results that introduction of synaptotagmin 1 C2AB domain into permeabilized acini inhibited Ca 2ϩ -dependent exocytosis by 35%. In contrast, constructs of synaptotagmin 3 had no effect. Confirmation of these findings was achieved by incubating intact acini with an antibody specific to the intraluminal domain of synaptotagmin 1, which is externalized following exocytosis. Externalized synaptotagmin 1 was detected exclusively along the apical membrane. Treatment with CCK-8 (100 pM, 5 min) enhanced immunoreactivity by fourfold, demonstrating that synaptotagmin is inserted into the apical membrane during ZG fusion. Collectively, these data indicate that acini express synaptotagmin 1 and support that it plays a functional role in secretion whereas synaptotagmin 3 has an alternative role in endolysosomal membrane trafficking. zymogen granules; exocytosis PANCREATIC ACINAR CELLS ARE a prototypical model of exocrine secretory cells that undergo stimulated exocytosis of digestive enzymes, which are necessary for the assimilation of the diet. Multiple intracellular signals including diacylglycerol, cAMP and inositol triphosphate (IP 3 )-mediated release of Ca 2ϩ have been shown to stimulate acinar exocytosis. However, the pivotal role of Ca 2ϩ in the overall secretory response is underscored by findings that double knockout of the type 2 and 3 IP 3 receptors fully abolished secretagogue-induced changes in cellular Ca 2ϩ and resulted in a complete lack of salivary and pancreatic acinar secretion (11). Although the critical importance of Ca 2ϩ signaling in acinar exocytosis is well established, the exocytic regulatory proteins that respond to elevated Ca 2ϩ in the cytoplasm are largely uncertain.Synaptotagmins are a family of membrane proteins composed of 15 known isoforms distributed widely in neuronal and nonneuronal tissues (for comprehensive reviews see Refs. 4,39,45). Family members have the same basic structure consisting of a short NH 2 -terminal intravesicular domain followed by a transmembrane domain and a large COOH-termina...

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Section: Discussionsupporting

confidence: 91%

“…Synaptotagmin 1 was highly localized to the apical membrane of enterocytes in the small intestine and was likewise present in a colon carcinoma cell line (Caco-2BBe) (36,37). Expression of synaptotagmin 1 was confirmed by RT-PCR, immunoblotting, small interfering RNA (siRNA) studies and by direct sequencing of the immunoprecipitated protein.…”

mentioning

confidence: 92%

Expression, localization, and functional role for synaptotagmins in pancreatic acinar cells

Falkowski¹,

Thomas²,

Messenger³

et al. 2011

American Journal of Physiology-Gastrointestinal and Liver Physi

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“…Calcium Inhibition of NHE3 Activity in Polarized OK Cells-NHE3 is acutely inhibited by elevation in the concentration of intracellular Ca 2ϩ by the cholinergic agonist carbachol (24 -26) as well as by serotonin (27), adenosine 1 receptor agonists (9), and thapsigargin and Ca 2ϩ ionophores (18,28,29). Decreased NHE3 activity also occurs in PS120 fibroblasts with Ca 2ϩ ionophores (10,11).…”

Section: Resultsmentioning

confidence: 99%

Elevated Calcium Acutely Regulates Dynamic Interactions of NHERF2 and NHE3 Proteins in Opossum Kidney (OK) Cell Microvilli

Zhu

Cha

Zachos

et al. 2011

Journal of Biological Chemistry

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Background: Na/H exchanger 3 is fixed to the BB cytoskeleton but is regulated by changes in trafficking. Results: Elevated Ca 2ϩ rapidly decreases BB NHE3 amount/activity and transiently decreases NHE3 association with NHERF2 and increases NHE3 BB mobility. Conclusion: There is coordinated regulation of trafficking and NHE3 BB cytoskeletal association. Significance: A newly recognized aspect of signal transduction in epithelial cells is dynamic/transient changes in BB transporter-cytoskeleton association.

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“…Clathrin-and caveolin-dependent pathways are well-defined cellular endocytic routes previously shown to be involved in the basal and regulated internalization of ion transporters (21,26,27,33,37). The endocytosis inhibitor dynasore inhibits dynamin, a 100-kDa GTPase required to pinch-off coated vesicles from the plasma membrane, and, thereby, prevents both clathrin-and caveolin-dependent endocytosis (23).…”

Section: Role Of Dynamin-dependent Endocytic Pathways In Regulating Amentioning

confidence: 99%

Mechanisms of DRA recycling in intestinal epithelial cells: effect of enteropathogenic E. coli

Gujral

Kumar

Priyamvada

et al. 2015

American Journal of Physiology-Cell Physiology

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opathogenic Escherichia coli (EPEC) is a food-borne pathogen that causes infantile diarrhea worldwide. EPEC decreases the activity and surface expression of the key intestinal Cl Ϫ /HCO 3 Ϫ exchanger SLC26A3 [downregulated in adenoma (DRA)], contributing to the pathophysiology of early diarrhea. Little is known about the mechanisms governing membrane recycling of DRA. In the current study, Caco-2 cells were used to investigate DRA trafficking under basal conditions and in response to EPEC. Apical Cl Ϫ /HCO 3 Ϫ exchange activity was measured as DIDS-sensitive 125 I Ϫ uptake. Cell surface biotinylation was performed to assess DRA endocytosis and exocytosis. Inhibition of clathrin-mediated endocytosis by chlorpromazine (60 M) increased apical Cl Ϫ /HCO 3 Ϫ exchange activity. Dynasore, a dynamin inhibitor, also increased function and surface levels of DRA via decreased endocytosis. Perturbation of microtubules by nocodazole revealed that intact microtubules are essential for basal exocytic (but not endocytic) DRA recycling. Mice treated with colchicine showed a decrease in DRA surface levels as visualized by confocal microscopy. In response to EPEC infection, DRA surface expression was reduced partly via an increase in DRA endocytosis and a decrease in exocytosis. These effects were dependent on the EPEC virulence genes espG1 and espG2. Intriguingly, the EPEC-induced decrease in DRA function was unaltered in the presence of dynasore, suggesting a clathrin-independent internalization of surface DRA. In conclusion, these studies establish the role of clathrin-mediated endocytosis and microtubules in the basal surface expression of DRA and demonstrate that the EPEC-mediated decrease in DRA function and apical expression in Caco-2 cells involves decreased exocytosis. DRA recycling; EPEC infection; endocytosis; exocytosis; clathrin; microtubules DOWNREGULATED IN ADENOMA (DRA), or SLC26A3, is an integral membrane transporter expressed on the apical membrane of epithelial cells, including the small intestine and colon. Mutations in the SLC26A3 gene are associated with congenital chloride diarrhea (CLD), characterized by voluminous diarrhea with high fecal chloride content (28). DRA knockout mice exhibit severe diarrhea and a phenotype similar to that of CLD patients (32). Emerging studies indicate that posttranslational modifications of DRA protein may underlie the pathophysiology of diarrheal diseases. For example, CLD-causing mutations induce diarrhea by causing loss of functional DRA protein at the plasma membrane, possibly via its misfolding and mistrafficking (25,36). Studies from our laboratory have shown that enteropathogenic Escherichia coli (EPEC) infection decreases DRA function and surface levels (7), which may underlie early diarrhea; however, the cellular recycling mechanisms of DRA under normal or diseased conditions are not well understood. Thus, investigation of the cellular pathways responsible for maintaining appropriate DRA levels on the apical membrane is important for understanding disease pathogenesis...

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Cyclic AMP-mediated endocytosis of intestinal epithelial NHE3 requires binding to synaptotagmin 1

Cited by 22 publications

References 48 publications

Expression, localization, and functional role for synaptotagmins in pancreatic acinar cells

Expression, localization, and functional role for synaptotagmins in pancreatic acinar cells

Elevated Calcium Acutely Regulates Dynamic Interactions of NHERF2 and NHE3 Proteins in Opossum Kidney (OK) Cell Microvilli

Mechanisms of DRA recycling in intestinal epithelial cells: effect of enteropathogenic E. coli

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