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. 2010 Feb;298(2):G203-11.
doi: 10.1152/ajpgi.00379.2009. Epub 2009 Nov 19.

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

Mark W Musch  1 , Donna L Arvans, Yunwei Wang, Yasushi Nakagawa, Elena Solomaha, Eugene B Chang
Affiliations

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

Mark W Musch et al. Am J Physiol Gastrointest Liver Physiol. 2010 Feb.

Abstract

The apical membrane Na(+)-H(+) exchanger (NHE)3 is regulated by cAMP-dependent phosphorylation, which inhibits its activity through membrane endocytosis. The clathrin complex adaptor protein synaptotagmin 1 (Syt 1) appears to be essential to this process, but little is known about its expression in intestinal epithelial cells or interaction with NHE3. The intestinal epithelial expression and apical location of Syt 1 were determined by Syt 1 mRNA profiling and immunolocalization. Tandem mass spectrometry was used for protein identification. Bis(sulfosuccinimidyl) suberate (BS(3)) cross linking suggested that NHE3 and Syt 1 were in a membrane complex following cAMP stimulation of Caco2BBE (Brush Border Expressions) cells. To investigate the regulation of NHE3 appearance in a Syt 1-containing membrane compartment, doxycycline-inducible hemaglutinin (HA)-tagged NHE3 was expressed in Caco2BBE cells. HA-NHE3 correctly targeted to the apical membrane, where, upon cAMP stimulation, it was internalized with a Syt 1-containing compartment. Site-directed mutagenesis of NHE3 showed that serine 605 (S605) was pivotal to NHE3 and Syt 1 association and internalization. Direct Syt 1 interaction with NHE3 was suggested by fluorescence resonance energy transfer (FRET) analysis. The physiological role of S552 was less clear. By FRET, this serine residue appeared to be involved in cAMP-induced Syt 1 binding of NHE3. However, when HA-tagged NHE3 S552A was expressed in Caco2 cells, the mutated construct was not inserted into the apical membrane. We conclude that intestinal epithelial Syt 1 plays an important role in cAMP-stimulated endocytosis of apical NHE3 through cAMP-dependent phosphorylation of S605 that is required for NHE3 and Syt 1 association.

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Figures

Fig. 1.
Fig. 1.
Synaptotagmin (Syt) 1 mRNA is expressed in mouse jejunum and brain and in human Caco2BBE cells. RNA was isolated from mouse jejunum (top, left) and Caco2BBE (top, right) monolayers (after 14 days confluence), reverse transcribed, and amplified using primers for mouse or human Syts. Bottom: RNA was isolated from Caco2BBE cells at 2 days (little differentiation) and 14 days (fully differentiated), mouse jejunum crypt or villus enterocytes (isolated by laser capture), whole mouse jejunal scrapings, and brain as a positive control. Arrows indicate PCR products isolated and sequenced. Images shown are representative of those of 3 separate experiments. SLP, Syt-like protein.
Fig. 2.
Fig. 2.
Tryptic peptides of immunoprecipitated Syt 1 confirm mouse intestinal and Caco2BBE expression. Syt 1 was immunoprecipitated from mouse jejunal brush-border membranes or Caco2BBE cell lysates, run on SDS-PAGE, eluted from the gel, digested with peptides, and analyzed by tandem mass spectrometry as described in materials and methods. Underlined amino acids are the identified peptides, and all had a confidence level of greater than 95% as predicted by Scaffold protein sequencing software.
Fig. 3.
Fig. 3.
Syt 1 and Na+/H+ exchanger (NHE)3 cross linking following cAMP stimulation. Caco2BBE cells were untreated or stimulated with cAMP (100 μM for 15 min) when appropriate. Cells were quickly chilled, and microsomal membranes were isolated and reacted with the 11.4 Angstrom cross linker bis(sulfosuccinimidyl) suberate (BS3) for 30 min at 4°C as designated. Membranes were solubilized and immunoprecipitated with either anti-Syt 1 or anti-NHE3 rabbit polyclonal antisera. Samples were washed and eluted, run on 5% SDS-PAGE for Western blots, and analyzed for Syt 1 or NHE3. An aliquot of each immunoprecipitation reaction was removed before addition of antibodies and analyzed for total Syt 1 or NHE3. Images are representative of those from 3 separate experiments.
Fig. 4.
Fig. 4.
Mutation of serine 605 to alanine (S605A) of NHE3 blocks its interaction with Syt 1 and endocytosis following cAMP stimulation. Histidine (His)-tagged, tet promoter regulated wild-type rat NHE3, and 2 mutant constructs (S505A and S605A) were transfected into Caco2BBE cells stably expressing the reverse tetracycline transactivator. For all 3 NHE3 cDNA, doxycycline (Dox) addition resulted in hemaglutinin (HA)-tagged proteins that could be biotinylated at the apical surface and had the same molecular mass as NHE3. Similar to the endogenous NHE3, the HA-tagged wild-type and S505A mutation were internalized upon cAMP stimulation, demonstrated by detection of the disulfide-cleaved, biotin-labeled NHE3 within the cell (middle row). These constructs could also be co-immunoprecipitated with Syt 1 (Co-IP). In contrast, the S605A NHE3 mutation failed to internalize or associate with Syt 1. Of note, thapsigargin (Thaps) (which increases cytosolic Ca), stimulated membrane endocytosis of all 3 transgene products as well as endogenous NHE3. Thus the S605 mutation is specific to cAMP-regulated NHE3 membrane trafficking. Images shown are representative of 4 separate experiments.
Fig. 5.
Fig. 5.
Syt 1 and NHE3 direct binding enables fluorescent resonance energy transfer. His-tagged full-length Syt 1 was labeled with (5-(((2-iodoacetyl)amino)ethyl)amino naphthalene-1-sulfonic acid) (IAEDANS), and His-tagged rat NHE3 COOH-terminal cytoplasmic tail (amino acids 436–831) was labeled with (4-(4-(dimethylamino) phenyl) azo)benzoic acid, succinimidyl ester (Dabcyl). IAEDANS-Syt 1 was excited at 295 nm, and emission was measured over the range 350–595 nm. NHE3 was phosphorylated in vitro by cAMP-dependent protein kinase with (left) or without ATP (right). Addition of all NHE3 constructs from reactions without ATP did not change the Syt 1 emission spectrum. However, addition of the wild-type (WT) NHE3 that had been phosphorylated by cAMP-dependent protein kinase caused a concentration-related shift in the emission spectral profile and decrease in emission intensity (top, left). Similar results were obtained with the NHE3 S505A mutation. When the PKA-phosphorylated S605A mutation was used, no changes were observed upon addition of the His-Dabcyl-tagged S605A NHE3. Addition of the NHE3 S552A mutation caused a concentration-dependent shift in the emission spectrum that differed from the wild-type or S505A mutation and slightly increased the emission maximum. Spectra shown were obtained on a Hitachi FluoroMax-3 fluorometer and are representative of those of 3 separate experiments with different reagents (IAEDANS-Syt 1 and Dabcyl-NHE3) in each experiment.
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