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

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.

SUBMITTER: Musch MW 

PROVIDER: S-EPMC2822502 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature