title: Synaptotagmin I binds intestinal epithelial NHE3 and mediates cAMP- and Ca2+-induced endocytosis by recruitment of AP2 and clathrin.
abstract: Apical membrane sodium hydrogen exchanger 3 (NHE3), a major pathway for non-nutrient-dependent intestinal Na(+) absorption, is tightly regulated by second messenger systems that affect its functional activity and membrane trafficking. However, the events and components involved in NHE3 regulation are only partially understood. We report that the adaptor protein synaptotagmin I (Syt I) plays a pivotal role in cAMP- and Ca(2+)-induced cargo recognition of NHE3 and initiation of its endocytosis. Both mouse small intestine (jejunum) and Caco-2BBe Syt I coimmunoprecipitated with NHE3, particularly following increases in cellular cAMP or Ca(2+). Following short interfering RNA (siRNA) suppression of Syt I expression, cAMP- and Ca(2+)-induced inhibition of NHE3 activity were still observed but NHE3 endocytosis was blocked, as assessed by (22)Na influx and apical membrane biotin labeling, respectively. Similar effects on NHE3 inhibition and endocytosis were observed by siRNA suppression of either the mu-subunit of the adaptor protein 2 (AP2) complex or the heavy chain of clathrin. Coimmunoprecipitation analyses of NHE3 with these adaptor proteins revealed that cAMP- and Ca(2+)-induced NHE3-Syt I interaction preceded and was required for recruitment of AP2 and the clathrin complex. Confocal microscopy confirmed both the time sequence and protein associations of these events. We conclude that Syt I plays a pivotal role in mediating cAMP- and Ca(2+)-induced endocytosis of NHE3 (but not in inhibition of activity) through cargo recognition of NHE3 and subsequent recruitment of AP2-clathrin assembly required for membrane endocytosis.
authors.affiliation: Dept. of Medicine, MC 6084, The Univ. of Chicago Hospitals, 5841 S. Maryland Ave., Chicago, IL 60637, USA.
authors.forename: Mark W
authors.initials: MW
authors.lastname: Musch
authors.forename: Donna L
authors.initials: DL
authors.lastname: Arvans
authors.forename: Margaret M
authors.initials: MM
authors.lastname: Walsh-Reitz
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Uchiyama
authors.forename: Mitsunori
authors.initials: M
authors.lastname: Fukuda
authors.forename: Eugene B
authors.initials: EB
authors.lastname: Chang
mesh_terms: D033962:Adaptor Protein Complex 2 / Q000235:genetics / Q000378:metabolism*; D033983:Adaptor Protein Complex mu Subunits / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D018938:Caco-2 Cells; D020013:Calcium Signaling; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D033922:Clathrin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D004705:Endocytosis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D007413:Intestinal Mucosa / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D018613:Microscopy, Confocal; D011485:Protein Binding; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D050863:Synaptotagmin I / Q000235:genetics / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection
chemical_list: D033962:Adaptor Protein Complex 2; D033983:Adaptor Protein Complex mu Subunits; D004791:Enzyme Inhibitors; D034741:RNA, Small Interfering; C000613385:SLC9A3 protein, human; C000613386:Slc9a3 protein, mouse; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D050863:Synaptotagmin I; C494311:Syt1 protein, mouse; D033922:Clathrin Heavy Chains; D019284:Thapsigargin; D012964:Sodium; D000242:Cyclic AMP; D000252:Calcium-Transporting ATPases