Synaptotagmin I binds intestinal epithelial NHE3 and mediates cAMP-and [Ca.sup.2+]-induced endocytosis by recruitment of AP2 and clathrin

Apical membrane sodium hydrogen exchanger 3 (NHE3), a major pathway for non-nutrient-dependent intestinal [Na.sup.+] 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.sup.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.sup.2+]. Following short interfering RNA (siRNA) suppression of Syt I expression, cAMP- and [Ca.sup.2+]-induced inhibition of NHE3 activity were still observed but NHE3 endocytosis was blocked, as assessed by [sup.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.sup.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.sup.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. sodium/hydrogen exchange; cyclic nucleotides; calcium; Caco-2BBe cells; membrane trafficking; water and electrolyte transport; intestinal Na absorption; diarrheal diseases; adaptor protein 2 doi: 10.1152/ajpgi.00388.2006