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MUCOSAL BIOLOGY

Synaptotagmin I binds intestinal epithelial NHE3 and mediates cAMP- and Ca²⁺-induced endocytosis by recruitment of AP2 and clathrin

¹Martin Boyer Laboratories, Department of Medicine, The University of Chicago, Illinois; and ²Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Miyagi, Japan

Submitted 21 August 2006 ; accepted in final form 9 February 2007

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 Ca²⁺-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 Ca²⁺. Following short interfering RNA (siRNA) suppression of Syt I expression, cAMP- and Ca²⁺-induced inhibition of NHE3 activity were still observed but NHE3 endocytosis was blocked, as assessed by ²²Na influx and apical membrane biotin labeling, respectively. Similar effects on NHE3 inhibition and endocytosis were observed by siRNA suppression of either the µ-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 Ca²⁺-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 Ca²⁺-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