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.

Functional coupling of the downregulated in adenoma Cl−/base exchanger DRA and the apical Na+/H+exchangers NHE2 and NHE3

“…Second messenger-induced DRA endocytosis is dependent on association with the adaptor protein Syt I with subsequent recruitment of AP2 and clathrin. The process therefore appears to be common with that observed for apical NHEs (34).…”

“…To determine whether DRA activity is regulated by second messenger cAMP or intracellular calcium, we treated DRA transgene-infected Caco2BBE monolayers with either the permeable 8-CPT-cAMP (100 M, 15 min) or the Ca 2ϩ -ATPase inhibitor thapsigargin (100 ng/ml, 15 min) to increase intracellular free Ca 2ϩ . These agents inhibit a large percentage of the apical Na ϩ influx mediated by NHE2 and NHE3 (34). Both of these second messenger pathway regulators also inhibited DRA activity (Fig.…”

“…Stealth oligonucleotides specific for human synaptotagmin I (Syt I; GenBank NM_005639, bases 613-637), the -subunit of adaptor protein 2 (AP2; NM_004068, bases 79 -103), or the heavy chain of clathrin (NM_004859, bases 3307-3331), or altered base controls for each, were used to silence these proteins involved in apical membrane protein trafficking as previously described (34). Two treatments of Stealth oligos were used, with the last at 12 h before experiments.…”

Myosin VI mediates the movement of NHE3 down the microvillus in intestinal epithelial cells

“…Basal microvillus NHE3 was increased by myosin VI KD, whereas the secondmessenger-induced (FSH and CCH) decrease in microvillus NHE3 activity and amount was blocked. Because these two agents have been shown to stimulate endocytosis without altering the rates of exocytosis (Gekle et al, 2002;Lee-Kwon et al, 2003;Musch et al, 2007;Zachos et al, 2013), we propose that both basal and stimulated endocytosis require myosin VI. Additionally, because EGF treatment of myosin VI KD cells still led to an increase in microvillar NHE3 activity, our results further indicate that myosin VI is not involved in the stimulated exocytosis of NHE3 in the Na + -absorptive cell.…”

Role for the Src Family Kinase Fyn in Sphingolipid Acquisition by Chlamydiae

“…Depletion of either synaptotagmin 1 or 2 resulted in increased sphingomyelin trafficking to the inclusion. Synaptotagmins are calcium binding proteins that are best known for their role in vesicular trafficking at synapses but are also expressed in nonsynaptic cells (40,43). Synaptotagmins have extensive roles in cellular signaling pathways, including regulating exocytic vesicles from the Golgi apparatus (57) and binding the MVB marker CD63 (18); both pathways are implicated in sphingomyelin trafficking to the inclusion (5,22,48).…”