Annexin A7 and SNAP23 interactions in alveolar type II cells and in vitro: A role for Ca2+ and PKC

Abstract

Lung surfactant secretion involves lamellar body docking and fusion with the plasma membrane in alveolar type II cells. Annexin A7 (A7) is postulated to play a role in membrane fusion during exocytosis. Our recent studies demonstrated increased co-localization of A7 with ABCA3 in lamellar bodies in type II cells stimulated with established secretagogues of lung surfactant. In this study, we investigated in vivo and in vitro interactions of A7 with the t-SNARE protein, SNAP23. Immuno-fluorescence studies showed time-dependent increases in co-localization of A7 with SNAP23 in PMA- and in A23187-stimulated cells. PMA and A23187 also caused a time-dependent increase in co-localization of ABCA3 with SNAP23. The relocation of A7 to SNAP23 domains was inhibited in the presence of PKC inhibitor, similar to that previously reported for co-localization of A7 with ABCA3. The interaction of A7 and SNAP23 was confirmed by affinity binding and by in vitro interaction of recombinant A7 and SNAP23 proteins. The in vitro binding of recombinant A7 (rA7) to GST-SNAP23 fusion protein was calcium-dependent. Phosphorylation of rA7 with PKC increased its in vitro binding to SNAP23 suggesting that a similar mechanism may operate during A7 relocation to t-SNARE domains. Thus, our studies demonstrate that annexin A7 may function in co-ordination with SNARE proteins and that protein kinase activation may be required for annexin A7 trafficking to the interacting membranes (lamellar bodies and plasma membrane) to facilitate membrane fusion during surfactant secretion.

Spectral phasor analysis of LAURDAN fluorescence in live A549 lung cells to study the hydration and time evolution of intracellular lamellar body-like structures

“…The molecular mechanism underlying exocytosis is a very specialized process that involves cytoskeletal machinery (F-Actin) [12] and particular proteins like Annexin 2 and 7 [13,14], SNAP-23 [15] and ABCA-3 [16]. As with many other secretory pathways, the LB secretory machinery uses Ca 2+ as a second messenger [17].…”

Annexins – insights from knockout mice

“…Like other annexins, AnxA7 translocates from the cytosol to these locations in a Ca 2+ -dependent manner (Kuijpers et al, 1994). Many reports support a role of AnxA7 in multiple aspects of Ca 2+ /GTP-dependent exocytic pathways (Srivastava et al, 1999;Caohuy and Pollard, 2002;Gerelsaikhan et al, 2012;Taniuchi et al, 2012;Chander et al, 2013). Related to Ca 2+ homeostasis, GTPase activity and possibly other cellular functions, AnxA7 participates in prostaglandin production, cardiac remodeling and inflammatory myopathies, but also cell survival and tumor growth (Voelkl et al, 2014;Luo et al, 2015).…”

Annexin A7 trafficking to alveolar type II cell surface: Possible roles for protein insertion into membranes and lamellar body secretion

“…The non-specific sites were blocked by equilibration for 30 min at 4°C with detergent-free blocking buffer containing 10% donkey/goat serum and 1% BSA. For permeabilizing protocol [23], the cells fixed in 4% PFA for 10 min at room temperature, washed (×3) and stored in PBS at 4°C until further processing. Before staining, the cells were simultaneously blocked and permeabilized for 30 min in blocking buffer supplemented with 0.1% Triton X-100 and 0.05% Tween 20 [23].…”

“…Stimulation of alveolar type II cells with several secretagogues of lung surfactant increases membrane-association of A7 [22]. The target membranes for A7 trafficking in stimulated type II cells include lamellar bodies and plasma membranes, as demonstrated by in vitro binding [21] and by immuno-fluorescence co-localization with ABCA3 [22] and SNAP23 [23]. Such trafficking is regulated by protein kinase-dependent phosphorylation of A7 [22, 23].…”