Physical and functional interactions of SNAP-23 with annexin A2.
Lung surfactant is secreted through the fusion of lamellar bodies with the plasma membrane of alveolar epithelial type II cells. Annexin A2, a Ca(2+)- and phospholipid-binding protein, promotes the fusion of lamellar bodies with the plasma membrane. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are known to have an essential role in surfactant secretion. We hypothesized that annexin A2 acts as a Ca(2+) sensor and mediates membrane fusion via its interaction with SNAREs. Both purified or endogenous annexin A2 in type II cells specifically bound with SNAP-23 in a Ca(2+)-dependent manner, as determined by pull-down experiments using recombinant glutathione S-transferase-tagged SNAP-23. A deletion study identified the cysteine-rich region (CRR) of SNAP-23 as the binding site for annexin A2. Mutations of cysteine residues in the CRR dramatically decreased the binding. SNAP-23 also co-immunoprecipitated with annexin A2; however, a SNAP-23 mutant failed to co-immunoprecipitate with annexin A2. Immunofluorescence revealed a co-localization of SNAP-23 and annexin A2 in type II cells. Furthermore, anti-SNAP-23 antibody significantly inhibited annexin A2-mediated fusion between lamellar bodies and the plasma membrane. These data suggest that annexin A2 and SNAP-23 are involved in the same pathway in the regulation of lung surfactant secretion.
Wang
Pengcheng
P
Department of Physiological Sciences, Oklahoma State University, 264 McElroy Hall, Stillwater, OK 74078, USA.
Chintagari
Narendranath Reddy
NR
Gou
Deming
D
Su
Lijing
L
Liu
Lin
L
D000595:Amino Acid Sequence; D000818:Animals; D017306:Annexin A2 / Q000302:isolation & purification / Q000378:metabolism*; D000906:Antibodies / Q000494:pharmacology; D001665:Binding Sites; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003545:Cysteine; D006801:Humans; D047468:Immunoprecipitation; D008322:Mammals; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D020033:Protein Isoforms / Q000378:metabolism; D011650:Pulmonary Alveoli / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017384:Sequence Deletion; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000378:metabolism*
D017306:Annexin A2; D000906:Antibodies; D050505:Mutant Proteins; D020033:Protein Isoforms; C498858:Snap23 protein, rat; D033921:Vesicular Transport Proteins; D003545:Cysteine