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1 Institute for Environmenal Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States
2 Institute for Environmental Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States
3 Institute for Environmental Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States; Philadelphia, Pennsylvania, United States
4 Dept. of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States; Institute for Environmenal Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States
* To whom correspondence should be addressed. E-mail: batekenn{at}mail.med.upenn.edu.
Surfactant protein (SP)-A binds to alveolar type II cells through a specific high affinity cell membrane receptor, although the molecular nature of this receptor is unclear. In the present study we have identified and characterized an SP-A cell surface binding protein by utilizing two chemical cross-linkers: Profound Sulfo-SBED protein-protein interaction reagent and Dithiobis succinimidylpropionate (DSP). Sulfo-SBED-biotinylated SP-A was cross-linked to the plasma membranes isolated from rat type II cells and the biotin label was transferred from SP-A to its receptor by reduction. The biotinylated SP-A-binding protein was identified on blots by using streptavidin-labeled horseradish peroxidase. By using DSP, we cross-linked SP-A to intact mouse type II cells and immunoprecipitated the SP-A-receptor complex using anti-SP-A antibody. Both of the cross-linking approaches showed a major band of 63 kDa under reduced conditions that was identified as the rat homologue of the human type II transmembrane protein p63 (CKAP4/ERGIC-63/CLIMP-63) by matrix-assisted laser desorption ionization and nano-electrospray tandem mass spectrometry of tryptic fragments. Thereafter, we confirmed the presence of p63 protein in the cross-linked SP-A-receptor complex by immuno-probing with p63 antibody. Co-immunoprecipitation experiments and functional assays confirmed specific interaction between SP-A and p63. Antibody to p63 could block SP-A-mediated inhibition of ATP-stimulated phospholipid secretion. Both intracellular and membrane localized pools of p63 were detected on type II cells by immunofluorescence and immunobloting. P63 co-localized with SP-A in early endosomes. Thus, p63 closely interacts with SP-A and may play a role in the trafficking or the biological function of the surfactant protein.
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