Identification and characterization of p63 (CKAP4/ERGIC-63/CLIMP-63), a surfactant protein A binding protein, on type II pneumocytes.
Surfactant protein A (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 homolog of the human type II transmembrane protein p63 (CKAP4/ERGIC-63/CLIMP-63) by matrix-assisted laser desorption ionization and nanoelectrospray tandem mass spectrometry of tryptic fragments. Thereafter, we confirmed the presence of p63 protein in the cross-linked SP-A-receptor complex by immunoprobing with p63 antibody. Coimmunoprecipitation 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 colocalized 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.
Institute for Environmental Medicine, University of Pennsylvania School of Medicine, Philadelphia, 19104, USA.
Nisha
N
Gupta
Yefim
Y
Manevich
Altaf S
AS
Kazi
Jian-Qin
JQ
Tao
Aron B
AB
Fisher
Sandra R
SR
Bates
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D047468:Immunoprecipitation; D008168:Lung / Q000166:cytology / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D011485:Protein Binding; D011650:Pulmonary Alveoli / Q000378:metabolism*; D037662:Pulmonary Surfactant-Associated Protein A / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism*
C081876:CKAP4 protein, human; D008565:Membrane Proteins; D037662:Pulmonary Surfactant-Associated Protein A; D011956:Receptors, Cell Surface; C067774:surfactant protein A receptor