Identification and characterization of p63 (CKAP4/ERGIC-63/CLIMP-63), a surfactant protein A binding protein, on type II pneumocytes

Abstract

) 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.lung; surfactant secretion; cross-linking; immunolocalization PULMONARY SURFACTANT IS A complex mixture of phospholipids and proteins. The major protein component, surfactant protein-A (SP-A), is a 34-to 36-kDa member of the calciumdependent lectin family of proteins that facilitates the surface tension-lowering properties of surfactant phospholipids in the alveolus; regulates surfactant phospholipid synthesis, secretion, and recycling; and also plays an important role in pulmonary host defense (18). The mechanism of SP-A binding to lung type II cells is not completely understood, yet it appears to be critical for the cell-associated functions of SP-A. Binding of SP-A to type II cells was found to be both saturable and specific, suggesting that this interaction is mediated through a receptor on the cell surface (24, 55).Thus far, investigators have identified SP-A receptor(s) by utilizing either anti-idiotypic antibodies (43, 46) or SP-A affinity column chromatography (9, 21). Using an idiotypic approach, one group has identified three proteins with apparent molecular mass values of 30, 52, and 60 kDa (44), and the 30-kDa protein was shown to be associated with the regulation of secretagogue-stimulated surfactant secretion (45, 46). On the other hand, another group ...

CKAP4 is a Dickkopf1 receptor and is involved in tumor progression

“…Taken together, these results suggest that expression of both proteins is necessary for cellular proliferation. SP-A and tPA were known to act as ligands for CKAP4 (27,28). SP-A was expressed little in S2-CP8 and A549 cells (Supplemental Figure 9A), and tPA was expressed in S2-CP8 but not A549 cells (Supplemental Figure 9B).…”

“…CKAP4 was originally discovered as a protein that mainly localized to the ER (25) and later shown to function as a ligand-specific receptor (27)(28)(29). SP-A blocks surfactant secretion from lamellar bodies in lung type II pneumocytes (28,43).…”

“…SP-A blocks surfactant secretion from lamellar bodies in lung type II pneumocytes (28,43). SP-A binds to CKAP4 to regulate surfactant clearance in pneumocytes, and CKAP4 knockdown suppresses SP-A-mediated inhibition of surfactant secretion (44).…”

“…It was originally discovered as a protein that is localized to the ER and binds to microtubules. Subsequently, CKAP4 was shown to be localized to the cell surface membrane of type II pneumocytes, bladder epithelial cells, and vascular smooth muscle cells, where it functions as a receptor for several ligands, including surfactant protein A (SP-A), tissue plasminogen activator (tPA), and anti-proliferating factor (APF) (27)(28)(29).…”

The serpentine path to a novel mechanism-based inhibitor of acute inflammatory lung injury

“…Secreted phospholipids that are bound in the alveolar space to surfactant protein A (SP-A) are endocytosed by type II cells utilizing a receptormediated process (48). We have recently described the SP-A receptor associated with this process as a protein called p63 (45). Intracellularly, the endocytosed phospholipids are sorted to either resecretion or degradative pathways; degradation products either are utilized for resynthesis of surfactant components, or they enter the general metabolic pool (13,30).…”