Isolation and Partial Characterization of a Receptor to Surfactant Protein A Expressed by Rat Type II Pneumocytes

Abstract

Surfactant protein A (SP-A), the most abundant protein component in pulmonary surfactant, has been shown to enhance surfactant phospholipid uptake by the type II alveolar epithelial cell. Recent evidence has shown that this process may be receptor-mediated. We undertook this study to isolate the putative receptor from type II cell membranes. We isolated two specific SP-A binding proteins from type II cells with apparent molecular weights (Mr) of 86 and > 200 kD under nonreducing conditions. Under reducing conditions, the higher-Mr protein was not present, but three proteins with apparent Mr of 65, 55, and 50 kD were visible, in addition to the 86-kD protein, indicating that the higher-Mr protein was composed of the smaller peptides which form disulfide bonds. The 86-kD protein is a glycoprotein with approximately 30% of its mass as carbohydrate. The 50-kD protein is also a glycoprotein (approximately 30% of its mass as carbohydrate), and SP-A binds to the protein core. Polyclonal and monoclonal antibodies to these peptides saturably bind to the surface of type II cells but not other lung cells, as shown by immunohistochemistry. SP-A competitively inhibits binding of one monoclonal antibody to type II cells, and the monoclonal antibody was able to block the effect of SP-A on phospholipid uptake by type II cells, indicating that this complex is a receptor to SP-A which is expressed on type II cells. This novel receptor is fundamental to the biology of surfactant metabolism in the lung.

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

“…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 using similar strategy identified a 55-kDa protein (43) shown to be involved in surfactant endocytosis by type II cells (53,55). Using ligand affinity chromatography, three proteins with apparent molecular mass values of 65, 55, and 50 kDa were identified by a third group on the type II cell surface, and the 50-kDa protein was shown to be involved in phospholipid uptake (21). A fourth group described a SP-A receptor from U937 macrophages with a molecular mass of 210 kDa using SP-A affinity column chromatography (9).…”

Role of the PI3-kinase signaling pathway in trafficking of the surfactant protein A receptor P63 (CKAP4) on type II pneumocytes

“…SP-A binding and uptake by the lung and type II cells in culture occur via a receptormediated clathrin-coated pit pathway (25,40). Thus our attention has been focused on the characterization of the SP-A receptor responsible for SP-A-mediated clearance of surfactant phospholipid.Several potential type II cell receptors for SP-A have been isolated and partially characterized (12,29,45,47). Recently, we have identified an SP-A-type II cell surface binding protein, P63, using chemical cross-linking techniques (22).…”

“…Several potential type II cell receptors for SP-A have been isolated and partially characterized (12,29,45,47). Recently, we have identified an SP-A-type II cell surface binding protein, P63, using chemical cross-linking techniques (22).…”

Surfactant protein-A plays an important role in lung surfactant clearance: evidence using the surfactant protein-A gene-targeted mouse

“…Clearance of SP-A from the lungs occurs via a classical receptor-mediated clathrin-coated pit pathway as uptake of SP-A by the lung was sensitive to clathrin inhibitors and gold-labeled SP-A was shown to bind to type II cells in coated plasma membrane invaginations (30,43). Several candidate SP-A receptors on the surface of type II cells have been described, including the transmembrane protein P63 (13,24,33,46,47). Since secretagogue treatment enhanced calcium-dependent SP-A binding and recruitment of SP-A receptors to the type II cell membrane (11), it has been proposed that SP-A enhancement of phospholipid uptake occurs through this clathrin-dependent mechanism.…”