Role of P63 (CKAP4) in binding of surfactant protein-A to type II pneumocytes

1 Institute for Environmental Medicine and 2 Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania Submitted 17 March 2008 ; accepted in final form 15 August 2008 We have recently described a putative receptor for lung surfactant protein-A (SP-A) on rat type II pneumocytes. The receptor, P63, is a 63-kDa type II transmembrane protein. Coincubation of type II cells with P63 antibody (Ab) reversed the inhibitory effect of SP-A on secretagogue-stimulated surfactant secretion from type II cells. To further characterize SP-A interactions with P63, we expressed recombinant P63 protein in Escherichia coli and generated antibodies to P63. Immunogold electron microscopy confirmed endoplasmic reticulum and plasma membrane localization of P63 in type II cells with prominent labeling of microvilli. Binding characteristics of iodinated SP-A to type II cells in the presence of P63 Ab were determined. Binding (4°C, 1 h) of 125 I-SP-A to type II cells demonstrated both specific (calcium-dependent) and nonspecific (calcium-independent) components. Ab to P63 protein blocked the specific binding of 125 I-SP-A to type II cells and did not change the nonspecific SP-A association. A549 cells, a pneumocyte model cell line, expressed substantial levels of P63 and demonstrated specific binding of 125 I-SP-A that was inhibited by the P63 Ab. The secretagogue (cAMP)-stimulated increase in calcium-dependent binding of SP-A to type II cells was blocked by the presence of P63 Ab. Transfection of type II cells with small interfering RNA to P63 reduced P63 protein expression, attenuated P63-specific SP-A binding, and reversed the ability of SP-A to prevent surfactant secretion from the cells. Our results further substantiate the role of P63 as an SP-A receptor protein localized on the surface of lung type II cells. lung; receptor; small interfering RNA; transmembrane protein; secretion Address for reprint requests and other correspondence: S. R. Bates, 1 John Morgan Bldg., Institute for Environmental Medicine, 3620 Hamilton Walk, Univ. of Pennsylvania, Philadelphia, PA 19104 (e-mail: batekenn{at}mail.med.upenn.edu )