Extracellular ATP binding proteins as potential receptors in mucociliary epithelium: Characterization using [32P]3?-O-(4-benzoyl)benzoyl ATP, a photoaffinity label

Abstract

3'-O-(4-benzoyl)benzoyl ATP (BzATP) was used as a photoaffinity analog of ATP to label potential ATP receptors in ciliated cells. Like ATP, without photoactivation, BzATP stimulated the ciliary beat frequency in tissue culture up to threefold. Irradiation of intact cells in the presence of [alpha-32P]BzATP followed by SDS-PAGE and autoradiography revealed two labeled proteins with molecular masses of 46 and 96 kDa (p46 and p96). Photolabeling of both proteins was susceptible to digestion with trypsin, implying that the labeled proteins are at least partially exposed on the extracellular surface of the plasma membrane. The dependence of 32P incorporation in both proteins on [alpha-32P]BzATP concentration was similar. Labeling of p46 but not p96 required Ca2+ or Mg2+. Various nucleotides stimulated the ciliary frequency, and inhibited the photolabeling of p46 and p96. The rank order of apparent affinity for p46 is: ATP approximately equal to ADP > GTP gamma S > ADP beta S, UTP, 2MeSATP, AMP-PNP > AMP-PCP > AMP > adenosine; for p96 it is: ADP approximately equal to ADP beta S > or = ATP >> AMP-PCP, AMP-PNP > GTP gamma S > or = AMP > 2MeSATP, UTP, adenosine. The rank of stimulation of ciliary beat frequency is: ADP beta S, UTP > or = 2MeSATP, GTP gamma S, AMP-PNP, ATP > or = ADP > AMP-PCP > adenosine > AMP. These results suggest the involvement of p46 in the stimulatory effect of extracellular ATP on the ciliary beat, as a P2 purinoceptor. On the other hand, p96 may represent a P2 purinoceptor or an ectonucleotidase.