CLONING AND EXPRESSION OF THE ALVEOLAR TYPE-II CELL P-2U-PURINERGIC RECEPTOR

Adenosine triphosphate (ATP) regulates surfactant phospholipid secretion from alveolar type II cells by interacting with P-2-purinoceptors on the alveolar type II cell surface. To further characterize regulation of surfactant secretion, we have cloned the type II cell P-2u-purinoceptor and expressed a functional receptor in an unrelated cell line. The coding sequence of the P-2u clone isolated from a type II cell cDNA library was 1.1 kb, encoding a putative protein of 374 amino acids. The putative protein demonstrated > 97% homology with the P-2u-purinoceptor previously identified in the hybrid neuroblastoma X glioma cell line, NG 108-15, 87% homology to the recently cloned human P-2u-purinoceptor, and 34% homology to the P-2u-purinoceptor cloned from chicken brain. The putative type II cell P-2u protein contains seven membrane-spanning domains, characteristic of G-protein-coupled receptors. The type II cell P-2u-purinoceptor nucleotide sequence also demonstrated > 95% homology to the nucleotide sequence of the NG 108-15 clone, However, the type II cell cDNA also demonstrated presence of an additional 208 bp insert in the 5' untranslated region, which was not present in the NG 108-15 clone. Using reverse transcriptase polymerase chain reaction, we examined expression of the two different sizes of mRNA in various rat tissues. Only the larger type II cell mRNA was expressed in rat heart, kidney, lung, spleen, and testis, with no expression of P-2u-purinoceptor mRNA noted in brain or liver. The smaller species of mRNA was only detected in mouse N18-TG2 cells, and these cells expressed a larger species as well, found in the rat tissues noted. The type II cell clone was ligated into a eukaryotic expression vector and transfected into A-10 cells not normally expressing P-2u-purinoceptors. Clonal lines derived from these transfections exhibited presence of a functional P-2u receptor as evidenced by Ca2+ mobilization in response to ATP or uridine triphosphate. Since characterization of purinoceptors has been hampered by lack of specific antagonists, this P-2u-purinoceptor clone, which represents the predominant species of mRNA expressed in rat tissues, should aid in further characterization of this important G-protein-coupled receptor.