Cloning and expression of the alveolar type II cell P2u-purinergic receptor.
Adenosine triphosphate (ATP) regulates surfactant phospholipid secretion from alveolar type II cells by interacting with P2-purinoceptors on the alveolar type II cell surface. To further characterize regulation of surfactant secretion, we have cloned the type II cell P2u-purinoceptor and expressed a functional receptor in an unrelated cell line. The coding sequence of the P2u 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 P2u-purinoceptor previously identified in the hybrid neuroblastoma x glioma cell line, NG 108-15, 87% homology to the recently cloned human P2u-purinoceptor, and 34% homology to the P2u-purinoceptor cloned from chicken brain. The putative type II cell P2u protein contains seven membrane-spanning domains, characteristic of G-protein-coupled receptors. The type II cell P2u-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 P2u-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.(ABSTRACT TRUNCATED AT 250 WORDS)
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Children's Hospital Medical Center, Division of Neonatology, Cincinnati, Ohio 45229-3039.
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Fiedeldey
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism; D009928:Organ Specificity; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011650:Pulmonary Alveoli / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D018048:Receptors, Purinergic P2 / Q000096:biosynthesis / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013047:Specific Pathogen-Free Organisms; D014162:Transfection
D018076:DNA, Complementary; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; C000717609:Tgm2 protein, rat; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D002118:Calcium