Cloning of the dopamine-1A (D1A) receptor gene expressed in porcine renal epithelial cells.
We sought to determine the molecular identify of the dopamine-1 (D1) receptor expressed in the porcine renal epithelial cell line LLC-PK1. We first isolated a partial cDNA by the reverse transcription-polymerase chain reaction procedure and then used the partial cDNA to isolate positive overlapping clones from a porcine genomic DNA library. Sequence analysis of the gene revealed that the longest open-reading frame encoded a 446 amino acid protein that was 95% identical to the human D1A receptor. Expression studies in mammalian cells were also consistent with the clones encoding a D1 receptor. Northern blot hybridizations with LLC-PK1 poly (A+) RNA were strongly positive. The porcine D1A gene has two exons and a short intron in the 5' untranslated region. The 5' flanking region lacks a TATA and CAAT box but is high in GC content (68%) and contains multiple Sp1 binding sites. The 5' flanking region also contains numerous other cis-acting elements for transcription factors. These results indicate that the D1A receptor is the major D1 receptor expressed in LLC-PK1 cells and further suggest that LLC-PK1 cells may be a useful model to study the regulation of renal D1A receptor gene transcription.
Healy
D P
DP
Department of Pharmacology, Mount Sinai School of Medicine, City University of New York, NY 10029, USA.
O'Rourke
D A
DA
Grenader
A C
AC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D018374:LLC-PK1 Cells; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D017447:Receptors, Dopamine D1 / Q000235:genetics*; D013552:Swine; D014158:Transcription, Genetic
D017931:DNA Primers; D017447:Receptors, Dopamine D1