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.