Cloning of the porcine D1A dopamine receptor gene expressed in renal epithelial LLC-PK1 cells.

The porcine renal epithelial cell line LLC-PK1 expresses a D1 dopamine receptor coupled to stimulation of adenylyl cyclase. The molecular identity of this receptor is unknown. We isolated a partial cDNA from LLC-PK1 poly(A)+ RNA by the reverse transcription-polymerase chain reaction procedure with degenerate D1 receptor oligonucleotide primers and used the partial cDNA to screen a porcine genomic library. One such genomic clone (lambda PGD1A.1) contained an open-reading frame that encoded a 446-amino-acid protein that is 95% identical to the human D1A receptor. The functional properties of the genomic clone transiently transfected into COS-7 cells were consistent with expression of a D1 receptor. RNA hybridization analyses with LLC-PK1 poly(A)+ RNA were positive. Primer extension analysis indicated that the primary transcription initiation site of the porcine D1A gene expressed in LLC-PK1 cells is 1,033 nucleotides upstream from the translation start site. The 5'-flanking region of the gene lacks a TATA and CAAT box but is high in GC content (68%) and contains multiple Sp1 binding sites. There is a 97-bp intron within the 5'-noncoding region, separating exons 1 and 2. These results add support to the view that the D1A receptor is the major D1 receptor expressed in kidney and further suggest that LLC-PK1 cells may be a useful model for study of the regulation of the renal D1A receptor gene.