Ac. Grenader et al., CLONING OF THE PORCINE D-1A DOPAMINE-RECEPTOR GENE EXPRESSED IN RENALEPITHELIAL LLC-PK1 CELLS, American journal of physiology. Renal, fluid and electrolyte physiology, 37(3), 1995, pp. 423-434
The porcine renal epithelial cell line LLC-PK1 expresses a D-1 dopamin
e receptor coupled to stimulation of adenylyl cyclase. The molecular i
dentity of this receptor is unknown. We isolated a partial cDNA from L
LC-PK1 poly(A)(+) RNA by the reverse transcription-polymerase chain re
action procedure with degenerate D-1 receptor oligonucleotide primers
and used the partial cDNA to screen a porcine genomic library. One suc
h genomic clone (lambda PGD1A.1) contained an open-reading frame that
encoded a 446-amino-acid protein that is 95% identical to the human D-
1A receptor. The functional properties of the genomic clone transientl
y transfected into COS-7 cells were consistent with expression of a D-
1 receptor. RNA hybridization analyses with LLC-PK1 poly(A)(+) RNA wer
e positive. Primer extension analysis indicated that the primary trans
cription initiation site of the porcine D-1A 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 hi
gh in GC content (68%) and contains multiple Sp1 binding sites. There
is a 97-bp intron within the 5'-noncoding region, separating exons 1 a
nd 2. These results add support to the view that the D-1A receptor is
the major D-1 receptor expressed in kidney and further suggest that LL
C-PK1 cells may be a useful model for study of the regulation of the r
enal D-1A receptor gene.