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.
Grenader
A C
AC
Department of Pharmacology, Mount Sinai School of Medicine, City University of New York, New York 10029.
O'Rourke
D A
DA
Healy
D P
DP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004848:Epithelium / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D018374:LLC-PK1 Cells; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017447:Receptors, Dopamine D1 / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013552:Swine; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D017447:Receptors, Dopamine D1