Cloning of the porcine D1A dopamine receptor gene expressed in renal epithelial LLC-PK1 cells.
Cloning of the porcine D1A dopamine receptor gene expressed in renal epithelial LLC-PK1 cells.
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肾上皮 LLC-PK1 细胞中表达的猪 D1A 多巴胺受体基因的克隆。
DOI：
10.1152/ajprenal.1995.268.3.f423
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Healy,DP
中科院分区：
文献类型：
--
作者：
Grenader,AC;O'Rourke,DA;Healy,DP
猪肾上皮细胞系LLC-PK 1表达与腺苷酸环化酶刺激偶联的D1多巴胺受体。这种受体的分子身份是未知的。本研究以LLC-PK 1 poly（A）+ RNA为模板，采用简并D1受体寡核苷酸引物，通过逆转录-聚合酶链反应（RT-PCR）技术，从LLC-PK 1 poly（A）+ RNA中扩增出部分cDNA，并将其用于猪基因组文库的筛选。一个这样的基因组克隆（λ PGD1A.1）包含一个开放阅读框，编码一个446个氨基酸的蛋白质，与人类D1 A受体有95%的相同性。瞬时转染到COS-7细胞中的基因组克隆的功能特性与D1受体的表达一致。用LLC-PK 1 poly（A）+ RNA进行的RNA杂交分析为阳性。引物延伸分析表明，在LLC-PK 1细胞中表达的猪D1 A基因的主要转录起始位点是翻译起始位点上游的1，033个核苷酸。该基因的5 '侧翼区缺乏TATA和CAAT盒，但GC含量高（68%），并含有多个Sp1结合位点。在5 '-非编码区内有一个97-bp的内含子，分隔外显子1和2。这些结果进一步支持了D1 A受体是肾脏中表达的主要D1受体的观点，并进一步表明LLC-PK 1细胞可能是研究肾脏D1 A受体基因调控的有用模型。
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.