Gerard N P, Garraway L A, Eddy R L, Shows T B, Iijima H, Paquet J L, Gerard C
Department of Medicine, Beth Israel Hospital, Boston, Massachusetts 02215.
Biochemistry. 1991 Nov 5;30(44):10640-6. doi: 10.1021/bi00108a006.
The gene for the human substance P receptor (NK-1) was cloned using cDNA probes made by the polymerase chain reaction from primers based on the rat sequence. The gene spans 45-60 kb and is contained in five exons, with introns interrupting at sites homologous to those in the NK-2 receptor gene. Analysis of restriction digests of genomic DNA from mouse/human cell hybrids indicates the NK-1 receptor is a single-copy gene located on human chromosome 2. Polymerase chain reaction using primers based on the 5' and 3' ends of the coding sequence was used to generate full-length cDNAs from human lung and from IM9 lymphoblast cells. When transfected into COS-7 cells, the NK-1 receptor binds 125I-BHSP with a Kd of 0.35 +/- 0.07 nM and mediates substance P induced phosphatidylinositol metabolism. The receptor is selective for substance P; the relative affinity for neurokinin A and neurokinin B is 100- and 500-fold lower, respectively. Human IM9 lymphoblast cells express relatively high levels of the NK-1 receptor, and Northern blot analysis indicates modulation of mRNA levels by glucocorticoids and growth factors, suggesting that this cell line may be useful as a model for studying the control of NK-1 receptor gene expression.
利用基于大鼠序列的引物通过聚合酶链反应制备的cDNA探针克隆了人P物质受体（NK-1）基因。该基因跨度为45 - 60 kb，包含在五个外显子中，内含子在与NK-2受体基因中同源的位点处中断。对来自小鼠/人细胞杂种的基因组DNA的限制性酶切分析表明，NK-1受体是位于人类2号染色体上的单拷贝基因。使用基于编码序列5'和3'末端的引物进行聚合酶链反应，以从人肺和IM9淋巴母细胞中产生全长cDNA。当转染到COS-7细胞中时，NK-1受体以0.35 +/- 0.07 nM的Kd结合125I-BHSP，并介导P物质诱导的磷脂酰肌醇代谢。该受体对P物质具有选择性；对神经激肽A和神经激肽B的相对亲和力分别低100倍和500倍。人IM9淋巴母细胞表达相对高水平的NK-1受体，Northern印迹分析表明糖皮质激素和生长因子可调节mRNA水平，提示该细胞系可用作研究NK-1受体基因表达调控的模型。
