CLONING OF GRF AND SOMATOSTATIN RECEPTOR cDNA : EXPRESSION IN PITUITARY ADENOMA OF ACROMEGALIC PATIENT.

基于已克隆的各种G蛋白偶联受体之间跨膜结构域中的cDNA序列的同源性，合成简并寡引物。在λ噬菌体中制备了从肢端肥大症患者中获得的产生GH的人垂体腺瘤的cDNA文库，所述肢端肥大症患者显示对GRF或生长抑素的GH反应。使用寡引物的各种组合通过PCR筛选cDNA文库。在一些实验中，还通过RT-PCR筛选从垂体瘤中提取的poly-A RNA。PCR扩增产物经凝胶电泳分离后，将其亚克隆到Bluescript载体上进行测序。经DNA序列分析，26个克隆中有5个克隆可能是未知G蛋白偶联受体的一部分。以这些PCR产物为探针，通过噬斑杂交再次筛选cDNA文库。5个噬菌斑为阳性，并进行DNA测序。其中之一是已经克隆的全长大麻素受体cDNA，然而，其配体仍然未知。有趣的是，大麻素受体在产生GH的垂体腺瘤中表达，这种表达的意义应进一步研究。然而，几个月前，其他实验室报道了克隆GRF和生长抑素受体。目前，我们正在分析其他4个克隆的DNA序列，并试图分离新的受体，因为垂体腺瘤可能表达各种未知的G蛋白偶联受体，调节肿瘤生长和GH分泌。

Degenerate oligoprimers were synthesized based on the homology of cDNA sequence in the transmembrane domain between various G-protein coupled receptors which are already cloned. cDNA libraries of GH producing human pituitary adenomas obtained from acromegalic patients who showed GH responses to GRF or somatostatin were prepared in lambda phage. cDNA libraries were screened by PCR using various combination of the oligoprimers. In some experiments, poly-A RNA extracted from the pituitary tumors were also screened by RT-PCR. PCR products, mainly 300-500bp, were size-fractionated by gel electrophoresis, then, subcloned into Bluescript vector for DNA sequencing. After analysis of DNA sequence, five out of 26 clones were found a possible part of unknown G-protein coupled receptor. Using these PCR products as a probe, cDNA libraries were screened again by plaque hybridization. Five plaques were positive and subjected to DNA sequence. One of them were full size cannabinoid receptor cDNA which was already cloned,however, its ligand was still unknown. It is interesting that the cannabinoid receptor is expressed in GH producing pituitary adenoma, and significance of this expression should be further investigated. However, cloning GRF and somatostatin receptors has been reported by other laboratories few month ago. Now, we are analyzing DNA sequence of other 4 clones and trying to isolate new receptors since the pituitary adenomas may express various unknown G-protein coupled receptors which regulates the tumor growth and GH secreation.