|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1992 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1991 : ¥1,300,000 (Direct Cost : ¥1,300,000)
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.