Research Project
3498303
The biochemical basis for stress induced diseases, such as immunosuppression, inhibition of growth, neuronal degeneration and skeletal muscle atrophy and for neuropsychiatric disorders such as amnesia and Alzeheimer's disease are poorly understood. Adrenocorticotropic hormone (ACTH) and its receptor are essential molecular components in the perception and mediation of the stress response. In the past fifteen years, we have developed the tools for the successful detection, characterization and identification of ACTH receptors (ACTH-R). We now propose to isolate the ACTH-receptor gene from bovine adrenal cortical complimentary-DNA library and use the bovine gene to isolate the human gene for ACTH-R. These goals will be attained by the following research strategy: a) development of ligand binding and functional assays for ACTH- R in Xenopus oocytes microinjected with mRNA from bovine adrenal cortex; b) size fractionation of mRNA to identify the active mRNA fraction which will be used for the construction of cDNA library in a vector containing T7 promoter; c) identification of the ACTH-R gene containing sub-clones by the combination of in vitro transcription and assay of expression of RNA microinjected into oocytes; d) determination of the nucleotide sequence of the active sub-clone. The ACTH receptor gene will then be expressed in several established cell lines. The possible heterogeneity of ACTH receptor subtypes in the three zones of adrenal cortex and in the various regions of the brain will be studied by Northern analysis, in situ hydribization and immunocytochemistry using antibodies raised against synthetic peptides corresponding to the receptor sequence. Potent antagonists will be developed using cell lines over expressing ACTH- receptor gene.
