Adrenocorticotropic hormone (ACTH) and angiotensin II (AII) stimulate steroidogenesis in bovine fasciculata cells but by different mechanism. ACTH stimulation is cAMP dependent, AII stimulation is cAMP independent and appear to operate by way of the protein kinase C pathway. We propose to apply the avidn-biotin technology to identify and characterize the receptor for both hormone from bovine adrenal cortical cells and membranes and to isolate the receptors from membranes. 125I-labeled dethiobiotinylated ACTH derivatives will serve as probes for ACTH receptors and their affinity for the receptors on the one hand and for succinoylavidin on the other will be exploited for receptor isolation. In one series of experiments, the probe we be covalently attached to adrenal membranes by the use of crosslinking reagents and the ensuing hormone-receptor complex will be isolated from the detergent solubilized membranes on columns of Sepharose 4B immobilized succinoylvidin. The crosslinked hormone-receptor complex will be removed from the affinity resin and will be examined by SDS-PAGE and autoradiography. The crosslinking experiments will be performed both in the absence and presence of unlabeled ACTH1-24 to determine the specificity of the crosslinking. The homogeneity of the isolated hormone-receptor complex will be assessed and it will be purified to homogeneity by conventional technique (HPLC). Using adrenal membranes as the source of receptor, we expect to produce sufficient hormone- receptor complex for sequence studies. The complex will be digested with trypsin and from the mixture of the resulting peptides, we expect to isolate a homogeneous peptide which will be subjected to microsequencing. An oligonucleotide corresponding to the peptide sequence will be synthesized and used as a probe to arrive at the cDNA and consequently, at the amino acid sequence of the ACTH receptor. A systematic study will be undertaken to solubilize the ACTH receptor of bovine adrenal membranes with retention of its ability to bind 1251-labeled dethiobiotinylated ACTH derivatives. If successful, we shall attempt to isolate the solubilized receptor by avidin-biotin technology. A parallel study aimed at isolation of AII receptors will be conducted simultaneously using the principles and techniques described for isolation of the ACTH receptor. Noteworthy is the fact that we have been successful in solubilizing the AII receptor with retention of binding affinity for I251-labeled biotinylated AII probes.
| Finn, F M; Hofmann, K (1990) Isolation and characterization of hormone receptors. Methods Enzymol 184:244-74 |
