EXPRESSION OF BIOLOGICALLY-ACTIVE PROCORTICOTROPHIN-RELEASING HORMONE(PROCRH) IN STABLY TRANSFECTED CHO-K1 CELLS - CHARACTERIZATION OF NUCLEAR PROCRH

Corticotrophin-releasing hormone (CRH) is a 41 amino acid neuropeptide which is cleaved at a pair of dibasic amino acids from a larger precu rsor molecule (pre-proCRH) by the action of endopeptidases. In cells p ossessing a regulated secretory pathway, sorting of proneuropeptides a nd prohormones occurs within the trans-Golgi network, where they are f inally packaged into secretory vesicles to be released in response to an external stimulus. Such cells also possess a constitutive secretory pathway, and neuropeptides are also translocated into this subcellula r compartment. We have recently established stably transfected CHO-K1 cells expressing the rat pre-proCRH cDNA, and shown that proCRH was lo calized within the secretory pathway and the nucleus of transfected ce lls, Both the cytoplasmic and nuclear species of IR-CRH displayed an a pparent molecular weight of approximately 19 kDa, consistent with the size of the uncleaved CRH precursor molecule, In this paper, we furthe r characterized the bitopological, i.e. nuclear and cytoplasmic locali zation of proCRH within transfected CHO-K1 cells. Immunoreactive nucle ar CRH was not extractable using detergents (Triton X-100 and CHAPS), 10 mM salt washes or RNase digestion but could be abolished by digesti on with DNase I, These results therefore suggest that nuclear proCRH i s in close association with DNA/chromatin. Treatment of transfected ce lls with inhibitors of protein and RNA synthesis for up to 24 h had no effect upon immunoreactive nuclear CRH, indicating that it is very st able with a long half life. Brefeldin A treatment had no effect upon t he nuclear translocation of newly synthesized proCRH, suggesting that late stages of the secretory pathway (i.e. post rough endoplasmic reti culum compartments) of the transfected cells do not play a role in pro CRH nuclear transport. We also demonstrate that proCRH synthesized wit hin stably transfected CHO-KI cells is capable of stimulating ACTH rel ease from primary cultures of anterior pituitary cells, therefore show ing for the first time that the intact precursor is also biologically active and could act as an ACTH secretagogue in-vivo.