Document Detail

Expression of biologically active procorticotrophin-releasing hormone (proCRH) in stably transfected CHO-K1 cells: characterization of nuclear proCRH.
MedLine Citation:
PMID:  7647768     Owner:  NLM     Status:  MEDLINE    
Corticotrophin-releasing hormone (CRH) is a 41 amino acid neuropeptide which is cleaved at a pair of dibasic amino acids from a larger precursor molecule (pre-proCRH) by the action of endopeptidases. In cells possessing a regulated secretory pathway, sorting of proneuropeptides and prohormones occurs within the trans-Golgi network, where they are finally 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 subcellular compartment. We have recently established stably transfected CHO-K1 cells expressing the rat pre-proCRH cDNA, and shown that proCRH was localized within the secretory pathway and the nucleus of transfected cells. Both the cytoplasmic and nuclear species of IR-CRH displayed an apparent molecular weight approximately 19 kDa, consistent with the size of the uncleaved CRH precursor molecule. In this paper, we further characterized the bitopological, i.e. nuclear and cytoplasmic localization of proCRH within transfected CHO-K1 cells. Immunoreactive nuclear CRH was not extractable using detergents (Triton X-100 and CHAPS), 10 mM salt washes or RNase digestion but could be abolished by digestion with DNase I. These results therefore suggest that nuclear proCRH is in close association with DNA/chromatin. Treatment of transfected cells with inhibitors of protein and RNA synthesis for up to 24 h had no effect upon immunoreactive nuclear CRH, indicating that it is very stable with a long half life. Brefeldin A treatment had no effect upon the nuclear translocation of newly synthesized proCRH, suggesting that late stages of the secretory pathway (i.e. post rough endoplasmic reticulum compartments) of the transfected cells do not play a role in proCRH nuclear transport. We also demonstrate that proCRH synthesized within stably transfected CHO-K1 cells is capable of stimulating ACTH release from primary cultures of anterior pituitary cells, therefore showing for the first time that the intact precursor is also biologically active and could act as an ACTH secretagogue in-vivo.
E Morrison; P Tomasec; E A Linton; P J Lowry; P R Lowenstein; M G Castro
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of neuroendocrinology     Volume:  7     ISSN:  0953-8194     ISO Abbreviation:  J. Neuroendocrinol.     Publication Date:  1995 Apr 
Date Detail:
Created Date:  1995-09-27     Completed Date:  1995-09-27     Revised Date:  2009-09-29    
Medline Journal Info:
Nlm Unique ID:  8913461     Medline TA:  J Neuroendocrinol     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  263-72     Citation Subset:  IM    
Department of Physiology, School of Molecular and Medical Biosciences, University of Wales College of Cardiff, UK.
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MeSH Terms
Adrenocorticotropic Hormone / secretion
CHO Cells
Cell Nucleus / chemistry*
Corticotropin-Releasing Hormone / analysis*
Cytoplasm / chemistry
Deoxyribonuclease I
Endoplasmic Reticulum / metabolism
Fluorescent Antibody Technique
Golgi Apparatus / drug effects
Pituitary Gland, Anterior / secretion
Protein Precursors / analysis*
Subcellular Fractions / chemistry
Transfection / genetics*
Grant Support
//Wellcome Trust
Reg. No./Substance:
0/Protein Precursors; 9002-60-2/Adrenocorticotropic Hormone; 9015-71-8/Corticotropin-Releasing Hormone; EC I

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