BRIEF REPORT |
Correspondence to: Maria G. Castro, Molecular Medicine Unit, Dept. of Medicine, U. of Manchester, Stopford Bldg., Rm. 1.302, Oxford Road, Manchester M139PT, UK. E-mail: mcastro@fs2.scg. man.ac.uk..
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Summary |
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We investigated the intracellular localization of CRH in transiently transfected COS-7 cells expressing the full-length rat corticotropin-releasing hormone (CRH) precursor cDNA. CRH synthesized by transfected COS-7 cells is mainly stored intracellularly. In contrast, CHO-K1 cells expressing the same CRH precursor stored and released equal amounts of immunoreactive (IR)-CRH. Ultrastructural analysis revealed that CRH is stored in electron-dense aggregates in the RER of transiently transfected COS-7 cells and does not migrate into the Golgi apparatus. On the basis of the different intracellular localization, storage, and release of CRH in COS-7 and CHO-K1 cells, we hypothesize that the intracellular trafficking of CRH within the constitutive secretory pathway for protein secretion not only depends on its primary amino acid sequence but might also be influenced by intracellular conditions or factors. (J Histochem Cytochem 46:11931197, 1998)
Key Words: corticotropin-releasing, hormone, intracellular trafficking, constitutive secretory pathway, COS-7, CHO
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Introduction |
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In the central nervous system, CRH is mainly expressed by and secreted from neurons of the hypothalamic paraventricular nucleus (
The establishment of the stably transfected CHO-K1 cell line expressing the rat CRH precursor has been previously described (
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To investigate the molecular forms of CRH synthesized by transiently transfected COS-7 cells, cell extracts were subjected to Western blotting as previously described (
To determine the time course of biosynthesis of CRH, COS-7 cells were metabolically labeled using [35S]-methionine/[35S]-cysteine at 48 hr post transfection. CRH was detected using immunoprecipitation followed by SDS-PAGE and autoradiography as described previously (
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To elucidate the intracellular localization of IR-CRH within transiently transfected COS-7 cells, we performed immunoelectron microscopy. Figure 2A shows a transfected COS-7 cell next to an untransfected cell. The transfected cell contains the electron-dense structures within tubular membrane-bound compartments (Figure 2A, Figure 2B, and Figure 2D). The membranes are studded with ribosomes, identifying the organelle as the RER. Immunogold particles surrounding electron-dense aggregates confirm that they are immunoreactive for CRH (Figure 2C). Figure 2D shows the Golgi apparatus (G), which does not contain any electron-dense structures (arrows). Therefore, in transfected COS-7 cells, CRH is present within electron-dense aggregates in the RER. Transiently transfected COS-7 cells were also examined with confocal immunofluorescence microscopy (data not shown). IR-CRH did not co-localize with the Golgi marker TGN38 but it did co-localize with ER resident proteins (
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Our EM data indicate that CRH condensation/aggregation occurs early within the secretory pathway of transfected COS-7 cells, i.e, the ER. This would also explain the relatively low levels of secreted IR-CRH detected in this study and the accumulation of intracellular IR-CRH within COS-7 cells. It has been previously shown that by starving and refeeding guinea pigs or injecting them with CoCl2 solution, the appearance of intracisternal granules (ICGs) within the RER of the exocrine pancreatic cells increases dramatically (
Our findings differ from previous studies on the release of secretory proteins from COS cells (
The molecular mechanisms that mediate the condensation/aggregation phenomenon encountered with the CRH precursor in COS-7 cells are at present unknown. Abnormal folding of the CRH precursor, as well as its possible association with cell proteins, might lead to aggregate formation within transfected COS-7 cells. Based on the differential behavior of CRH within both COS-7 and CHO-K1 transfected cells, we hypothesize that trafficking of the CRH precursor within the constitutive secretory pathway of COS-7 and CHO-K1 cells might be influenced by association with chaperones and/or the intracellular milieu.
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Footnotes |
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1 Should be considered first authors.
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Acknowledgments |
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Supported by project grants from the Biotechnology and Biological Sciences Research Council (Chemicals and Pharmaceuticals Directorate) and The Welcome Trust. We acknowledge the support from the Medical Research Council (MRC, UK), the Sir Halley Stewart Trust, the Cancer Research Campaign, the Royal Society, the Parkinson's Disease Society, REMEDI, and the Department of Medicine, University of Manchester. PRL is a Research Fellow of The Lister Institute of Preventive Medicine, UK.
We thank Dr B. Eipper for the TGN38 antibody and Dr E. Coudrier and Dr D. Louvard for the ER resident protein antibody.
Received for publication February 11, 1998; accepted May 26, 1998.
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