title: Failed export of the adrenocorticotrophin receptor from the endoplasmic reticulum in non-adrenal cells: evidence in support of a requirement for a specific adrenal accessory factor.
abstract: Difficulty in expressing the adrenocorticotrophin (ACTH) receptor (melanocortin 2 receptor; MC2R) after transfection of various MC2R expression vectors has been experienced by many researchers. Reproducible evidence for expression has been obtained only in the Y6/OS3 corticoadrenal cell lines or in cells expressing endogenous melanocortin receptors. In order to determine the cause of this failure of expression we have undertaken the following studies. An MC2R expression plasmid was constructed in which the green fluorescent protein (GFP) coding region had been added to the C-terminus of the mature protein. Transfection of this plasmid into Y6 cells with a cAMP-responsive reporter plasmid demonstrated normal function of this receptor. Imaging of CHO cells expressing MC2R-GFP revealed perinuclear expression, although a cholecystokinin receptor (CCKR)-GFP construct was efficiently expressed at the cell surface. Y6 cells, in contrast, showed cell surface fluorescence after transfection with MC2R-GFP. Several other cell types showed a similar pattern of GFP distribution characteristic of retention in the endoplasmic reticulum. Counterstaining with an anti-KDEL antibody confirmed this location. Co-expression of the MC2R and the CCKR-GFP did not impair CCKR trafficking to the cell surface, implying a receptor-specific impairment to trafficking in the CHO cell which was absent in the Y6 cell.
authors.affiliation: Department of Endocrinology, Barts and the London, Queen Mary School of Medicine, West Smithfield, University of London, UK.
authors.forename: L A
authors.initials: LA
authors.lastname: Noon
authors.forename: J M
authors.initials: JM
authors.lastname: Franklin
authors.forename: P J
authors.initials: PJ
authors.lastname: King
authors.forename: N J
authors.initials: NJ
authors.lastname: Goulding
authors.forename: L
authors.initials: L
authors.lastname: Hunyady
authors.forename: A J L
authors.initials: AJ
authors.lastname: Clark
mesh_terms: D000324:Adrenocorticotropic Hormone / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D044103:Receptor, Melanocortin, Type 2; D011949:Receptors, Cholecystokinin / Q000378:metabolism; D018043:Receptors, Corticotropin / Q000378:metabolism*; D014162:Transfection
chemical_list: D008164:Luminescent Proteins; D044103:Receptor, Melanocortin, Type 2; D011949:Receptors, Cholecystokinin; D018043:Receptors, Corticotropin; D049452:Green Fluorescent Proteins; D000324:Adrenocorticotropic Hormone