Cell-surface expression of RhD blood group polypeptide is posttranscriptionally regulated by the RhAG glycoprotein.
In most cases, the lack of Rh in Rh(null) red cells is associated with RHAG gene mutations. We explored the role of RhAG in the surface expression of Rh. Nonerythroid HEK293 cells, which lack Rh and RhAG, or erythroid K562 cells, which endogenously express RhAG but not Rh, were transfected with RhD and/or RhAG cDNAs using cytomegalovirus (CMV) promoter-based expression vectors. In HEK293 cells, a low but significant expression of RhD was obtained only when RhAG was expressed at a high level. In K562 cells, as expected from the opposite effects of the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) on erythroid and CMV promoters, the levels of endogenous RhAG and recombinant RhD transcripts were substantially decreased and enhanced upon TPA treatment of RhD-transfected cells (K562/RhD), respectively. However, flow cytometry and fluorescence microscopy analysis revealed a decreased cell-surface expression of both RhAG and RhD proteins. Conversely, TPA treatment of RhAG-transfected cells increased both the transcript and surface expression levels of RhAG. When K562/RhD cells were cotransfected by the RhAG cDNA, the TPA-mediated induction of recombinant RhAG and RhD transcription was associated with an increased membrane expression of both RhAG and RhD proteins. These results demonstrate the role of RhAG as a strictly required posttranscriptional factor regulating Rh membrane expression. In addition, because the postulated 2:2 stoichiometry between Rh and RhAG observed in the native red cell membrane could not be obtained in cotransfected K562 cells, our study also suggests that as yet unidentified protein(s) might be involved for optimal membrane expression of Rh.
Institut National de la Santé et de la Recherche Médicale U76, Faculté de Médecine Xavier Bichat, Paris, France.
Isabelle
I
Mouro-Chanteloup
Anne Marie
AM
D'Ambrosio
Pierre
P
Gane
Caroline
C
Le Van Kim
Virginie
V
Raynal
Didier
D
Dhermy
Jean-Pierre
JP
Cartron
Yves
Y
Colin
D000954:Antigens, Surface / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D001798:Blood Proteins; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006801:Humans; D020014:K562 Cells; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008856:Microscopy, Fluorescence; D021381:Protein Transport / Q000187:drug effects; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012204:Rh-Hr Blood-Group System / Q000737:chemistry / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D000954:Antigens, Surface; D001798:Blood Proteins; D008562:Membrane Glycoproteins; C498523:RHAG protein, human; D011994:Recombinant Proteins; D012204:Rh-Hr Blood-Group System; D013755:Tetradecanoylphorbol Acetate