Sialylation of human thyrotropin receptor improves and prolongs its cell-surface expression.
Glycosylation of the thyrotropin receptor (TSHR) has been shown to be essential for correct protein folding and for cell-surface targeting. In a recent study, we detected increased expression of beta-galactoside alpha(2,6)-sialyltransferase (SIAT1) in toxic thyroid adenomas where gain-of-function mutations of the TSHR have been invoked as one of the major causes. To investigate the physiological meaning of these findings, we designed experiments to evaluate the consequences of sialylation for the expression of the TSHR. Hence, we investigated the effect of coexpressing the TSHR and different sialyltransferases (SIAT1, SIAT4a, and SIAT8a) for cell-surface expression of the receptor. Coexpression of each of the three SIAT isoforms and the TSHR in COS-7 cells increased TSHR expression on the cell surface in the range of 50 to 100%. Moreover, Western blot analysis with lectins specific for alpha(2,3) and alpha(2,6)-linked sialic acids and lectin-binding enzyme-linked immunosorbent assay support a direct effect on TSHR cell-surface expression mediated by sialic acid transfer to the TSHR. Finally, we treated living COS-7 cells after cotransfection of TSHR and SIAT8a with neuraminidase for 30 min to remove covalently linked sialic acid. Subsequent loss of TSHR cell-surface expression suggests that sialylation prolongs the resting time of the TSHR on the cell surface. Our data demonstrate for the first time that the transfer of sialic acid can improve and prolong cell-surface expression of a transmembrane receptor.
Frenzel
Romy
R
Medical Department, University of Leipzig, D-04103 Leipzig, Germany.
Krohn
Knut
K
Eszlinger
Markus
M
Tönjes
Anke
A
Paschke
Ralf
R
D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D011869:Radioligand Assay; D011989:Receptors, Thyrotropin / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012799:Sialyltransferases / Q000235:genetics
D012333:RNA, Messenger; D011989:Receptors, Thyrotropin; D000242:Cyclic AMP; D012799:Sialyltransferases; D019158:N-Acetylneuraminic Acid