Requirement of N-glycosylation of the prostaglandin E2 receptor EP3beta for correct sorting to the plasma membrane but not for correct folding.
Eight heptahelical receptors have been characterized for prostaglandin (PG) D(2), PGE(2), PGF(2alpha), prostacyclin and thromboxane A(2). They share a sequence identity of 40%. All of them have potential N-glycosylation sites. The current study analysed the role of the two N-glycosylation sites in the rat EP3beta-subtype PGE(2) receptor for protein folding and sorting. The N-glycosylation consensus sequences were eliminated by site-directed mutagenesis and receptors expressed in HEK-293 cells. Both potential N-glycosylation sites were used. Their joint elimination resulted in the synthesis of a receptor protein with full binding competence, biological activity and no reduction of affinity; however, the half-life of the non-glycosylated receptor was slightly reduced. Ligand binding to intact stably transfected cells and confocal laser microscopic immunocytochemistry showed that the glycosylated receptor was correctly inserted into the plasma membrane to a much larger extent than the non-glycosylated receptor, which tended to accumulate in the perinuclear zone of the endoplasmic reticulum. Inhibition of N-glycosylation with tunicamycin resulted in a similar perinuclear distribution of the wild-type receptor. Therefore, glycosylation of the EP3beta receptor seems not to be necessary for correct folding of the receptor protein but for the efficient transport of the receptor protein to the plasma membrane. This contrasts with a previous finding which described a reduction of the affinity for PGE(2) of the EP3alpha receptor by elimination of the distal glycosylation site when the receptor protein was expressed in insect cells.
Böer
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Universität Potsdam, Institut für Ernährungswissenschaft, Arthur-Scheunert-Allee 114-116, D-14558 Bergholz-Rehbrücke, Germany.
Neuschäfer-Rube
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F
Möller
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Püschel
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GP
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers; D006031:Glycosylation; D006801:Humans; D007150:Immunohistochemistry; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D021382:Protein Sorting Signals; D018078:Receptors, Prostaglandin E / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D021382:Protein Sorting Signals; D018078:Receptors, Prostaglandin E