Requirement of N-glycosylation of the prostaglandin E-2 receptor EP3 beta 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(2 alpha), 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 t he rat EP3 beta-subtype PGE, receptor for protein folding and sorting. The N-glycosylation consensus sequences were eliminated by site-directed mutage nesis and receptors expressed in HEK-293 cells. Both potential N-glycosylat ion 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 recep tor was slightly reduced. Ligand binding to intact stably transfected cells and confocal laser microscopic immunocytochemistry showed that the glycosy lated receptor was correctly inserted into the plasma membrane to a much la rger extent than the nonglycosylated receptor, which tended to accumulate i n the perinuclear zone of the endoplasmic reticulum. Inhibition of N-glycos ylation with tunicamycin resulted in a similar perinuclear distribution of the wild-type receptor. Therefore, glycosylation of the EP3 beta receptor s eems not to be necessary for correct folding of the receptor protein but fo r the efficient transport of the receptor protein to the plasma membrane. T his contrasts with a previous finding which described a reduction of the af finity for PGE(2) of the EP3 alpha receptor by elimination of the distal gl ycosylation site when the receptor protein was expressed in insect cells.