Abstract
Cyclooxygenases (COXs) catalyze the committed step in prostaglandin (PG) biosynthesis. COX-1 is constitutively expressed and stable, whereas COX-2 is inducible and short lived. COX-2 is degraded via endoplasmic reticulum (ER)-associated degradation (ERAD) following post-translational glycosylation of Asn-594. COX-1 and COX-2 are found in abundance on the luminal surfaces of the ER and inner membrane of the nuclear envelope. Using confocal immunocytofluorescence, we detected both COX-2 and microsomal PGE synthase-1 (mPGES-1) but not COX-1 in the Golgi apparatus. Inhibition of trafficking between the ER and Golgi retarded COX-2 ERAD. COX-2 has a C-terminal STEL sequence, which is an inefficient ER retention signal. Substituting this sequence with KDEL, a robust ER retention signal, concentrated COX-2 in the ER where it was stable and slowly glycosylated on Asn-594. Native COX-2 and a recombinant COX-2 having a Golgi targeting signal but not native COX-1 exhibited efficient catalytic coupling to mPGES-1. We conclude that N-glycosylation of Asn-594 of COX-2 occurs in the ER, leading to anterograde movement of COX-2 to the Golgi where the Asn-594-linked glycan is trimmed prior to retrograde COX-2 transport to the ER for ERAD. Having an inefficient ER retention signal leads to sluggish Golgi to ER transit of COX-2. This permits significant Golgi residence time during which COX-2 can function catalytically. Cytosolic phospholipase A2α, which mobilizes arachidonic acid for PG synthesis, preferentially translocates to the Golgi in response to physiologic Ca(2+) mobilization. We propose that cytosolic phospholipase A2α, COX-2, and mPGES-1 in the Golgi comprise a dedicated system for COX-2-dependent PGE2 biosynthesis.
Highlights
When cyclooxygenases-1 and -2 (COXs-1 and -2) are co-expressed, COX-2 can function, whereas COX-1 is latent
We speculate that because COX-1 lacks an efficient endoplasmic reticulum (ER) to Golgi we examined the subcellular location of human trafficking signal this isoform resides microsomal PGE synthase-1 (mPGES-1) expressed heterologously in HEK293 cells and primarily in ER
The results suggest that cPLA2␣, COX-2, and mPGES-1 in the Golgi provide an efficient system for PGE2 formation
Summary
When cyclooxygenases-1 and -2 (COXs-1 and -2) are co-expressed, COX-2 can function, whereas COX-1 is latent. We identified a 27-amino acid instability motif involving residues Glu-586 to Lys-612 of COX-2 that acts as a degradation signal or “degron” [39] This degron includes the unique 19-amino acid sequence near the C terminus of COX-2 that is essential for regulating N-glycosylation of Asn-594 and mediating COX-2 entry into the ERAD pathway. During the process of distinguishing between terminally misfolded proteins and properly folded intermediates, ␣-mannosidases remove mannoses to trim the Asn-linked Man9(GlcNAc) oligosaccharides, generating an appropriate ERAD signal [38, 43, 44] This occurs with the N-glycosyl moiety linked to Asn-594 of COX-2. These findings in combination with a previous observation that cytosolic (c) PLA2␣ undergoes a Ca2ϩ-dependent translocation to the Golgi [46] led us to conclude that there is a COX-2-dependent PGE2 biosynthetic system located in the Golgi apparatus
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