A cyclooxygenase-2-dependent prostaglandin E2 biosynthetic system in the Golgi apparatus

J Biol Chem. 2015 Feb 27;290(9):5606-20. doi: 10.1074/jbc.M114.632463. Epub 2014 Dec 29.

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.

Keywords: Arachidonic Acid (AA) (ARA); Aspirin; COPII; Coxib; Cyclooxygenase (COX); Degron; Endoplasmic Reticulum-associated Protein Degradation (ERAD); Glycoprotein; NSAID; Prostaglandin.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Asparagine / genetics
  • Asparagine / metabolism
  • Cyclooxygenase 2 / genetics
  • Cyclooxygenase 2 / metabolism*
  • Cysteine Proteinase Inhibitors / pharmacology
  • Dinoprostone / biosynthesis*
  • Endoplasmic Reticulum / metabolism*
  • Endoplasmic Reticulum-Associated Degradation / drug effects
  • Fibroblasts / cytology
  • Fibroblasts / metabolism
  • Glycosylation
  • Golgi Apparatus / metabolism*
  • Group IV Phospholipases A2 / metabolism
  • HEK293 Cells
  • Humans
  • Immunoblotting
  • Intramolecular Oxidoreductases / genetics
  • Intramolecular Oxidoreductases / metabolism
  • Isoquinolines / pharmacology
  • Leupeptins / pharmacology
  • Mice
  • Microscopy, Confocal
  • Mutation
  • NIH 3T3 Cells
  • Prostaglandin-E Synthases
  • Protein Kinase Inhibitors / pharmacology
  • Protein Transport / drug effects
  • Sulfonamides / pharmacology

Substances

  • Cysteine Proteinase Inhibitors
  • Isoquinolines
  • Leupeptins
  • Protein Kinase Inhibitors
  • Sulfonamides
  • Asparagine
  • Cyclooxygenase 2
  • Group IV Phospholipases A2
  • Intramolecular Oxidoreductases
  • PTGES protein, human
  • Prostaglandin-E Synthases
  • Ptges protein, mouse
  • Dinoprostone
  • N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide
  • benzyloxycarbonylleucyl-leucyl-leucine aldehyde