Subcellular Localization of Prostaglandin Endoperoxide Synthase-2 in Murine 3T3 Cells

doi:10.1006/abbi.1993.1168

Archives of Biochemistry and Biophysics

Volume 301, Issue 2, March 1993, Pages 439-444

https://doi.org/10.1006/abbi.1993.1168 Get rights and content

Abstract

Polyclonal antisera specific for prostaglandin endoperoxide (PGH) synthases-1 and -2 were used to determine the subcellular locations of each PGH synthase isozyme in detergent-permeabilized mouse 3T3 fibroblasts by indirect immunocytofluorescence. Antiserum to PGH synthase- 1 demonstrated a mottled pattern of cytoplasmic and perinuclear staining of both serum-starved and serum-stimulated 3T3 cells. This pattern of staining is consistent with the results of earlier studies which demonstrated that PGH synthase-1 is associated with the endoplasmic reticulum and nuclear envelope of these cells. As expected, antibodies directed against a peptide unique to PGH synthase-2 failed to stain serum-starved cells, which lack appreciable levels of this second form of the enzyme. However, serum-stimulated 3T3 cells, which do express PGH synthase-2, showed the same pattern of staining with PGH synthase-2 antibodies as was observed with anti-PGH synthase-1 serum-mottled cytoplasmic staining and perinuclear staining. We conclude that the subcellular location of PGH synthase-2 is the same as PGH synthase-1 in murine 3T3 cells. Thus, the notable differences in the primary amino acid sequence-the signal peptide and the additional 18 amino acid C-terminal segment in PGH synthase-2-do not cause a change in localization. Colocalization of PGH synthases-1 and -2 implies that the source of arachidonate substrate, the site of PGH₂ and prostanoid formation, and the mechanism of product transport from the inside to the outside of the cell are the same for these isozymes.

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Citation Excerpt :
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The two cyclooxygenase (COX) enzymes catalyze the oxygenation of arachidonic acid to prostaglandin endoperoxides, which are the common intermediates in the biosynthesis of the bioactive lipids prostaglandins and thromboxane. COX-1 and COX-2 are ∼60% identical in amino acid sequence, exhibit highly homologous three-dimensional structures, and appear functionally similar at the biochemical level. Recent work has uncovered a subtle functional difference between the two enzymes, namely the ability of COX-2 to efficiently utilize neutral derivatives (esters and amides) of arachidonic acid as substrates. Foremost among these neutral substrates are the endocannabinoids 2-arachidonoylglycerol and arachidonoylethanolamide. This raises the possibility that COX-2 oxygenation plays a role in a novel signaling pathway dependent on agonist-induced release of endocannabinoids and their selective oxygenation by COX-2. Among the products of COX-2 oxygenation of endocannabinoids are glyceryl prostaglandins, some of which (e.g. glyceryl prostaglandin E₂ and glyceryl prostaglandin I₂) exhibit interesting biological activities in inflammatory, neurological, and vascular systems. These compounds are produced in intact cells stimulated with physiological agonists and have been isolated from in vivo sources. Important concepts relevant to the hypothesis of a COX-2-selective signaling pathway are presented.
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Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step in the prostanoid biosynthesis pathway, converting arachidonic acid into prostaglandin H₂. COX-2 exists as 72 and 74 kDa glycoforms, the latter resulting from an additional oligosaccharide chain at residue Asn⁵⁸⁰. In this study, Asn⁵⁸⁰ was mutated to determine the biological significance of this variable glycosylation. COS-1 cells transfected with the mutant gene were unable to express the 74 kDa glycoform and were found to accumulate more COX-2 protein and have five times greater COX-2 activity than cells expressing both glycoforms. Thus, COX-2 turnover appears to depend upon glycosylation of the 72 kDa glycoform.
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The eicosanoids are a diverse family of molecules that have powerful effects on cell function. They are best known as intercellular messengers, having autocrine and paracrine effects following their secretion from the cells that synthesize them. Many of the eicosanoids are produced from one polyunsaturated fatty acid, arachidonic acid. The diversity of possible products that can be synthesized from arachidonic acid is due, in part to the variety of enzymes that can act on it. Over the past 15 years, studies have placed many, but not all, of these enzymes at or inside the nucleus. In some cases, the nuclear import or export of arachidonic acid-processing enzymes is highly regulated. Furthermore, nuclear receptors that are activated by specific eicosanoids are known to exist. Taken together, these findings indicate that the enzymatic conversion of arachidonic acid to specific signaling molecules can occur in the nucleus, that it is regulated, and that the synthesized products may act within the nucleus. The objectives of this commentary are to review what is known about the metabolism of arachidonic acid to eicosanoids within the nucleus and to point to important areas for future discovery.
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Regular ArticleSubcellular Localization of Prostaglandin Endoperoxide Synthase-2 in Murine 3T3 Cells

Abstract

Regular Article
Subcellular Localization of Prostaglandin Endoperoxide Synthase-2 in Murine 3T3 Cells