Karen M. Lakkides

Prostanoid receptors of murine NIH 3T3 and RAW 264.7 cells. Structure and expression of the murine prostaglandin EP4 receptor gene.

Prostaglandin endoperoxide H synthase-1 (PGHS-1) is expressed constitutively in murine NIH 3T3 cells and RAW 264.7 cells. PGHS-2 is inducibly expressed in these cells following stimulation with serum or bacterial lipopolysaccharide (LPS), respectively. Reverse transcription-polymerase chain reaction (RT-PCR) analysis established that a variety of G protein-linked and peroxisomal proliferator-activated prostanoid receptors are expressed in both of these cell types. The levels of the EP2 and EP4 prostaglandin E2 (PGE2) receptors and the prostaglandin I2 receptor were changed in these cells by serum or LPS stimulation. Quantitative RT-PCR indicated that the mRNA for the murine EP4 receptor, the butaprost-insensitive PGE2 receptor that couples to Gs, increases 1.5-3-fold in response to serum (NIH 3T3) or LPS (RAW 264.7) with a time course approximating the induction of PGHS-2 expression. To study expression of the EP4 receptor we isolated the mouse EP4 receptor gene; the gene is 10 kilobase pairs (kb) in length and, like other known prostanoid receptor genes, contains three exons and two introns. The first intron is 0.5 kb and is located 16 base pairs (bp) downstream of the translational start site. This is a different location than that of the first introns of other prostanoid receptor genes. The second intron is located immediately following the sixth transmembrane domain at the same position as the second intron of the thromboxane A2 receptor, prostaglandin D2 receptor, prostaglandin I2 receptor, and one of the PGE2 (EP1) receptor genes. A major transcriptional start was detected at −142 bp upstream of the translational start. There are a variety of putative cis-acting elements within 1.5 kb upstream of the translational start site and within the first intron. Promoter analyses of the EP4 receptor gene promoter in RAW 264.7 cells indicated that there is a constitutive negative regulatory region between −992 and −928 bp, a constitutive positive region between −928 and −554 bp, and an LPS/serum-responsive region between −554 and −116 bp.

Pgg 2 11r-Hpete and 15r/S-Hpete are Formed From Different Conformers of Arachidonic Acid in the Prostaglandin Endoperoxide H Synthase-1 Cyclooxygenase Site

Prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and -2) catalyze the committed step in the formation of prostanoids (prostaglandins, thromboxane A2 (l-6). PGHSs catalyze two separate reactions: a cyclooxygenase reaction in which arachidonate is converted to prostaglandin G2 (PGG2) and a peroxidase reaction in which PGG2undergoes a two-electron reduction to PGH2. The cyclooxygenase reaction begins with a rate-limiting abstraction of the 13-proS hydrogen from arachidonate to yield an arachidonyl radical (7,8). This is followed by sequential oxygen additions at C-11 and C-15 to yield the prostaglandin endoperoxide PGG2. PGHSs exhibit some lipoxygenase activity producing small amounts of 11-hydroperoxy-5Z,8Z,12E,14Z-eicosatetraenoic acid (11-HPETE) and 15-hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-HPETE) from arachidonic acid (9, 10). Aspirin-acetylated PGHS-2, which has no cyclooxygenase activity, synthesizes 15R-HPETE (10,11). Studies comparing native and aspirinacetylated PGHS-2 have raised the possibility that arachidonate can bind in distinct orientations in the PGHS-2 active site to produce either PGG2, 1 l R-HPETE or 15RHPETE (10). Here we develop the concept that arachidonate can be bound in the cyclooxygenase active site of ovine (o)PGHS-1 in at least three different, catalytically competent arrangements that lead to PGG2, 11R-HPETE, and 15R/S-HPETE, respectively, and that these three arrangements of arachidonate occur subsequent to its entry into the cyclooxygenase active site.

Fatty acid binding to cyclooxygenases

Abstract Prostaglandin endoperoxide H synthase-1 and -2 (PGHS-1 and -2) convert arachidonic acid (AA) to prostaglandin H 2 (PGH 2 ) in the committed step in prostaglandin biosynthesis. Although the cyclooxygenase activity favors AA as the substrate, both isoforms will oxygenate a variety of 18–22 carbon fatty acids with reduced efficiencies. In this review, we discuss how the fatty acid substrates AA and dihomo-γ-linolenic acid (DHLA; 20:3 ω−6) are bound in the cyclooxygenase active site of ovine (o) PGHS-1. Based on the conformations of the fatty acids within the active site, we describe the key roles that Val349 and Ser530 play as determinants of fatty acid substrate specificity for oPGHS-1.

Substrate Interactions in the Cyclooxygenase-1 Active Site

Prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and -2) catalyze the conversion of arachidonic acid, two molecules of O2 and two electrons to PGH2. This is the committed step in the formation of prostaglandins and thromboxane A2 [1]. Crystallographic studies of enzyme inhibitor complexes have suggested that the cyclooxygenase active sites of PGHSs are hydrophobic channels that protrude into the body of the major globular domain of the enzymes [2]. We have now determined the structure of arachidonic acid (AA) bound within the cyclooxygenase active site of ovine (o)PGHS-1 [3]. AA is bound in an extended L-shaped conformation and makes a total of 49 hydrophobic contacts (i.e. 2.5–4.0 A) and two hydrophilic contacts with the protein involving a total of 19 different residues (Figures 1,2). Although AA can assume some 107 low energy conformations [4], only three of these are catalytically competent [5]. One conformation leads to PGG2, one leads to 11R-HPETE, and a third leads to 15R- plus 15S-HPETE. Previous mutational studies have established the importance of Arg120 in AA binding to PGHS-1 and -2 [6–8], the role of Tyr385 in abstraction of the 13-proS-hydrogen from AA [9,10] and the importance of Ser530 and I1e523 as determinants of inhibitor specificity [11–13]. We have performed mutational analyses of a number of the residues that line the cyclooxygenase channel to determine their functional importance in AA binding and oxygenation. Substitutions of several cyclooxygenase site residues lead to large increases in 11-HPETE or 15-HPETE formation but with small changes in the Km for AA. Our results suggest that individually and collectively the hydrophobic residues function primarily to position AA in a specific conformation that optimizes its conversion to PGG2.