title: Distinct influences of carboxyl terminal segment structure on function in the two isoforms of prostaglandin H synthase.
abstract: The cyclooxygenase activity of the two prostaglandin H synthase (PGHS) isoforms, PGHS-1 and -2, is a major control element in prostanoid biosynthesis. The two PGHS isoforms have 60% amino acid identity, with prominent differences near the C-terminus, where PGHS-2 has an additional 18-residue insert. Some mutations of the C-terminal residue in PGHS-1 and -2 have been found to disrupt catalytic activity and/or intracellular targeting of the proteins, but the relationship between C-terminal structure and function in the two isoforms has been poorly defined. Crystallographic data indicate the PGHS-1 and -2 C-termini are positioned to interact with the endoplasmic reticulum (ER) membrane, although the C-terminal segment structure was not resolved for either isoform. We constructed a series of C-terminal substitution, deletion, and insertion mutants of human PGHS-1 and -2 and evaluated the effects on cyclooxygenase activity and intracellular targeting in transfected COS-1 cells expressing the recombinant proteins. PGHS-1 cyclooxygenase activity was strongly disrupted by C-terminal substitutions and deletions, but not by elongation of the C-terminal segment, even when the ultimate residue was altered. Similar alterations to PGHS-2 had markedly less effect on cyclooxygenase activity. The results indicate that the functioning of the longer C-terminal segment in PGHS-2 is distinctly more tolerant of structural change than the shorter PGHS-1 C-terminal segment. C-Terminal substitutions or deletions did not change the subcellular localization of either isoform, even at short times after transfection, indicating that neither C-terminal segment contains indispensable intracellular targeting signals.
authors.affiliation: Department of Internal Medicine, University of Texas Health Science Center at Houston, 77030, USA.
authors.forename: Q
authors.initials: Q
authors.lastname: Guo
authors.forename: R J
authors.initials: RJ
authors.lastname: Kulmacz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D021381:Protein Transport; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D007527:Isoenzymes; D008565:Membrane Proteins; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases