Cloning, expression, and functional characterization of human cyclooxygenase-1 splicing variants: evidence for intron 1 retention

J Pharmacol Exp Ther. 2005 Dec;315(3):1298-305. doi: 10.1124/jpet.105.090944. Epub 2005 Sep 1.

Abstract

Recently, a splicing variant of cyclooxygenase (COX)-1, arising via the retention of its intron 1, was identified in canine. It was called COX-3 and was reported to be differentially sensitive to inhibition by various nonsteroidal anti-inflammatory drugs (NSAIDs) as well as acetaminophen (Chandrasekharan et al., 2002). However, the existence of an orthologous splicing variant in human tissues has been questioned due to a reading frame shift and premature termination. In this study, we first confirmed the existence of intron 1-retained COX-1 in certain human tissues at both the mRNA and protein levels. Molecular biology studies revealed that three distinct COX-1 splicing variants exist in human tissues. The most prevalent of these variants, called COX-1b1, arises via retention of the entire 94 base pair (bp) of intron 1, leading to a shift in the reading frame and termination at bp 249. However, the other two variant types, called COX-1b2 and COX-1b3, retain entire intron 1, but they are missing a nucleotide in one of two different positions, thereby encoding predicted full-length and likely COX-active proteins. Functional studies revealed that the COX-1b2 is able to catalyze the synthesis of prostaglandin F2alpha from arachidonic acid with Km and Vmax values of 0.54 microM and 3.07 pmol/mg/min, respectively. However, no significant differential selectivity for inhibition by selected NSAIDs was observed. Accordingly, we conclude that intron 1-retained human COX-1 is not likely to be the therapeutic target of acetaminophen or a candidate of COX-3.

Publication types

  • Comparative Study

MeSH terms

  • Acetaminophen / pharmacology
  • Amino Acid Sequence
  • Animals
  • Anti-Inflammatory Agents, Non-Steroidal / pharmacology
  • Base Sequence
  • Blotting, Western
  • Cloning, Molecular*
  • Cyclooxygenase 1 / analysis
  • Cyclooxygenase 1 / chemistry
  • Cyclooxygenase 1 / genetics*
  • Cyclooxygenase 1 / metabolism*
  • Gene Expression*
  • Genetic Variation
  • Humans
  • Insecta / cytology
  • Introns*
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Kinetics
  • Molecular Sequence Data
  • RNA Splicing*
  • RNA, Messenger / genetics
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Spodoptera / genetics
  • Tissue Distribution

Substances

  • Anti-Inflammatory Agents, Non-Steroidal
  • Isoenzymes
  • RNA, Messenger
  • Recombinant Proteins
  • Acetaminophen
  • Cyclooxygenase 1