MONOTOPIC MEMBRANE ANCHORS IN PGHS SYNTHASES 1 AND 2

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The major focus of this grant is to examine the membrane interactions of the prostaglandin endoperoxide synthases-1 and -2 (PGHS-1 and PGHS-2) (also called cyclooxygenases; COX-1 and COX-2). The PGHSs have been studied extensively because of their essential and regulatory role in prostaglandin synthesis and also because they are the site of action for non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin and ibuprofen. Aspirin and other NSAIDs have also been effective in reducing the incidence of colon cancer in man, and in inhibiting tumor formation in animal models of colon cancer, suggesting that prostaglandins are important regulators of proliferation and/or transformation. However, the protective effects of NSAIDs, both in inflammation and cancer, appear to be mediated through inhibition of PGHS-2 and not PGHS-1. To better understand the mechanism for differential signaling by PGHS-1 and PGHS-2, we will examine their physical interactions with membranes. These isoenzymes do not contain trans-membrane sequences, but instead have four short contiguous amphipathic helices, or membrane binding domains, that are thought to anchor these proteins within the lipid bilayer. Our main hypothesis is that differences in the membrane interaction of these two isozymes affects their biological signaling properties. Our primary goal will be to characterize the structural features of a functional PGHS membrane binding domain, and to determine whether differences in the membrane binding domain sequences of PGHS-1 and PGHS-2 result in changes in the orientation of the two isozymes within membranes that may relate to their unique biological properties. Our first objective will be to optimize previously developed methods for the reconstitution of active PGHS-1 and PGHS-2 into lipid vesicles. Reconstitution experiments will allow us to determine the membrane topography of PGHS-1 and PGHS-2 using three different biochemical and physical methods: (a) site specific labeling of membrane associated protein sequences; (b) EPR spectroscopy of spin-labeled PGHSs; and (c) neutron diffraction scattering analysis of crystals of the PGHS-1 and PGHS-2 in deuterated detergents.