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J. Biol. Chem., Vol. 282, Issue 7, 4821-4829, February 16, 2007

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Oligomerization of the UDP-glucuronosyltransferase 1A Proteins

HOMO- AND HETERODIMERIZATION ANALYSIS BY FLUORESCENCE RESONANCE ENERGY TRANSFER AND CO-IMMUNOPRECIPITATION^*

From the Departments of Chemistry & Biochemistry and Pharmacology, Laboratory of Environmental Toxicology, University of California, San Diego, La Jolla, California 92093

UDP-glucuronosyltransferases (UGTs) are membrane-bound proteins localized to the endoplasmic reticulum and catalyze the formation of -D-glucopyranosiduronic acids (glucuronides) using UDP-glucuronic acid and acceptor substrates such as drugs, steroids, bile acids, xenobiotics, and dietary nutrients. Recent biochemical evidence indicates that the UGT proteins may oligomerize in the membrane, but conclusive evidence is still lacking. In the present study, we have used fluorescence resonance energy transfer (FRET) to study UGT1A oligomerization in live cells. This technique demonstrated that UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10 self-oligomerize (homodimerize). Heterodimer interactions were also explored, and it was determined that UGT1A1 was capable of binding with UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10. In addition to the in vivo FRET analysis, UGT1A protein-protein interactions were demonstrated through co-immunoprecipitation experiments. Co-expression of hemagglutinin-tagged and cyan fluorescent protein-tagged UGT1A proteins, followed by immunoprecipitation with anti-hemagglutinin beads, illustrated the potential of each UGT1A protein to homodimerize. Co-immunoprecipitation results also confirmed that UGT1A1 was capable of forming heterodimer complexes with all of the UGT1A proteins, corroborating the FRET results in live cells. These preliminary studies suggest that the UGT1A family of proteins form oligomerized complexes in the membrane, a property that may influence function and substrate selectivity.

Received for publication, October 5, 2006 , and in revised form, November 30, 2006.

^* This work was supported in part by U. S. Public Health Service Grant GM49135. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Depts. of Chemistry & Biochemistry and Pharmacology, Laboratory of Environmental Toxicology, Leichtag Biomedical Research Bldg., Rm. 211, University of California, San Diego, La Jolla, CA 92093-0722. Tel.: 858-822-0286; Fax: 858-822-0363; E-mail: rtukey{at}ucsd.edu.

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