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Oligomerization of the fifth transmembrane domain from the adenosine A_2A receptor

Department of Chemistry and Biochemistry, Department of Chemical Engineering, Delaware Biotechnology Institute, University of Delaware, Newark, Delaware 19711, USA

(RECEIVED February 11, 2005; FINAL REVISION April 26, 2005; ACCEPTED May 6, 2005)

The human adenosine A_2A receptor (A_2AR) belongs to one of the largest family of membrane proteins, the G-protein coupled receptors (GPCRs), characterized by seven transmembrane (TM) helices. Little is known about the determinants of their structures, folding, assembly, activation mechanisms, and oligomeric states. Previous studies in our group showed that peptides corresponding to all seven TM domains form stable helical structures in detergent micelles and lipid vesicles. However, the peptides behave differently; TM5 is the only peptide to have a ratio []₂₂₂/[]₂₀₈ obtained by circular dichroism (CD) spectroscopy>1. This finding suggested to us that TM5 might self-associate. In the present study, we investigate the unique properties of the TM5 domain. We performed detailed analyses of TM5 peptide behavior in membrane-mimetic environments using CD spectroscopy, fluorescence spectroscopy and Förster resonance energy transfer, and gel electrophoresis. We find that TM5 peptide has the ability to self-associate to form oligomeric structures in various hydrophobic milieus and that these oligomers are highly resistant to temperature and chemical denaturation. We also find that mutation of the full-length A_2AR at position M193, which is located in the fifth TM domain, noticeably alters A_2AR monomer: dimer ratio as observed on SDS-PAGE. Our results suggest that parallel association of TM5 dimers may play a role in the known adenosine A_2A receptor dimerization. This study represents the first evidence of an individual GPCR transmembrane domain self-association.

Keywords: membrane proteins; GPCR dimerization; helix association; transmembrane peptide; FRET

Abbreviations: GPCR, G-protein coupled receptor • TM, transmembrane • A_2AR, adenosine A_2A receptor • SDS, sodium dodecyl sulfate • DMPC, dimyristoyl phosphatidylcholine • CD, circular dichroism • PAGE, Polyacrylamide gel electrophoresis • FRET, Förster resonance energy transfer • PFO, perfluorooctanoic acid • T_m, temperature of phase transition • TCEP, Tris(2-carboxyethyl)phosphine

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051409205.

Reprint requests to: Clifford R. Robinson, Department of Chemistry and Biochemistry, and Delaware Biotechnology Institute, 15 Innovation Way, University of Delaware, Newark, DE 19711, USA; e-mail: robinson{at}dbi.udel.edu; fax: (302) 831-3447.

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