Oligomerization of the fifth transmembrane domain from the adenosine A2A receptor.
The human adenosine A2A receptor (A(2A)R) 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 [theta]222/[theta]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(2A)R at position M193, which is located in the fifth TM domain, noticeably alters A(2A)R 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 A2A receptor dimerization. This study represents the first evidence of an individual GPCR transmembrane domain self-association.
Department of Chemistry and Biochemistry, Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711, USA.
Damien
D
Thévenin
Tzvetana
T
Lazarova
Matthew F
MF
Roberts
Clifford R
CR
Robinson
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D043705:Receptor, Adenosine A2A / Q000737:chemistry* / Q000235:genetics; D019163:Reducing Agents / Q000494:pharmacology
D043705:Receptor, Adenosine A2A; D019163:Reducing Agents