D2 Dopamine Receptor Homodimerization Is Mediated by Multiple Sites of Interaction, Including an Intermolecular Interaction Involving Transmembrane Domain 4

• Published in: Biochemistry (Easton) Vol. 42; no. 37; pp. 11023 - 11031
• Main Authors: Lee, Samuel P, O'Dowd, Brian F, Rajaram, Ryan D, Nguyen, Tuan, George, Susan R
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 23.09.2003
• Subjects: Animals; Binding Sites; Cell Line; Cell Membrane - metabolism; COS Cells; Dimerization; Disulfides; DNA, Complementary - metabolism; Gene Deletion; Genetic Vectors; Glycosylation; Humans; Immunoblotting; Insecta; Kinetics; Mutation; Protein Binding; Protein Isoforms; Protein Structure, Tertiary; Receptors, Dopamine - chemistry; Reducing Agents - pharmacology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0345539

In this study, we examined the mechanisms of intermolecular interaction involved in D2 dopamine receptor dimer formation to develop an understanding of the quaternary structure of G protein-coupled receptors. The potential role of two mechanisms was investigated:  disulfide bridges and hydrophobic interactions between transmembrane domains. D2 dopamine receptor oligomers were unaffected by treatment with a reducing agent; however, oligomers of the D1 dopamine receptor dissociated following a similar treatment. This observation suggested that other forces such as hydrophobic interactions were more robust in the D2 receptor than in the D1 receptor in maintaining oligomerization. To elucidate which transmembrane domains were involved in the intermolecular hydrophobic interactions, truncation mutants were generated by successive deletion of transmembrane domains from amino and/or carboxyl portions of the D2 dopamine receptor. Immunoblot analyses revealed that all the fragments were well expressed but only fragments containing transmembrane domain 4 were able to self-associate, suggesting that critical areas for receptor dimerization resided within this transmembrane domain. Disruption of the helical structure of transmembrane domain 4 in a truncated receptor capable of forming dimers interfered with its ability to self-associate; however, a similar disruption of the transmembrane domain 4 helix structure in the full-length receptor did not significantly affect dimerization. These results indicated that there are other sites of interaction involved in D2 receptor oligomer assembly in addition to transmembrane domain 4.