Preferential Formation of MT 1 / MT 2 Melatonin Receptor Heterodimers with Distinct Ligand Interaction Properties Compared with MT 2 Homodimers

Heterodimerization has been documented for several members of the G protein-coupled receptor (GPCR) superfamily, including the closely related MT1 and MT2 melatonin receptors. However, the relative abundance of heteroversus homodimers and the specific properties, which can be attributed to each form, are difficult to determine. Using a bioluminescence resonance energy transfer (BRET) donor saturation assay, we show that half-maximal MT1/MT2 heterodimer formation is reached for expression levels as low as 4000 receptors per cell. The relative propensity of MT1 homodimer and MT1/MT2 heterodimer formation are similar, whereas that for the MT2 homodimer formation is 3to 4-fold lower. These data indicate that both the relative expression level of each receptor isoform and the affinities between monomers may determine the actual proportion of homoand heterodimers. The specific interaction of ligands with the MT1/MT2 heterodimer was studied using a BRET-based assay as a readout for the conformational changes of the heterodimer. An MT1/MT2 heterodimer-specific profile and ligands selective for the MT1/MT2 heterodimer compared with the MT2 homodimer could be identified. Classic radioligand binding and BRET studies suggest that heterodimers contain two functional ligand binding sites that maintain their respective selectivity for MT1 and MT2 ligands. Occupation of either binding site is sufficient to induce a conformational change within the heterodimer. Taken together, these results show that the probability of GPCR heterodimer formation may be equal to or even higher than that of the corresponding homodimers and that specific properties of heterodimers can be revealed using a BRETbased ligand/receptor interaction assay. A growing number of observations suggest that G proteincoupled receptors (GPCR) form homodimers and heterodimerize with other members of the same receptor superfamily. Heterodimerization may have important consequences in terms of receptor function, because significant changes in ligand binding, signaling, or trafficking were observed for several heterodimers (Gazi et al., 2002). Considering that multiple receptors are expressed simultaneously in tissues and cells, it is reasonable to assume that most cells coexpress several different GPCRs that may be engaged in heterodimeric complexes. So far, little is known about the rules that govern homoand heterodimer formation. The homo-/heterodimer ratio is expected to depend on the relative affinity of receptor subtypes for each other and on the expression level of the interacting partners. Most studies on GPCR heterodimerization did not examine the proportion of heterodimers versus homodimers. In addition, these studies were principally performed in cells expressing supraphysiological levels of recombinant receptors, in which the formation of GPCR heterodimers might be overestimated, compared with normal tissues that express endogenous levels of receptors. Recent quantitative BRET-based studies in transfected cells expressing high concentrations (1–10 pmol/mg of protein) of 1and 2-adrenergic receptors (Mercier et al., 2002) or oxytocin and vasopressin receptors (Terrillon et This work was supported by the Institut de Recherche Servier, the Association pour la Recherche sur le Cancer grant 7537, and grants from the Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, and the Université Paris V. M.A.A. is supported by the IRS. □S The online version of this article contains supplemental figures. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. DOI: 10.1124/mol.104.000398. ABBREVIATIONS: GPCR, G protein-coupled receptor; BRET, bioluminescence resonance energy transfer; Rluc, Renilla reniformis luciferase; YFP, yellow fluorescent protein, I-MLT, 2[I]iodomelatonin; S20098, N-[2-(7-methoxynapht-1-yl)ethyl]acetamide; S20928, N-[2-(1-naphtyl) ethyl]cyclobutanecarboxamide; S22153, N-[2-(5-ethylbenzo[b]thiophen-3-yl)ethyl]acetamide; S24773, N-{2-[3-(3-aminophenyl)-7-methoxy-1naphtyl]ethyl}acetamide; S26284, N-(2-{7-[4-({8-[2-acetylamino)ethyl]-2-naphtyl}oxy)butoxy]-1-naphtyl}ethyl)acetamide; 4P-PDOT, 4-phenyl-2proprionamidotetraline; HEK, human embryonic kidney; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; BRET50, half-maximal bioluminescence resonance energy transfer; 1D-AR, 1D-adrenergic receptor. 0026-895X/04/6602-312–321$20.00 MOLECULAR PHARMACOLOGY Vol. 66, No. 2 Copyright © 2004 The American Society for Pharmacology and Experimental Therapeutics 398/1166334 Mol Pharmacol 66:312–321, 2004 Printed in U.S.A. 312 http://molpharm.aspetjournals. org/content/suppl/2004/08/02/mol.104. 000398.DC1 Supplemental material to this article can be found at: at A PE T Jornals on Jne 6, 2017 m oharm . aspeurnals.org D ow nladed from al., 2003) suggested that the probability of forming homoand heterodimers is similar. Ligand binding properties of receptors may change when they are engaged in heterodimeric complexes (Jordan and Devi, 1999). Because GPCRs are major pharmacological targets, the discovery of specific ligand binding profiles for heterodimers may have important implications for the development and screening of new drugs. However, the determination of a specific binding profile for heterodimers is difficult to establish using classic radioligand competition binding assays. This limitation is particularly true for heterodimers composed of two receptors, which display similar affinities for the same