Although homo-oligomerization has been reported for several G protein-coupled receptors, this phenomenon was not studied at low concentrations of receptors. Furthermore, it is not clear whether homo-oligomerization corresponds to an intrinsic property of nascent receptors or if it is a consequence of receptor activation. Here CCR5 receptor oligomerization was studied by bioluminescence resonance energy transfer (BRET) in cells expressing physiological levels of receptors. A strong energy transfer could be observed, in the absence of ligands, in whole cells and in both endoplasmic reticulum and plasma membrane subfractions, supporting the hypothesis of a constitutive oligomerization that occurs early after biosynthesis. No change in BRET was observed upon agonist binding, indicating that the extent of oligomerization is unrelated to the activation state of the receptor. In contrast, a robust increase of BRET, induced by a monoclonal antibody known to promote receptor clustering, suggests that microaggregation of preformed receptor homo-oligomers can occur. Taken together, our data indicate that constitutive receptor homo-oligomerization has a biologically relevant significance and might be involved in the process of receptor biosynthesis.G protein-coupled receptors (GPCRs) 1 constitute the largest family of membrane receptors. They are involved in the regulation of most biological functions and represent, collectively, one of the most important targets for therapeutic intervention. A rapidly growing number of studies indicate that GPCRs may be organized as oligomers (1). The biological function of this phenomenon was questioned for some time until it was reported that the hetero-oligomerization of two isoforms of the GABA B receptor was indispensable for the formation of functional GABA B binding sites in tissues (2-4). Hetero-oligomerization of other GPCRs has also been proposed as a mean to increase pharmacological diversity and expand signaling modes for this class of receptors (5, 6). However, very little is known about the role that receptor homo-oligomerization could play. The fact that homo-oligomerization has been studied mainly with receptors overexpressed in heterologous systems has even raised concerns about the biological relevance of this phenomenon. Whether oligomeric complexes form at the plasma membrane or in other subcellular compartments and whether or not oligomers result from a dynamic, regulated, and reversible assembly of monomers following receptor activation are two other important and still open questions. The latter question, in particular, was the object of several studies using different experimental approaches that gave different interpretations. For example, it was concluded that agonists could increase homo-oligomerization of ␤ 2 -adrenergic (7) and TRH (8) receptors, decrease the homo-dimerization of the ␦-opioid receptor (9), or have no effect on the oligomeric state of M3-muscarinic receptors (10).The issue of receptor oligomerization and its potential regulation by agonists is...

The neuropeptide neurotensin (NT) elicits hypothermic and naloxone-insensitive analgesic responses after brain injection. Recent pharmacological evidence obtained with NT agonists and antagonists suggests that these effects are mediated by a receptor distinct from the initially cloned high-affinity NT receptor (NTR1). The recent cloning of a second NT receptor (NTR2) prompted us to evaluate its role in NT-induced analgesia. Intracerebroventricular injections in mice of two different antisense oligodeoxynucleotides from the NTR2 markedly decreased NTR2 mRNA and protein and reduced NT-induced analgesia.This effect was specific, because NTR1 levels were unaffected, and sense or scramble oligodeoxynucleotides had no effect. Structure-activity studies revealed a close correlation between the analgesic potency of NT analogs and their affinity for the NTR2 and disclosed potent and selective agonists of this receptor. These data confirm that NTR1 is involved in the NTelicited turning behavior and demonstrate that the NTR2 mediates NT-induced analgesia.