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The first and arguably most important question that could be asked about the biology of any protein is: does it function alone? Cell surface receptors present special problems for stoichiometric analysis because, being located within lipid bilayers, they are often very hydrophobic, which means that once isolated they can exhibit a strong tendency to aggregate. A very welcome development, therefore, has been the advent of in situ methods for probing receptor organization, the most important of which are presently based on resonance energy transfer. Our first bioluminescence resonance energy transfer (BRET) experiments were, however, inconclusive since both monomeric and dimeric receptors gave high levels of energy transfer (James et al., 2006). It was only with the application of theoretical principles first developed for (Fung and Stryer, 1978; Wolber and Hudson, 1979), and then used in (Kenworthy and Edidin, 1998), Forster resonance energy transfer (FRET) experiments that we could use BRET to confidently distinguish between monomers and dimers.  We were very keen to test G protein-coupled receptors (GPCRs) using the new approach given the great interest in these important proteins forming constitutive oligomeric complexes (Angers et al., 2000; Ramsay et al., 2002; Babcock et al., 2003). This seemed unlikely to us firstly because, structurally, GPCRs are ideally configured for functioning autonomously (Meng and Bourne, 2001) and, secondly, because functional autonomy explains the remarkable evolutionary success (Schioth and Fredriksson, 2005) of this very large family of receptors. We were initially ignorant of the extent to which BRET was used to buttress the “GPCRs as oligomers” concept (Pfleger and Eidne, 2005), but when our initial analyses of human β2-adrenergic (β2AR) and mouse cannabinoid (mCannR2) receptors yielded the “BRET signatures” of monomers (James et al., 2006), we had to confront this body of data. The resulting controversy (Bouvier et al., 2007; James and Davis, 2007a,b; Salahpour and Masri, 2007) seems to have prompted the development of other, more complicated approaches. Here, we describe our experiences using BRET and briefly consider the merits of these alternative approaches.

Unraveling Receptor Stoichiometry Using Bret