Here, we presented a new approach to analyze protein-protein interaction at the cell surface based on a combination of TR-FRET and snap-tag technologies. We validated this approach using well recognized GPCR dimers, the GABAB and mGlu1 receptors, and confirm that both class A and B receptors also form dimers or larger oligomers at the cell surface. When associated with a quality control system allowing the labeling of a single subunit, we show that whereas only strict dimers of mGluR1 could be observed, the GABAB receptor assembles into at least dimers of dimers.
Although FRET and BRET have been widely used to analyze the oligomeric state of membrane proteins, the low signal to noise ratio made difficult the use of such techniques in screening assays. Moreover, it was still difficult to prove that the signals obtained originate from the cell surface. Indeed, even though a nice and saturable BRET signal could be measured between GABAB1
in the presence of GABAB2
(supp Fig. H
), we could not exclude that this signal originate from intracellular GABAB1
. Although, imaging techniques and total internal reflection fluorescence microscopy can be used to examine FRET at the plasma membrane, such approaches are not compatible with systematic and quantitative assessments of the interaction. In contrast, the TR-FRET snap-tag technology, by allowing an easy assessment of the protein proximity at the cell surface enables a clear demonstration of the specificity of the interaction. Indeed, the assay was conducted in 96 well plates, and can easily be adapted to 384 plates as many other TR-FRET cellular assays. Of note, a very low emission of the acceptor is observed when non-interacting proteins are being studied, showing that even with over-expressed proteins, very low FRET occurs due to random collision at the cell surface. This suggests that the high non-specific “by slander” FRET or BRET measured with GFP or Rluc fused membrane proteins likely originate from the intracellular proteins.
Within the last 10 years, a large number of studies reported that GPCRs can form oligomers, but it was still not known whether such complexes were limited to dimers or whether higher-order oligomers could form6,7,27
. By taking advantage of an optimized quality control system, we show here that mGlul dimers cannot on their own self associate, demonstrating that a dimeric organization of these receptors is sufficient for function. One of course cannot exclude the possibility that, in their native environment, these mGlu dimers can associate into larger complexes through interaction with scaffolding proteins. To our surprise however, we found that the GABAB
receptor heterodimer can form larger oligomers through GABAB1
interaction. Because no close proximity between GABAB2
subunits was observed, it is likely that these oligomers are limited to dimers of dimers. Accordingly, as for any other GPCR homodimer such a quaternary organization of the GABAB
receptor possesses two agonists binding sites, and two possible G-protein coupling domains. Importantly, this organization of the GABAB
receptor can be observed over a wide range of receptor density at the cell surface, including that reported for this receptor in the brain23
. Since the receptor density is expected to be even higher in the specific micro domains where this receptor is targeted in neurons (dendritic spines and pre-synaptic terminals), this makes likely that what is observed here in transfected cells can also occur in vivo,
unless specific interacting proteins absent in HEK or COS cells prevent this.
By preventing GABAB
heterodimers to associate, using a minimal domain of GABAB1
, we also provide a functional evidence in favor of the dimer of dimer organization of the GABAB
receptor. Our data are consistent with a lower G protein-coupling efficacy of the GABAB
receptor when associated into dimers of dimers. Of interest, such a dimerization of the GABAB
heterodimer reproduces what has been recently shown for GPCR homodimers. Indeed, in GPCR homodimers, a single subunit can activate a G-protein at a time2,28–30
. Since GPCR monomers can effectively activate G-proteins2–5
, then two separated monomers are expected to activate more G-proteins than a homodimer. This has been recently demonstrated for both rhodopsin2
and the neurotensinl receptor30
. Such a process could be a way to modulate coupling efficacy in vivo,
or to allow simultaneous coupling to both G protein-dependent and G protein-independent pathways, but more work is required to validate this proposal.
In conclusion, we showed here that the combined use of snap-tag and TR-FRET allows a rapid, easy and quantitative assessment of cell surface protein interactions. This approach confirmed the oligomeric assembly of GPCRs at the cell surface and allowed us to analyze the stoichiometry of class C GPCR oligomers. This technology will certainly be useful to study the dynamics of any cell surface protein complexes, and to identify drugs that modulate these.