Evidence for domain swapping in GPCRs from the evolutionary trace method?

Georgios V Gkoutos and Christopher A Reynolds

Department of Biological Sciences, Central Campus, Wivenhoe Park, Colchester, Essex, CO4 3SQ, United Kingdom

 

Introduction

The functional significance of dimerisation in G-protein coupled receptors is currently the subject of much debate. A number of studies have shown that the activity of defective receptors can be rescued by a second receptor which is also defective in a different way1-3. Disulphide-linked receptor dimers have been identified for both the metabotrophic glutamate receptor4 and the calcium sensing receptor5. Concurrent inhibition of activity and dimer formation by a peptide derived from helix 6 has been observed for the b 2-adrenergic receptor6 (b 2-AR), and similar but less extensive studies have been carried out on the dopamine D2 receptor7. In addition, Cvejic and Devi8 and also Ciruela et al.9 have identified dimers in the d -opiod and angiotensin II receptors respectively. The observation of bell-shaped dose-response curves may also be indicative of dimerisation in GPCRs10, since it is indicative of dimerisation in other receptors11. Additional experimental evidence for dimerisation is discussed and interpreted in references12-17, which also include theoretical studies relevant to G-protein coupled receptor dimerisation. In these theoretical studies it was proposed12-15 that the dimerisation may proceed via a domain swapping18,19 mechanism, as shown in figure 1. In this mechanism, new interactions are generated at the interfaces between helices 5 and 6.

(a) The proposed domain-swapping rearrangement (b) For clarity, the dimerisation is also illustrated using Maggio's chimeric adrenergic (black) - muscarinic (grey) receptors. Note that the transmembrane helices in the A domain (N-terminus and helices 1 – 5) and the B domain (helices 5 and 6 through to the C-terminus) may retain their relative orientation throughout the rearrangement

Recent studies, notably by Pazos et al.20 and by Lichtarge et al.21-23 have implied that functional interfaces can be identified from sequence data. Pazos used correlated mutation analysis to identify correctly docked protein heterodimers from incorrectly docked structures. Elsewhere it has been shown that correlated mutations tend to accumulate at the interface between helices 5 and 6 of the b 2-adrenergic receptor dimer13 and that correlated mutations also appear amongst the external (lipid-facing) residues of other receptors, including the neurokinin, dopamine and muscarinic receptors15,17. This may indicate that the receptors form interactions with other proteins and so is consistent with dimerisation. An extensive analysis of the evidence of domain swapping in GPCRs is given here. The evolutionary trace method of Lichtarge is attractive because it has the potential to identify the dimerisation site of one protein from its sequence in the absence of the other protein.Consequently, we have built a model of the b 2-adrenergic receptor from the recent low resolution cryo electronmicroscopy structure of Unger et al.24 which has been interpreted by Baldwin to generate an alpha carbon backbone25.  Click here to view the 3D structure of this model. Using this model we have been able to apply the evolutionary trace method using the currently available sequences for the adrenergic receptors and to observe whether the results are consistent with current hypotheses regarding the possible dimerisation interface in the G-protein coupled receptors.

 

References

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