© 2

011

Nat

ure

Am

eric

a, In

c. A

ll ri

gh

ts r

eser

ved

.

578 nature chemical biology | VOL 7 | SEPTEMBER 2011 | www.nature.com/naturechemicalbiology

news & views

GPCRs are transmembrane receptors that are extremely important as drug targets and have functional

complexity that appears virtually unlimited because of their conformational plasticity and ability to homo- and heterodimerize1. Although several GPCRs function as obligate heterodimers (GPCR class C)2, others (class A) have been shown to be able to activate G protein both in monomeric3 and in various dimeric forms4. Recently, allosteric communication between the protomers in class A GPCRs was shown to regulate agonist binding affinities5,6. It has been speculated that such allosteric interactions may be involved in functional selectivity, that is, the observation that agonist binding induces unique conformations that can differentially modulate the G protein coupling process7. Urizar et al.8 have developed an elegant approach to directly measure G protein activation by specific GPCR dimers and used it to investigate the functional selectivity of the D1 and D2 dopamine receptor heterodimer. They demonstrated that allosteric interactions in the D1-D2 heterodimer are responsible for functional selectivity observed for the D2 receptor agonist R-(−)-propylnorapomorphine (NPA).

GPCRs are seven-transmembrane-helix proteins that interact with and activate intracellular heterotrimeric G proteins (classified as Gi, Gs, Gq and G12/13) and thereby determine the downstream signaling cascades. Expressed individually, the D1 and D2 dopamine receptors form homodimers that specifically activate Gs and Gi, respectively. When D1 and D2 receptors are coexpressed, homomeric and heteromeric species may form, and signaling can result from any combination of these receptor complexes. Functional selectivity has been observed for several GPCRs9, but so far it has not been directly linked to allosteric effects of GPCR dimerization. The major obstacle to quantitatively assessing the functional importance of dimerization between class A GPCRs has been the methodological limitation to

control the identity of the components comprising the signaling unit. In order to selectively analyze signal activation from D1-D2 receptor heterodimers, Urizar et al. adapted a reporter assay for conformational changes in the GPCR-G protein complex by

bioluminescence resonance energy transfer (BRET)8,10. BRET is based on the distance-dependent efficiency of energy transfer from a bioluminescence donor molecule (for example, Renilla luciferase, Rluc) to a proximal fluorescent protein (for example,

GPCRs

Caught in a spectroscopic trapHomo- and heterodimerization of G protein–coupled receptors (GPCRs) have been described for numerous receptors, but their functional role has remained elusive. With a new spectroscopic assay based on protein fragment complementation, GPCR heterodimerization was demonstrated to contribute to a phenomenon called ‘functional selectivity’.

Jacob Piehler

Figure 1 | Selectivity of GPCR activation assays by BRET and CODA-RET. (a,b) BRET assays using a D2–Renilla luciferase (Rluc) fusion and a G protein subunit (Gαi)-mVenus fusion. (a) This method selectively probes the activation of the heterotrimeric G protein Gαi but not that of Gαs. (b) The activation of homo- and heterodimers containing D2 cannot be distinguished. (c) The CODA-RET assay includes the formation of functional Rluc by complementation of protein fragments fused to the D1 and the D2 receptors. Thus, activation of Gαi through the D1-D2 heterodimer can be probed selectively.

a D1 D1D2

D1 D2

D2

D2 D2

BRET

mVenus

RLuc

αi βγ

αs βγ

b

BRETBRET

mVenus

αiβ

γ

mVenus

αi βγ

αiβ

γ

D1 D2 D2 D2c

BRET

RLuc

αi βγ

RLuc

© 2

011

Nat

ure

Am

eric

a, In

c. A

ll ri

gh

ts r

eser

ved

.

nature chemical biology | VOL 7 | SEPTEMBER 2011 | www.nature.com/naturechemicalbiology 579

news & views

mVenus). Fusing Rluc to a GPCR and mVenus to a Gα protein makes it possible to detect GPCR activation as a change in the BRET efficiency due to a conformational change in the GPCR–G protein complex. Thus, activation of specific Gα proteins at a specific GPCR can be probed (Fig. 1a), but their activation by homo- or heterodimers cannot be discriminated (Fig. 1b).

In order to allow BRET only from specific GPCR heterodimers, Rluc was split into two inactive N- and C-terminal fragments, which were fused to the D1 and D2 receptors, respectively. Functional Rluc is formed by the complementation of both fragments due to GPCR heterodimerization. Thus, the BRET donor is available only in D1-D2 receptor heterodimers (Fig. 1c), whereas all other dimeric species remain ‘in the dark’—that is, they do not contribute to the BRET signal. This technique, termed ‘complemented donor-acceptor resonance energy transfer’ (CODA-RET), enabled the authors to quantitatively probe the agonist-induced response from D1-D2 receptor heterodimers directly at the level of G protein activation rather than further downstream, where different receptor species may contribute as a result of integration from other signaling pathways.

The authors used CODA-RET to further explore receptor activation by different D1 and D2 receptor agonists. For NPA, a ten-fold increase in the potency of Gi activation by the D2 receptor was observed upon coexpression of the D1 receptor but was not observed upon stimulation

with other D2 receptor agonists. CODA-RET experiments with NPA and other D2 receptor agonists confirmed that this selective increase in potency was indeed caused by G protein activation by the D1-D2 receptor heterodimer, whereas no change in potency was observed for the D2-D2 receptor homodimer. Thus, allosteric communication between the protomers in the D1-D2 receptor heterodimer specifically affects NPA potency, possibly by increasing the binding affinity. This is the first time that such functional selectivity could be related to allosteric interactions in a GPCR dimer, and this observation will have important implications in fundamental GPCR research as well as for GPCR drug design. The critical question now is how to identify the structural basis of differential allostery in different GPCR dimersa question that will require perseverance to answer definitively, given the notorious difficulty of obtaining high-resolution structures of GPCRs. However, with the powerful CODA-RET assay in hand, mutational analysis will provide a first glimpse into this important question. The CODA-RET assay will also allow more systematic structure-function analysis of the NPA agonist, which will help to exploit functional selectivity for tailoring GPCR agonists.

The future is bright for CODA-RET in GPCR research and beyond, as it generically resolves the notorious problem of quantitatively probing interactions and conformational changes involving highly promiscuous interaction partners.

In addition to its application to other GPCRs and GPCR effector proteins such as arrestins, CODA-RET can also be used to study signaling by other types of promiscuous dimeric receptors, such as receptor tyrosine kinases or cytokine receptors and their effector proteins. Another exciting possibility is to extend CODA-RET to Förster resonance energy transfer (FRET) by using split fluorescent proteins as energy donors. An advantage of FRET compared to BRET is that it allows direct visualization of signaling complexes using fluorescence microscopy techniques and thus can be used to assess functional heterogeneity on the plasma membrane down to the level of individual receptor complexes. ■

Jacob Piehler is in the Biology Department at the University of Osnabrück in Osnabrück, Germany. e-mail: piehler@uos.de

References1. Smith, N.J. & Milligan, G. Pharmacol. Rev. 62, 701–725 (2010). 2. Kniazeff, J., Prezeau, L., Rondard, P., Pin, J.P. & Goudet, C.

Pharmacol. Ther. 130, 9–25 (2011). 3. Whorton, M.R. et al. Proc. Natl. Acad. Sci. USA 104, 7682–7687

(2007). 4. Albizu, L. et al. Nat. Chem. Biol. 6, 587–594 (2010). 5. Vilardaga, J.P. et al. Nat. Chem. Biol. 4, 126–131 (2008). 6. Han, Y., Moreira, I.S., Urizar, E., Weinstein, H. & Javitch, J.A. Nat.

Chem. Biol. 5, 688–695 (2009). 7. Gilchrist, A. Trends Pharmacol. Sci. 28, 431–437 (2007). 8. Urizar, E. et al. Nat. Chem. Biol. 7, 624–630 (2011).9. Smith, N.J., Bennett, K.A. & Milligan, G. Mol. Cell. Endocrinol.

331, 241–247 (2011). 10. Galés, C. et al. Nat. Struct. Mol. Biol. 13, 778–786 (2006).

Competing financial interestsThe author declares no competing financial interests.

Go to a hardware store and buy every glue on the shelf; then bring them to the beach and try sticking something

to a wet rock. You will find almost no bonding at all. This failure of man-made glues is one of the reasons that we are so fascinated by marine bioadhesives. Mussels, barnacles and oysters are a few examples of the many creatures that produce adhesives and cements to affix themselves to surfaces. At this time, we still have much to learn about how these animals stick. On page 588 of this issue, Yu and colleagues have pried a

key insight from mussel glue1: these shellfish appear to be regulating redox equilibria in the name of adhesion.

The rare amino acid 3,4-dihydroxy-phenylalanine (dopa) is essential for generating the mussel’s adhesive matrix of cross-linked proteins (Fig. 1). Post-translational oxidation of tyrosine gives rise to this cross-linkable residue found in all mussel foot proteins (mfps). Dopa is susceptible to air oxidation, especially at the slightly basic pH of sea water. How do dopa proteins bond to surfaces? What chemistry

might be involved in protein cross-linking to form this glue?

To address these questions, Yu et al. examined an isolated mfp (mfp-3) with a surface forces apparatus to show that protein adhesion energy decreases significantly with increasing pH, a consequence of dopa oxidation at the higher pH1. Supporting the notion that oxidation is detrimental to adhesion energy, an analogous loss of adhesion occurred upon addition of periodate, a potent oxidizer1. If typical marine conditions will oxidize dopa but

BIOMATERIALS

Redox and adhesion on the rocksMan-made adhesives cannot match the ability of a marine mussel to affix itself to a wet rock. New insights help to describe the protein-surface bonding central to this feat of biological materials engineering.

Jonathan J Wilker