NATURE METHODS | VOL.2 NO.3 | MARCH 2005 | 163

NEWS AND VIEWS

New eyes to see texture in ligand efficacyTerry P Kenakin

Two different but complementary resonance energy transfer techniques present researchers with new tools to visualize ligand-mediated activation of G protein–coupled receptors in living cells.

Two ways to see new value in known things are to increase the resolving power of the tools used to look at them, and to change your vantage point (look at them from a different point of view). In this issue, the exceptional papers by Hoffmann et al.1 and Gales et al.2 illustrate how both of these approaches can greatly enrich our understanding of G protein–coupled receptor (GPCR) function in living cells.

Hoffmann et al.1 increased the resolv-ing power of an assay to measure con-formational changes in GPCRs through the innovative use of a fluorescein-based biarsenical dye (FlAsH) in fluorescence resonance energy transfer (FRET) stud-ies. The FRET technique is used to moni-tor the transfer of energy between two fluorophores bound in close proximity to each other. The change in energy is dependent on the distance between the donor and acceptor fluorophores to the sixth power, and therefore the method is sensitive to very small changes in distance (on the order of those that occur with changes in protein conformation). But a drawback of previous assays has been the large size of the fluorophores. The use of the green fluorescent protein variants CFP and YFP attached to GPCRs allowed mea-surement of changes in conformation but caused a loss of normal GPCR signaling function. To alleviate this problem with CFP and YFP, Hoffmann et al.1 replaced YFP with the much smaller FlAsH dye

binding motif. This preserved the signal-ing function of GPCRs and resulted in an increase in the signal. Using time-resolved signals from this new system, the authors monitored changes consistent with agonist-induced conformational changes in receptors. In practice, increasing the amplitude of the signal and minimizing the effect on the test protein increased the resolution power of the FRET technology. Furthermore, the increased amplitude of the signal may allow the quantitative study of agonist-specific receptor con-formations, which may lead to increased selectivity for agonists.

Taking the approach of changing the vantage point, the authors of the second paper, Gales et al.2, showed how biolu-minescence resonance energy transfer (BRET) can be used to directly moni-tor GPCR–G protein interaction in real time. This method is used to monitor energy transfer between a biolumines-cent donor (Renilla reniformis luciferase fused to the receptor) and a fluorescent acceptor (GFP variant fused to G-protein subunits) as the two proteins are brought together through agonist activation. No excitation light source is required, there-by eliminating high background signals sometimes observed with FRET. This technique is ideal for monitoring the real-time interaction of GPCR coupling to G proteins, the kinetics of early interactions between receptors and G proteins, and the

desensitization of receptors. The biosen-sor signal is extremely dependent upon the nature of the GPCR and G-protein subunit interaction; therefore, this approach can be used to probe the identity of the G pro-tein–receptor interaction. This theoreti-cally allows the quantification of agonist-directed stimulus trafficking (formation of agonist-specific receptor states that select for distinct G-protein types).

It has been through methods such as these that the concept of efficacy has been expanded. This term, first coined by the pharmacologist R.P. Stephenson, describes the ability of molecules to produce a physi-ological response (in Stephenson’s case, a contraction of guinea pig ileum). Because Stephenson’s vantage point was, singularly, tissue response, this was the only defini-tion originally available for efficacy.

Can a change in vantage point really change one’s impression of what is being seen? Well, the flat Earth of Ptolemy looked quite different once we possessed the technology to observe it from the moon. Parallel technological advances in biological research have provided new vantage points that have allowed the discovery of new behaviors of GPCRs. It is now known that GPCRs possess a rich library of behaviors ranging from pleiotropic interaction with G proteins and with other cellular proteins such as RAMPs and RCP3,4 to internalization5, production of homo- and heterodimers6 and desensitization7,8, among others.

Continuing with the previous astro-nomical allusion, it is easy to see how increasing the resolving power of obser-vational tools can allow us to see features previously undetected. The mountains on the moon did not spring into being in 1609; they simply became evident to Galileo after he constructed the first astronomical telescope. Similarly, ligands can be thought to have a number of ‘efficacies’, and using new tools such as BRET and FRET allows these behaviors to be observed. With these observations comes a necessarily new definition of efficacy. Realizing that GPCRs possess a number of behaviors mediated both spon-

Terry P. Kenakin is in the Department of Assay Development and Compound Profiling, GlaxoSmithKline Research and Development, Research Triangle Park, North Carolina 27709, USA.e-mail: terry.p.kenakin@gsk.com

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164 | VOL.2 NO.3 | MARCH 2005 | NATURE METHODS

NEWS AND VIEWS

taneously and through ligand interaction, efficacy is better defined as ‘that property of molecule that causes the receptor to change its behavior toward the cell’.

The ability of ligands to induce these GPCR behaviors need not necessarily be linear in sequence9,10. For example, whereas receptor internalization often follows receptor activation, there are cases in which ligands induce internalization without first causing activation11. The ability to identify collateral efficacy is a major new advance in the drug discovery process because, at least theoretically, it allows for the better definition of thera-peutic target molecules. New ways to see GPCR behavior allow the construction of more discerning pharmacological assays that, in turn, can lead to more selective drugs. This may be especially relevant in an era of drug discovery for cases in which it is more cost effective to rescreen chemi-cal libraries in different ways to determine hidden value rather than to simply make

more libraries. The key is to develop more ‘eyes to see’ receptor function in the form of new receptor assays. The application of BRET and FRET analyses to this end is a very appropriate advancement in the sci-ence of receptor activation and new drug discovery.

1. Hoffmann, C. et al. Nat. Methods 2, 171–176 (2005).

2. Galés, C. et al. Nat. Methods 2, 177–184 (2005).

3. Brady, A.E. & Limbird, L.E. Cell. Signal. 14, 297–309 (2002).

4. Lanier, S.M. Biol. Cell. 96, 369–372 (2004).5. Mack, M. et al. J. Exp. Med. 187, 1215–1224

(1998).6. George, S.R., O’Dowd, B.F. & Lee, S.P. Nat. Rev.

Drug Discov. 1, 808–820 (2002).7. Kohout, T.A. & Lefkowitz, R.J. Mol. Pharmacol.

63, 9–18 (2003).8. Sneddon, W.B. et al. J. Biol. Chem. 278,

43787–43796 (2003).9. Kenakin, T.P. Nat. Rev. Drug Discov. 1, 103–110

(2002).10. Kenakin, T.P. Nat. Rev. Drug Discov. 2, 429–438

(2003).11. Roettger, B.F. et al. Mol. Pharmacol. 51, 357–

362 (1997).

Stem cell culture, one step at a timeMartin F Pera

Introducing inhibitors of bone morphogenic proteins along with basic fibroblast growth factor (bFGF) into culture medium for human embryonic stem (ES) cells facilitates long-term maintenance of these cells in the pluripotent state, coming one step closer to the goal of a fully defined system for human ES cell (hESC) culture free of animal products.

When the derivation of hESCs was first reported, it was clear that substantial advances in cell culture methodology would be essen-tial to realize their enormous potential in research and regenerative medicine. Over six years later, researchers are still working out culture issues, step by step, and one devel-opment likely to be important is described in a report by Xu et al.1 in this issue of Nature Methods.

The original cell culture system used for the derivation and maintenance of

hESCs, which incorporated mouse feeder cell layer support and fetal calf serum as a media supplement, was not very different from that used in the isolation of mouse ES cells in 1981. Its limitations include the presence of several undefined components in serum and the feeder cell layer. These can have untoward effects on the growth and differentiation of hESC, and the use of products derived from animal sources carries the risk of pathogen transmis-sion. Furthermore this system only poorly

supports the clonal growth of hESCs (which is a major drawback for many experimental protocols requiring growth or selection of single cells).

Xu et al. based their study on the most widely used system for hESC cultivation today, which uses a proprietary serum replacement and bFGF as media addi-tives2. Many workers use these additives along with a mouse feeder cell layer to grow hESCs, but others have reported that conditioned medium (medium harvested from a culture of feeder cells), in combina-tion with a basement membrane extract known as Matrigel, is sufficient to support stem cell maintenance3. Xu et al. aimed to eliminate the requirement for both feeder cells and conditioned medium. The mechanism whereby feeder cells support hESCs is completely unclear and is almost certainly multifactorial.

Bone morphogenetic proteins (BMPs) are a group of proteins in the transform-ing growth factor- (TGF- ) superfamily with critical roles in several developmen-tal processes. BMPs had previously been shown to induce differentiation of hESCs, though the outcome of differentiation was highly dependent on other components of the cell culture environment4,5. Xu et al. noted that the medium containing the proprietary serum substitute had BMP-like activity, and they observed that after this medium was conditioned by several days of feeder cell cultivation, this BMP-like activity was reduced. The team then found that addition of the BMP antagonist noggin to the culture medium, along with an increase in the dose of bFGF, enabled the growth of hESC in the complete absence of a feeder cell layer or feeder cell–conditioned medium. The method sup-ported long-term propagation of stem cells in the undifferentiated state, and notably, these cells maintained pluripotency and a normal diploid karyotype. The new tech-nique eliminates any need to use a feeder cell layer and conditioned medium, both sources of undefined components and potential biohazards, in hESC culture.

But, as the authors note, their sys-tem still requires the proprietary serum replacement and Matrigel and is thus reliant on the use of complex animal pro-ducts. The culture system also does not support clonal growth of hESCs. Many important protocols for manipulation of cultured cells, such as the isolation of cells incorporating DNA constructs,

Martin F. Pera is at the Monash Institute of Medical Research, Monash University, and the Australian Stem Cell Centre, Victoria, Australia. email: martin.pera@med.monash.edu.au

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