The secret lives of GPCRs

As described in a recent review byGraham Milligan [1], new technologiessuch as ‘high-content assays’ haveenabled us to see previously unknown,or at least not easily quantified,G-protein-coupled receptor (GPCR)behaviours. Historically, drug efficacywas defined in terms of tissue response,because this was the only meansavailable to detect ligand-inducedchanges in receptors. However, newassay techniques have shown that, in addition to activating G-proteins,GPCRs display a range of behaviours,including homodimerization,heterodimerization, internalization and interaction with a host of cytosolicand membrane proteins. Many of thesebehaviours are associated with anagonistic response but not necessarilywith G-protein activation [2]. In fact,dissociations have been observed – for example, the cholecystokinin (CCK)receptor antagonist D-Tyr-Gly-[(Nle28,31,D-Trp30)cholecystokinin-26-32]-phenethyl ester does not producereceptor activation but does produce aprofound receptor internalization [3].

There are at least two reasons whyexpanded detection systems forligand-induced changes in receptorbehaviour are potentially beneficial fordrug discovery. The first is that,thermodynamically, there is reason tobelieve that ligand binding to receptorsalso biases the conformations of thereceptor (i.e. affinity and efficacy arecorrelated) [4]. For instance, the newtechnique, fluorescence lifetimespectroscopy, shows Gaussiandistributions for the halftime of acovalently bound fluorescent probe onthe β2-adrenoceptor [5], indicating anever-changing protein environment (an ensemble of tertiary conformations).The presence of the neutral antagonist,alprenolol, changes the distribution oflifetimes (β2-adrenoceptor conformations)

indicating that, although no effect ofthis ligand is seen with conventionalfunctional assays, protein conformation isstill changed by the binding of the ligand.In other words, if a given assay does notdetect altered receptor efficacy, this doesnot necessarily mean that no change hasoccurred. A dramatic example of this isthe prevalence of inverse agonism,a phenomenon thought to be rare whenfirst discovered by Costa and Hertz [6],and now becoming extremely commonwith the increasing availability of theassay required to view the effect(constitutively active receptor systems).

The second reason why expandeddetection might be of benefit is thatsome of the non-G-protein-associatedactivities of GPCRs could betherapeutically useful. For example,internalization of the chemokinereceptor, CCR5, is theoretically anexcellent strategy for the prevention ofHIV-1 infection [7]. By screening with theexpanded eyes of high-content assays,ligand effects on the secret lives of GPCRswill hopefully lead to new generations oftherapeutically useful drugs.

References1 Milligan, G. (2003) High-content assays for

ligand regulation of G-protein-coupledreceptors. Drug Discov Today 8, 579–585

2 Kenakin, T.P. (2002) Efficacy at G proteincoupled receptors. Annu. Rev. Pharmacol.Toxicol. 42, 349–379

3 Roettger, B.F. et. al (1997)Antagonist-stimulated internalization of theG protein-coupled cholecystokinin receptor. Mol. Pharmacol. 51, 357–362

4 Kenakin, T. P. and Onaran, O. (2002) The ligand paradox between affinity andefficacy: can you be there and not make adifference? Trends Pharmacol. Sci. 23,275–280

5 Ghanouni, P. et. al (2001) Functionallydifferent agonists induce distinctconformations in the G protein couplingdomain of the β2 adrenergic receptor. J. Biol. Chem. 276, 24433–24436

6 Costa, T. and Herz, A. (1989) Antagonistswith negative intrinsic activity at δ-opioidreceptors coupled to GTP-binding proteins.Proc. Natl. Acad. Sci. U. S. A. 86, 7321-7325

7 Wells, T.N. et. al (1996) Chemokine receptors– the new frontier for AIDS research. Chem. Biol. 3, 603–609

Terry P. KenakinSystems Research 3-2104.2B

GlaxoSmithKline5 Moore Drive

Research Triangle Park, NC 27709, USA

update discussion forum DDT Vol. 8, No. 15 August 2003

1359-6446/03/$ – see front matter ©2003 Elsevier Science Ltd. All rights reserved. PII: S1359-6446(03)02783-1674 www.drugdiscoverytoday.com

The BioMedNet Magazine

The online-only BioMedNet Magazine contains a range of topical articles currently available in Current Opinion and Trends journals,

and offers the latest information and observations of direct and vitalinterest to researchers.

You can elect to receive the BioMedNet Magazine delivered directly to your email address, for a regular and convenient survey of at what’s

happening outside your lab, your department, your specialty.

Issue by issue, the BioMedNet Magazine provides an array of some of the finest material available on BioMedNet, dealing with matters of dailyimportance: careers, funding policies, current controversy and changing

regulations in the practice of research.

Don’t miss out!

Join the challenge at the start: register now athttp://news.bmn.com/magazine to receive our regular editions.

Written with you in mind – the BioMedNet Magazine.