G protein coupled receptors – exploiting flexible conformations

Posted: 3 September 2012 |

G-protein coupled receptors (GPCRs) are a diverse super-family of proteins located within the plasma membrane of eukaryotic cells which have a common architecture consisting of seven-transmembrane (7-TM) segments, connected by extracellular (ECL) and intracellular (ICL) loops. They differ from other 7-TM proteins in their ability to activate guanine-nucleotide binding proteins or β-arrestin and so initiate a signalling cascade. They have a wide range of physiological roles and provide many successful drug targets, playing a role in disorders including allergies, cardiovascular dysfunction, depression, obesity, cancer, pain, diabetes and a variety of central nervous system conditions. This review will give a general overview of GPCRs and how their structures and activities can be used in drug discovery…

Figure 1: Classification Scheme of GPCRs. R (Rhodopsin-like), S (Secretin-like), G (Glutamate-like), Others (Adhesion, Frizzled, Taste type-2, unclassified)

Figure 1: Classification Scheme of GPCRs. R (Rhodopsin-like), S (Secretin-like), G (Glutamate-like), Others (Adhesion, Frizzled, Taste type-2, unclassified)

G-protein coupled receptors (GPCRs) are a diverse super-family of proteins located within the plasma membrane of eukaryotic cells which have a common architecture consisting of seven-transmembrane (7-TM) segments, connected by extracellular (ECL) and intracellular (ICL) loops. They differ from other 7-TM proteins in their ability to activate guanine-nucleotide binding proteins or β-arrestin and so initiate a signalling cascade. They have a wide range of physiological roles and provide many successful drug targets, playing a role in disorders including allergies, cardiovascular dysfunction, depression, obesity, cancer, pain, diabetes and a variety of central nervous system conditions1-3. This review will give a general overview of GPCRs and how their structures and activities can be used in drug discovery.

GPCRs may be classified into five major families with the Rhodopsin-like being the largest (672 human family members) followed by the Frizzled / Taste (36 members), Adhesion (33), Glutamate (22), and Secretin (15) sub-groups (Figure 1)4. While the Rhodopsin-like family remains the most widely studied, the Adhesion family of GPCRs is particularly fascinating. They have an extended cleavable N-terminal domain thought to be involved in cell-cell contact5 and almost all are still orphan receptors. There are significant associations between the Adhesion group and human disease but as yet, there are no specific small molecules which address their function. Frizzled receptors play a role in governing cell polarity, embryonic development, formation of neural synapses, cell proliferation and many other processes in developing and adult organisms6.

The small Secretin-receptor family is structurally and functionally diverse and includes receptors for polypeptide hormones, and for Drosophila proteins that regulate stress responses and longevity7. Glutamate receptors have a large extracellular N-terminus (often likened to a clam) which binds the orthosteric (endogenous) ligand. Several allosteric ligands to these receptors have been identified and these appear to bind within the seven transmembrane region. Many of the receptors have accessory proteins which either facilitate trafficking to the plasma membrane or influence the specificity of the ligand8.

GPCRs respond to various ligands extracellularly, such as amines, peptides, hydrophobic effectors, hormones, small proteins and volatiles, which allows the receptors to activate the G-proteins intracellularly. Pharmacological agents that act at GPCRs can broadly be categorised into four main classes: agonists, inverse agonists, antagonists and allosteric modulators. Nearly 50 per cent of currently marketed drugs2,9-11 are targeted at less than 20 per cent of these receptors (non-odorant group). This number of drugs is expected to increase as new functions / ligands for GPCRs are discovered and so GPCR targets will remain a very active on-going focus in drug discovery efforts12,13.

Figure 1: Classification Scheme of GPCRs. R (Rhodopsin-like), S (Secretin-like), G (Glutamate-like), Others (Adhesion, Frizzled, Taste type-2, unclassified)

Figure 1: Classification Scheme of GPCRs. R (Rhodopsin-like), S (Secretin-like), G (Glutamate-like), Others (Adhesion, Frizzled, Taste type-2, unclassified)

GPCR structure and in silico ligand design

Traditionally, pharmacophore-based app – roaches, where the most relevant structural features of active compounds are collected, have been applied to GPCR ligand design14-16. However, exploitation of the 3D structures of GPCRs is now preferable due to the elucidation of the structure of rhodopsin17, followed by those of the beta118, beta219, adrenergic, adenosine A2A20, dopamine D321, CXCR4 chemokine22, histamine H1 receptors23 and recently sphingosine 1-phosphate receptor24 and the M2 muscarinic acetylcholine receptor25. These GPCRs tend to accommodate their small molecule effectors with similar spatial arrangement, but the particular interactions with amino acid side chains are quite different. These crystal structures have presented the real possibility of using information on the ligandbinding pockets to allow in silico screening for novel small-molecule ligands.

Structure based virtual screening (VS) is typically performed by docking a molecule into the binding site and determining the optimal orientation. Subsequent scoring of these complexes allows assessment of the binding modes and ranking by affinity, prioritisation and biological testing. Carlsson et al26 utilised molecular docking to computationally screen a 1.4 million compound database against the adenosine A2A receptor20. Of those tested, 35 per cent showed substantial activity with affinities between 200 nM and 9 μM with over 50-fold specificity for the adenosine A2A versus the related A1 and A3 adenosine receptor subtypes. Katritch et al27 also screened four million commercially available compounds against the same crystal structure. Out of 56 high ranking compounds tested in adenosine A2A receptor binding assays, 23 showed affinities under 10 μM, 11 of those had sub-μM affinities and two under 60 nM indicating the success of such an approach.

The structures of activated and/or agonistbound GPCRs have also been determined28-31 and can now be utilised in drug design efforts. The biggest structural change observed is a movement outwards from the bilayer of the cytoplasmic parts of the 5th and 6th transmembrane (TM) helices. The structure of activated beta-2 adrenergic receptor in complex with Gs confirmed that the Gα binds to a cavity created by this movement in the vicinity of the 5/6 intracellular loop32.

Homology modelling of GPCRs from X-ray structures is also now more accurate with the use of multi-template models33, the incorporation of knowledge-based constraints and molecular dynamics simulations34-36 and by refining docking poses37. These approaches can indicate potential structural features not revealed by X-ray methods. The feasibility of docking screens against modelled GPCRs was considered in the recent study of Carlsson et al38 on 3.3 million molecules docked into a homology model of the D3 dopamine receptor and subsequently on the released crystal structure. For the homology model, 26 were tested for binding and six had affinities ranging from 0.2 to 3.1 μM. Of the 25 from the crystal structure, five had affinities ranging from 0.3 to 3.0 μM.

GPCR – G protein Assays

Historically, 7-TM receptors were reported to couple to one type of G-protein: e.g. Gi, Gs, Go or Gq, and the method chosen to measure function was linked to the G-protein classification.

Due to the low expression levels of GPCRs in native tissue, the development of recombinant technology has greatly enhanced the way in which we are able to measure GPCR activity. However, this has come at a price. Data from recombinant cell lines can suffer from lack of the appropriate associating proteins, or again, from altered post-translation modification compared to native systems, and thus the receptors can exhibit non-physiological pharmacology39. In addition, recombinant cell lines have high levels of receptor reserve and, therefore, partial agonists or antagonists may appear as full agonists or demonstrate agonism respectively. For instance, N-des-methyl clozapine (NDMC), has been shown to act as a potent partial agonist of the M1 receptor in recombinant systems40-42, but behaves as an antagonist with human tissue43. It is suggested that recombinant systems more efficiently couple to the G-protein, and thus can reach full activation through lower occupancy39,44.

If partial agonists cannot be identified early in drug discovery, this will have an impact on the success of the programme depending on the mode of action required for therapeutic efficacy, since partial agonists will actually lower endogenous agonist response. One successful way of being able to fully characterise full, partial and inverse agonism is by measuring G-protein [35S] GTPγS binding, in preference to reporter gene, calcium flux or downstream kinase assessment assays45. The latter are generally not sensitive enough to report efficacy (Emax) values since they are reliant on accumulation of product, but, [35S] GTPγS binding is a direct measure of GPCR activity.

Recent developments of specific anti-Gs and Gq probes have allowed, in conjunction with scintillation proximity assay (SPA) bead technology46-48, the direct ‘capture’ of Gs and Gq as well as Gi signals49,50 in plate-based formats. These have been used successfully for both recombinant and native tissue systems where knock-out mice and chemical tools are available to validate the system for particular GPCR subtypes50. Recently, label free technologies51 have been developed to measure GPCR efficacy related to specific G-proteins as well as measuring the response in real time, thus capturing all events rather than in a particular time interval assay such as with [35S] GTPγS, where time-dependent events may be missed (reviewed in52).

β-arrestin activity

Label free assays are measures of impedance or optical changes for live cell responses to ligands, which can detect changes in cellular features including adhesion, proliferation, migration and cell death52. GPCRs may not signal by just one process but be functionally selective53. Each transduction system is thought to demonstrate a distinct temporal profile54. For example, agonists may differentially activate G-proteins and/or β-arrestin (biased ligands, Figure 3). Conventionally, the measurement of each event requires a different cell line and cell specific affects cannot be excluded over biased signalling. However, label free technology holds the promise of identifying ligand biased affects in the same cell type. Watts et al55 have demonstrated, for example, CXCL9 as a Gprotein specific agonist of the chemokine receptor CXCR3 compared to previous evidence which suggested a role in desensitisation56. Rational design of such ‘biased’ or ‘functionally selective’ ligands requires a deep structural understanding of the different conformations a GPCR can assume and of how each con – formation influences various downstream G-protein and arrestin signalling pathways – a very challenging objective57.

Figure 3: Currently, GPCRs are considered to utilize two primary types of transducers: G proteins and β- arrestins. Accessory proteins can either facilitate trafficking to the plasma membrane or influence the specificity of the ligand. Homodimerisation, heterodimeristion and transactivation may aid in increasing the diversity of signalling pathways

Figure 3: Currently, GPCRs are considered to utilize two primary types of transducers: G proteins and β- arrestins. Accessory proteins can either facilitate trafficking to the plasma membrane or influence the specificity of the ligand. Homodimerisation, heterodimeristion and transactivation may aid in increasing the diversity of signalling pathways


It has generally been thought that one agonist molecule binds one GPCR molecule; which in turn activates one G-protein subtype. However, gathering evidence suggests that this is not always (or maybe even never has been) the case53. One example is seen with the melanocortin 4 receptor (MC4R) which we have demonstrated forms homodimers when recombinantly expressed in the HEK-293 cell line (unpublished data). When a mutant receptor that lacked the ability to bind the agonist was co-expressed with a receptor that could not signal, MC4R signalling was restored. Such transactivation58 has been further demonstrated in vivo54, whereby, in a transgenic mouse model expressing binding-deficient luteinising hormone receptor (LHR) and signalling deficient LHR, normal signalling occurred59.

Attention now is focusing not only on homo-dimerisation and its functional consequence but also on hetero-dimerisation where such distinct heterodimers should be classed as a molecular target rather than the individual proteins60,61 (Figure 3). Likewise, GPCRs come under a number of allosteric pressures from accessory proteins; for example we have demonstrated that the G-protein negatively modulates agonist binding in the MC4R (unpublished data) and in the absence of the G-protein (high GTPγS concentrations), agonist occupancy increases. Likewise, Secretin GPCRs are well documented to require accessory proteins such as RAMPs for function62,63. This kind of observation, alongside the premise of receptor reserve oligomerisation, cell specific G-protein coupling and accessory protein requirement is driving pharmacological investigation away from simple recombinant systems towards more ‘native-like’ systems for studying GPCR function in the hope of higher success rates in the identification of novel therapeutics.

GPCR pharmacology

Changes in ‘typical’ GPCR pharmacology are also reflected in the types of ligands that are being sought. Identification of allosteric modulation of GPCRs is not novel, but the pharmaceutical search for an alternative mechanism to activate GPCR function has expanded into this space. Positive allosteric modulators have typically enhanced endogenous agonist activity. However, it is as yet unclear whether the ‘alternative’ binding site nevertheless still resides in the penumbra of the agonist site as has been suggested by mutagenesis. The recent interest in dimerisation / complementation and accessory proteins has employed allosterism not only for modulating the orthosteric site, but also protein interactions that modulate GPCR function.

Dualsteric ligands represent a novel mode of targeting GPCRs by addressing simul – taneously both the orthosteric and an allosteric binding site e.g. the design of dualsteric muscarinic agonists64. To date, discovery efforts for several GPCR subtypes have failed to deliver really selective drug candidates65 but potentially, with a dualsteric approach, orthosteric receptor activation may be linked with allosteric subtype-selectivity and intracellular signalling pathway selectivity in a single molecular entity. Such an approach may also simultaneously target more than one GPCR, e.g. bivalent beta2- adrenergic and adenosine A1 receptor ligands66.

Pharmacological chaperones

Mutated versions of membrane proteins can result in local miss-folding, preventing efficient trafficking to the plasma membrane and exposure to circulating ligands. This can result in disease. Effective trafficking can be rescued using pharmacological chaperones (small hydro – phobic molecules that can penetrate the cell membrane) which bind to the nascent GPCR, and presumably restore the native fold. Conn and Janovick67 have pioneered the use of cell-permeant small molecule Gonadotropinreleasing hormone (GnRH) receptor antagonists to rescue poorly expressed GnRH mutant receptors. Recently, a set of MC4R mutants, linked with an obese phenotype, has been successfully rescued to the plasma membrane by the MC4R inverse agonist ML0025376468. To examine the potential effect that these mutations may have on the local receptor structure and the binding poses of the identified pharmacological chaperones with MC4R, we developed a model of the ‘inactive form’ of MC4R and the three mutants V50M, S58C and I137T (unpublished work). From our computational analysis, it is evident that such point mutations can result in changed interactions which may alter helical-helical packing and effect receptor stability and/or activation steps without directly affecting the ligand binding pocket. Additionally, the local change induced with each of the three mutants is different and further supports a hypothesis that each is retained by a different molecular mechanism. Exposure to putative pharmacological chaperones was shown to increase the plasma membrane expression of the wild type and the three mutant MC4Rs, V40M, S58C and I137T (unpublished work).


This short review has attempted to capture the essence of the family of GPCRs as we currently understand them. Increasing knowledge of their structural and functional characteristics has opened up new avenues for intervention strategies for GPCR-related human conditions. In particular, their apparent atypical conformational flexibility has been intriguing, giving rise to the prospect of new therapeutic approaches. At the same time, however, it will continue to be important to develop assay systems that address this dynamic behaviour, not only with respect to different conformational states but also probably different forms of quaternary associations.



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About the authors

Dr Kathryn Chapman is a senior assay development scientist and a member of Imperial College Academic Drug Discovery Centre. She is involved in a number of drug discovery programmes focusing on early hit discovery and pharmacological profiling.

Professor John Findlay studies membrane proteins which are hugely important for investigations ranging from fundamental studies on structure and cellular biology, to humanitarian and commercial applications in areas such as drug discovery and the emergent field of biosensors.

Dr Gemma Kinsella is a Health Research Board (HRB) postdoctoral research fellow in the Membrane Protein Lab, of the Department of Biology, National University of Ireland Maynooth. Her research focuses on protein structure prediction and the early stages of drug development for diseases encompassing a number of GPCR targets.