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STRANGE Lab for GPcR Research

The group works on G protein coupled receptors aiming to understand their structure and function. We have concentrated on receptors for neurotransmitters, particularly dopamine and serotonin, because of the importance of these as targets for drugs used to treat brain disorders. More recently, we have begun to examine chemokine receptors owing to their role in facilitating the infection of cells by HIV. Current projects are as follows :- The basis and importance of GPCR dimerisation/oligomerisation Understanding Receptor/G protein signalling The mechanism of action of antipsychotic drugs The action of chemokines on the chemokine receptor CCR5 Analytical Pharmacology

Current Projects
(i)The basis and importance of GPCR dimerisation/oligomerisation This is a phenomenon for GPCR's that is beginning to be accepted^30 but the functional importance of dimer formation is not yet well defined. For the D2 dopamine receptor we have shown using biochemical techniques such as immunoprecipitation of two epitope tagged forms of the receptor and using Fluorescence Resonance Energy Transfer (FRET) that the receptor is a constitutive dimer/oligomer^33. The importance of this oligomer formation for the function of the receptor has been studied using ligand binding. For the receptor expressed in either CHO cells or in Sf 9 insect cells we have shown that the maximal binding (Bmax) for different radioligands is different and may be manipulated by changing the concentration of sodium ions in the assay buffer ^24,48. These data are not consistent with a monomeric receptor and may be interpreted in terms of binding to an oligomeric receptor with negatively cooperative interactions between successive molecules of ligand for binding to the receptor. It seems that drugs can interact at the D2 receptor in two ways, either by competitive inhibition or by allosteric/non-competitive interaction across a dimeric/oligomeric receptor. Previous work in the lab had shown that the D2 receptor was subject to allosteric regulation by amiloride analogues ^4,19 so this may all be part of a general picture that is emerging whereby drugs can act on GPCR's either at the primary ligand binding site or via allosteric interactions. If, as seems be apparent, GPCRs are oligomers it is important now to examine whether this has further functional consequences.	(ii) Understanding receptor/G protein signalling in relation to the mechanism of agonists and inverse agonists. Here we are taking several approaches. CHO cells expressing D2 dopamine and 5HT1A serotonin receptors are being examined. Assays have been developed for the activation of G proteins using the binding of [^35S]GTPgS and the system is being manipulated in various ways and effects on agonist signalling determined ^1,6,9,31. We are using these systems to ask questions about how agonists and inverse agonists signal through G protein coupled receptors in order to understand the mechanisms of ligand efficacy. For example, we have performed several investigations in to the mechanisms of agonist action ^6,9,29. For some groups of compounds the stabilisation of the ternary complex of agonist/receptor/G protein can account for agonist efficacy whereas for other this is not the case and the system seems more complex. One possibility is that for full and partial agonists the rate determining step in the G protein cycle is different and we have some evidence to support this idea ^38. In order to examine this further we are developing assays for each of the steps in the G protein cycle so that effects of full and partial agonists may be examined^54. We are also using the insect cell/baculovirus system in order to express specified combinations of D2 receptor and different G proteins in order to examine R/G specificity and the effects of changing the R/G ratio. Using this system we have shown that agonists signal more effectively through a D2/Go combination as compared to a D2/Gi2 combination ^25. This work has been extended to examine interactions between D2 and Gi1, Gi2, Gi3 and Go ^32,34. It seems that each R/G combination has its own pharmacological signature. Much of this work is backed up by simulations using models for R/G interaction and a number of theoretical papers have been published on this topic and models generated.
(iii) The mechanism of action of antipsychotic drugs. We published the first report that these drugs are inverse agonists at D2 dopamine receptors and we are examining the importance of this inverse agonism for the actions of these drugs ^5. We also showed that inverse agonism was a property of both the typical and the atypical antipsychotic drugs ^44. Additionally, we have been able to show inverse agonist effects at the level of inhibition of [^35S]GTPgS binding and this work has shown that some of the drugs achieve their inverse agonist effects by converting the receptor to an inactive state unable to signal, whereas others prevent formation of the ternary complex ^46. We have also studied a range of partial agonist drugs such as aripiprazole, a new antipsychotic that conflicts with the accepted paradigm that antispsychotics must be antagonists or inverse agonists at D2 receptors. Using the [^35S]GTPgS binding assay we have shown that this drug is indeed a partial agonist and we have developed conditions for increasing the signals for such partial agonists in this assay^50. This has allowed us to profile a range of drugs at this receptor for their activity and develop an efficacy scale^50, 56.	(iv) The actions of chemokines on the chemokine receptor CCR5. This receptor is of importance because as well as being an important target for chemokine action it is a co-receptor for the entry of HIV in to lymphocytes. We are examining a number of events that follow CCR5 activation (G protein activation, calcium release, inhibition of cAMP production, receptor phosphorylation, receptor internalisation, receptor dimerisation) and the ability of a range of natural chemokines to trigger these different events. The panel of chemokines used exhibit different abilities to affect the different signalling events ^27, 49, 53. We have also examined the mechanisms of internalisation of CCR5 in cells and have provided evidence that CCR5 enters cells through coated pit and caveloae mechanisms ^26. This work has been extended to examine the intracellular pathways involved in receptor internalisation and recycling and it appears that actin microfilaments and Rho proteins may be involved ^40. A series of drugs directed at CCR5 has been examined and shown to be inverse agonists against constitutive activity from the native receptor. These drugs are also allosteric antagonists of chemokine activation of CCR5^52.
(v) Analytical Pharmacology. Much of our work is backed up by simulations of data. Recently, however, a study has been published that addresses issues about the definition of agonist affinity and agonist efficacy^57. It seems that for many GPCRs, providing G protein coupling is suppressed, good estimates of agonist affinity may be obtained using ligand binding and functional assays. New methods for assessing agonist efficacy are also proposed.

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Last updated Feb. 2008