An Investigation into the Pharmacology of the Ghrelin Receptor Kirstie Ann Bennett (BSc Hons) Submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy Department of Neuroscience and Molecular Pharmacology Faculty of Biomedical and Life Sciences University of Glasgow August 2009 2 Abstract The ghrelin receptor (GRLN-R) was cloned in 1996 after the discovery that a series of synthetic growth hormone-releasing compounds (the growth hormone secretagogues) acted through a receptor distinct from the growth hormone-releasing hormone receptor. In 1999 the endogenous ligand of the receptor, ghrelin, was discovered. As well as stimulating growth hormone release, ghrelin has been shown to be involved in many other processes such as appetite stimulation and the regulation of energy homeostasis, making the ghrelin/GRLN-R system an attractive pharmaceutical target for the treatment of disorders such as growth hormone deficiency, cachexia and obesity. The GRLN-R displays a high level of ligand-independent (constitutive) activity and has been suggested to couple to Gαq/11 , G αi/o , Gαs and G α12/13 G protein pathways, although little is known about the signalling of ghrelin and the growth hormone secretagogues in all but the G αq/11 pathway. Two of the growth hormone secretagogues, GHRP-6 and L-692,429, have been described as ‘ago-allosteric modulators’ of the GRLN-R as, when co-administered with ghrelin, GHRP-6 and L-692,429 were reported to act both as co-agonists (increasing the efficacy of the ghrelin response) and as negative or positive (respectively) regulators of the potency of ghrelin. This study sought to investigate the pharmacology of the GRLN-R through the Gαi/o pathway. 35 [ S]GTP γS binding assays were used to measure activation of the G αi/o pathway, demonstrating that GHRP-6, L-692,585 (a commercially available analogue of L-692,429) and a third growth hormone secretagogues, MK-677, acted with higher efficacy than ghrelin. At least in the system tested, upon co-administration with ghrelin each of the growth hormone secretagogues acted in a simple competitive fashion with ghrelin. Radioligand binding experiments showed that the dissociation kinetics of [His[ 125 I]]- ghrelin from the GRLN-R were not altered by co-administration of the growth hormone secretagogues. Fitting data to a modified operational model of allosterism demonstrated that GHRP-6, L-692,585 and MK-677 were not ago-allosteric modulators of the GRLN-R but simple orthosteric agonists. In order to further examine the receptor-specific effects of ghrelin and the growth hormone secretagogues, a Flp-In™ T-REx™ HEK293 cell line expressing the GRLN-R was constructed. [35 S]GTP γS binding assays confirmed that the GRLN-R was constitutively active through both the G αq/11 and G αi/o pathways, demonstrated by an increase in [35 S]GTP γS loading upon receptor expression which could be reduced by the 3 administration of the GRLN-R inverse agonist SPA. Upon expression of the GRLN-R a considerable level of cell detachment was observed with the remaining cells appearing rounded compared to parental HEK293 cells, an affect that appeared to be mediated by the constitutive activity of the receptor. 35 35 In contrast to the [ S]GTP γS assays used to measure activation of G αi/o, [ S]GTP γS assays with a G αq-immunoprecipitation step demonstrated that the growth hormone secretagogues acted with equal efficacies to that of ghrelin, demonstrating functional selectivity at the GRLN-R. Intact cell assays were also used to measure Gαq/11 and G αi/o responses, however, a G αi/o -mediated response could not be measured in cAMP accumulation assays, suggesting that the GRLN-R could signal via activation of G αo but not G αi1-3. Although the activation of the G αs pathway by the GRLN-R remains controversial, in this study ghrelin and the growth secretagogues could evoke a G αs-mediated cAMP response (although L-692,585 acted with a lower efficacy than ghrelin). Finally, two naturally occurring missense mutations of the GRLN-R (A204E in the second extracellular loop and I134T in the third transmembrane helix) were analysed to investigate whether these mutations led to retention of the receptor within the endoplasmic reticulum and to investigate whether the mutations affected the ability of the GRLN-R to signal to the growth hormone secretagogues. The A204E mutation caused partial retention of the GRLN-R within the endoplasmic reticulum, whilst receptor that was transported to the plasma membrane did not display any measurable constitutive activity. In contrast, the I134T mutation did not alter receptor localisation, nor did it have any effect on the constitutive or ligand-induced activation of the GRLN-R, however, it appeared to lower the efficacy of the inverse agonist SPA. 4 Contents 1 Introduction .............................................................................................................19 1.1 Introduction to ghrelin and the ghrelin receptor ................................................19 1.2 Ghrelin tissue distribution.................................................................................24 1.3 GRLN-R tissue distribution..............................................................................24 1.4 Biological actions of the GHs-R1b ...................................................................25 1.5 Biological actions of ghrelin, the growth hormone secretagogues and the GRLN- R .........................................................................................................................25 1.5.1 GH release................................................................................................25 1.5.2 Appetite and energy homeostasis ..............................................................26 1.5.3 Other ghrelin/GRLN-R effects..................................................................28 1.5.4 Existence of receptors for ghrelin/the growth hormone secretagogues other than the GRLN-R.....................................................................................................29 1.6 Ghrelin-null or GRLN-R-null models...............................................................31 1.7 Clinical trials and therapeutic potential of GRLN-R agonists/antagonists..........33 1.7.1 Cachexia...................................................................................................33 1.7.2 GH deficiency...........................................................................................34 1.7.3 Obesity.....................................................................................................35 1.8 GPCR structure ................................................................................................36 1.8.1 Introduction to the G protein-coupled receptor superfamily.......................36 1.8.2 GPCR classification..................................................................................36 1.8.3 GPCR crystal structure .............................................................................38 1.8.4 GRLN-R structure ....................................................................................39 1.8.5 Residues contributing to the constitutive activity of the GRLN- R ............43 1.8.6 Ligand-binding pocket of the GRLN-R.....................................................45 1.8.7 Ligand-binding pocket for SPA ................................................................46 1.9 Dimerisation.....................................................................................................48 1.9.1 Introduction to dimerisation......................................................................48 1.9.2 Dimerisation of the GRLN-R....................................................................49 1.10 GPCR signalling...............................................................................................50 1.10.1 Introduction to G proteins .........................................................................50 1.10.2 G protein coupling of the GRLN-R ...........................................................51 1.10.3 G protein-independent signalling ..............................................................55 1.10.4 Desensitisation and endocytosis................................................................56 1.10.5 Constitutive activity..................................................................................60 1.10.6 Allosterism...............................................................................................65 1.11 Project aims......................................................................................................67 2 Materials and methods .............................................................................................68 2.1 General reagents, enzymes and kits ..................................................................68 2.2 Antibodies and antisera ....................................................................................70 2.3 Pharmacological compounds ............................................................................71 2.4 Radiochemicals................................................................................................72 2.5 Tissue culture disposables and reagents ............................................................72 2.6 Buffers and solutions........................................................................................73 2.6.1 Molecular biology buffers and solutions ...................................................73