An Investigation into the pharmacology of the ghrelin receptor

Bennett, Kirstie Ann (2009) An Investigation into the pharmacology of the ghrelin receptor. PhD thesis, University of Glasgow.

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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
Gaq/11, Gai/o, Gas and Ga12/13 G protein pathways, although little is known about the signalling of ghrelin and the growth hormone secretagogues in all but the Gaq/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
Gai/o pathway.

[35S]GTPyS binding assays were used to measure activation of the Gai/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[125I]]-
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. [35S]GTPyS binding assays confirmed that the GRLN-R was constitutively
active through both the Gaq/11 and Gai/o pathways, demonstrated by an increase in [35S]GTPyS loading upon receptor expression which could be reduced by the 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.

In contrast to the [35S]GTPyS assays used to measure activation of Gai/o, [35S]GTPyS assays
with a Gaq-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 Gaq/11 and Gai/o responses, however, a Gai/o-mediated response could not be measured in cAMP accumulation assays, suggesting that the GRLN-R could signal via activation of Gao but not Gai1-3. Although the activation of the Gas pathway by the GRLN-R remains controversial, in this study ghrelin and the growth secretagogues could evoke a Gas-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.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: ghrelin, GPCR, GTPyS, pharmacology, allosterism, ghrelin receptor, constitutive activity
Subjects: Q Science > QH Natural history > QH301 Biology
Q Science > QP Physiology
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
Supervisor's Name: Milligan, Prof. Graeme and Wise, Dr. Alan
Date of Award: 2009
Depositing User: Dr Kirstie Ann Bennett
Unique ID: glathesis:2009-1047
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Aug 2009
Last Modified: 10 Dec 2012 13:31

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