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The molecular basis of cannabinoid CB1 receptor regulation of the sensitivity of orexin-1 receptor signalling

Reference	BB/D001951/1
Principal Investigator / Supervisor	Professor Graeme Milligan
Co-Investigators / Co-Supervisors
Institution	University of Glasgow
Department	Institute of Biomedical & Life Sciences
Funding type	Research
Value (£)	197,433
Status	Completed
Type	Research Grant
Start date	01/11/2005
End date	31/08/2009
Duration	46 months

Abstract

Hetero-dimerisation between certain GPCR pairs is increasingly appreciated to occur in physiological settings and is know to be able to alter receptor function and pharmacology. It is difficult to establish cell lines that stably and constitutively express certain GPCRs. The orexin-1 receptor is an example. To overcome this we have used the HEK293 Flp-In-T-rex cell line, in which an introduced cDNA is transcribed to generate protein from a single chromosomal integration site and in an inducible manner. By so doing we have been able to establish cell lines that express one GPCR constitutively and the second only upon induction. We will use these cells, and generate a number of others on the same basis, to examine the ability and propensity of the cannabinoid CB1 and the orexin-1 receptors to interact, using both FRET and BRET-based approaches, and hence to alter the sensitivity of orexin-1 receptor signalling. We have noted that only upon co-expression, the orexin-1 receptor internalises in an agonist-independent manner. Mutants of the CB1 receptor have been described that are unable to internalise in response to agonist ligands and probably will also fail to do so in an agonist-independent manner. When these forms of the CB1 receptor are co-expressed with the orexin-1 receptor we will examine if this prevents agonist-independent internalisation of the orexin-1 receptor and if so, the effects of antagonist ligands at each receptor on spontaneous recycling of the co-expressed wild type receptors and sensitisation of orexin-1 receptor signalling. SiRNA-based approaches will also be use to reduce beta-arrestin levels if spontaneous recycling of the two GPCRs is shown to be beta-arrestin-dependent and the effect of this on signal transduction will also be measured.

Summary

It is now well established that not only are G protein-coupled receptors (GPCRs) able to form constitutive homo-dimers but that at least certain pairs of GPCRs are also able to form hetero-dimers. This is not a random, stochastic process and occurs in physiological settings as well as in transfected cells. In different examples hetero-dimerisation has been shown to alter ligand pharmacology, signal capacity and the nature of the G protein activated by receptors. Although it is widely believed that GPCRs are located at the cell surface until stimulated by an agonist, constitutive, agonist-independent internalisation and recycling to the cell surface has recently been noted for a significant number of GPCRs. Although, when expressed alone the cannabinoid CB1 receptor is able to internalise in such a ligand-independent manner, the orexin-1 receptor does not. However, when co-expressed with the CB1-receptor the orexin-1 receptor displays this phenotype. The ability of an agonist-occupied GPCR to cause internalisation of a co-expressed GPCR for which the agonist has no significant affinity has previously been used to support concepts of hetero-dimerisation but this is the first example where internalisation of a potential partner GPCR occurs without activation of the other GPCR. Interestingly, it has previously been shown that co-expression of the CB1-receptor and the orexin-1 receptor results in hyper-sensitisation of phosphorylation of the ERK MAP kinases in response to orexin peptides and this is prevented by a CB1 receptor antagonist that is currently under clinical evaluation. We will explore if the basic phenomenon we observe reflects high affinity protein-protein interactions between these two GPCRs and if modulation of this phenotype eliminates the hypersensitivity of orexin-1 receptor signalling.

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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