Agonist stimulus trafficking by human prostanoid CRTH2 (DP2) receptors.

McArthur Wilson, Richard John (2007) Agonist stimulus trafficking by human prostanoid CRTH2 (DP2) receptors. PhD thesis, University of Nottingham.



Agonists of hormone receptors possess affinity (the ability to bind) & efficacy (the ability to stimulate effect). In this thesis, alternative expressions of efficacy by recombinant prostanoid Chemoattractant Receptor Homologous molecule of TH2 cell (hCRTH2) receptors have been studied using a variety of assays and pharmacological techniques.

When expressed in CHO cells, either with or without co-expression of chimeric G alpha 16z49 G-proteins, CRTH2 receptor-mediated calcium mobilisation pharmacology was found to be as published. Coupling of receptor activation to calcium elevation involved G beta gamma i/o mediated PLC beta -dependent mobilisation of both intra- & extra- calcium. In chimera-expressing cells, an additional coupling mechanism was observed which was presumably G alpha 16z49-mediated. The relative expression of receptor and G-protein molecules in both cell types was investigated but because of deficiencies in the methods employed the relative expression is essentially unknown. Because G alpha 16z49 & G beta gamma i/o represent different classes of PLC beta -activating G-proteins, simultaneous activation of them may have produced a synergistic response in chimera-expressing cells which may have affected the observed receptor pharmacology.

When the G alpha 16z49 component was isolated in PTX-treated chimera-expressing CHO G alpha 16z49 cells, reversals of potency order were observed with respect to responses in untreated cells. These were most striking for 17 phenyl PGD2, 15 R 15 methyl PGF2 alpha, 15 deoxy delta 12,14 PGJ2 and 15 R 15methyl PGF2 alpha. Alterations of potency order were also observed in non-chimeric cells (G beta gamma i/o coupling) compared with PTX treated chimera-expressing cells. These were most striking for indomethacin, 16,16 dimethyl PGD2, delta 12 PGJ2 and 9,10 dihydro 15 deoxy delta 12,14 PGJ2.

In [35S]-GTP gamma S accumulation assays using membranes prepared from non-chimeric cells and presumably reporting G alpha i/o coupling, agonist pharmacology was similar to G alpha 16z49 mediated calcium mobilisation data. However, the data were markedly different from G beta gamma i/o-mediated calcium mobilisation data generated in non-chimeric cells. These differences were most apparent for 13,14 dihydro 15 keto PGD2, 15 deoxy delta 12,14 PGJ2 and indomethacin.

Desensitisation of agonist-stimulated calcium mobilisation was also studied. PGD2 produced rapid & long-lasting desensitisation of hCRTH2 receptors in a biphasic manner suggesting that two desensitisation mechanisms may operate. At low concentrations of PGD2 desensitisation was PTX-insensitive suggesting that a non-Gi/o-protein mediated mechanism may be responsible. Other CRTH2 receptor agonists inhibited responses to subsequent PGD2 EC80 exposure in calcium mobilisation assays. Interestingly, a group of molecules devoid of agonism in the calcium assay also inhibited PGD2 responses. This group of molecules included 19 hydroxy prostaglandins A2, E2 & F2 alpha , and PGE2 and appeared to mediate their effects through a mechanism that did not involve a competitive interaction with PGD2.

The data generated here show that CRTH2 receptor agonist pharmacology is critically dependent on G-protein coupling partner and assay methodology, and are strongly indicative of agonist-directed stimulus trafficking. The data are consistent with the notion that G beta gamma subunit activation is not a passive "on-off" event but is rather an active event triggered by agonist- and GTP-dependent conformation changes in both receptor and G alpha subunit molecules.

Item Type:Thesis (PhD)
Supervisors:Hill, Stephen
Uncontrolled Keywords:Pharmacology, 7TM, 7 transmembrane, receptor, GPCR, G-protein coupled receptor, CRTH2, chemottractant receptor like molecule of TH2 cells, DP2, prostaglandin, G-protein, coupling, stimulus, trafficking, agonist, antagonist, conformation
Faculties/Schools:UK Campuses > Faculty of Medicine and Health Sciences > School of Biomedical Sciences
ID Code:308
Deposited By:Richard John McArthur Wilson
Deposited On:20 Mar 2008
Last Modified:06 Feb 2009 14:43

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