G protein signalling characteristics of human IP prostanoid receptors

Fong, Chee-Wai (1999) G protein signalling characteristics of human IP prostanoid receptors. PhD thesis, University of Glasgow.

Abstract

The functional assay of agonists acting on G protein coupled receptors (GPCRs) coupled to the Gsalpha subunit usually involves the measurement of effector (adenylate cyclase) activity. However, the activity of adenylate cyclase can be modulated by proteins other than Gsalpha, and their levels and subtypes vary between cell lines. As such, measurement of agonist efficacy at the level of the G protein would be most ideal. This is currently not possible with traditional assays such as the [35S]GTPalphaS binding and high affinity GTPase assays, since activated Gsalpha has low rates of GTP exchange and hydrolysis (Wieland et al. 1994; Gierschik et al. 1994). A FLAG(TM)-tagged form of the human IP prostanoid receptor (a Gsalpha-coupied GPCR) was expressed stably in HEK293 cells and bound [3H]iloprost with high affinity and stimulated cAMP production when exposed to agonist. A cDNA encoding the Gi1alpha sequence but with the carboxyl-terminal six amino acids of Gsalpha was also constructed. Co-expression of this chimeric G protein Gi1/Gs6alpha, but not Gsalpha or Gi1alpha, resulted in robust stimulation by iloprost. This significantly high levels of agonist-stimulated [35S]GTPalphaS binding and high affinity GTPase were not abolished by treatment with both cholera and pertussis toxins. This correlated with the loss of both cholera (arginine 201 of Gsalpha(L)) and pertussis (cysteine 351 of Gi1alpha) toxin-susceptible sites in the Gi1 /Gs6alpha protein. This clearly demonstrated the utility of chimeric G proteins to combine the high GTP exchange and hydrolysis capacity of Gi1a with the ability to couple to a Gsalpha-coupled GPCR. The stoichiometry of GPCR to Galpha can have a direct impact on the signalling cascades of GPCRs (Kenakin 1995a; 1997). In addition, there is evidence that GPCR and Galpha may not be localised in the same microdomain of the plasma membrane (Neubig 1994). Through the use of a fusion protein between the alpha2-adrenergic receptor and Gsalpha, Bertin et al. (1994) demonstrated productive interactions between the fused partners. A fusion protein of the FLAG(TM)-tagged human IP prostanoid receptor with Gsalpha(L)(HA) was therefore generated and stably expressed in HEK293 cells. These cells bound [3H]iloprost with high affinity and also stimulated adenylate cyclase upon addition of agonist. When compared to the freely interacting IP prostanoid receptor, the fusion protein FhlPR-Gsalpha exhibited enhanced agonist-stimulated activities in both the [35S]GTPalphaS binding and high affinity GTPase assays. Furthermore, cholera toxin treatment diminished its capacity to hydrolyse GTP but not the incorporation of [35S]GTPalphaS. The fidelity of signalling in GPCR-Galpha fusion proteins was established by studying the Ga activity of a series of FhlPR-Galpha fusions. When stably expressed in HEK293 cells and stimulated by iloprost, the FhlPR-Gi1alpha protein failed to elevate the low levels of high affinity GTPase and [35S]GTPalphaS binding activity. These low levels of activity were shown to be derived from activation of endogenous Gsalpha but not receptor-linked Gi1alpha. Substituting the carboxyl-terminal six amino acids of FhlPR-Gi1a with Gsalpha resulted in the production of the FhlPR- Gi1/Gs6alpha fusion protein. This protein produced substantial elevation of both high affinity GTPase and [35S]GTPalphaS binding activity upon stimulation by iloprost. In addition, these activities were resistant to both cholera and pertussis toxin treatments, as was observed for the freely interacting Gi1/Gs6alpha protein. This clearly demonstrated that fusing the GPCR and Galpha did not alter their individual characteristics. The assay of agonist activity at the G protein level for a Gsalpha-coupled GPCR is now possible by using the chimeric Gi1/Gs6alpha protein or GPCR-Galpha fusions. The GPCR-Galpha fusion approach is superior to the chimeric protein as the stoichiometry of GPCR to Galpha is fixed at 1:1 and the interacting partners are co-targeted to the same microdomain of the plasma membrane.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Endocrinology.
• Subjects: Q Science > QH Natural history > QH345 Biochemistry
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: Milligan, Professor Graeme
• Date of Award: 1999
• Depositing User: Enlighten Team
• Unique ID: glathesis:1999-71304
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 10 May 2019 10:49
• Last Modified: 18 Oct 2022 14:37
• Thesis DOI: 10.5525/gla.thesis.71304
• URI: https://theses.gla.ac.uk/id/eprint/71304