The use of receptor chimeras to study the function of the carboxyl terminal domain in prostacyclin receptor signalling

Murdoch, Hannah (2002) The use of receptor chimeras to study the function of the carboxyl terminal domain in prostacyclin receptor signalling. PhD thesis, University of Glasgow.

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To investigate the role of the carboxyl terminal in the regulation of the prostacyclin (IP) receptor, chimeric receptors expressing the carboxyl termini of either the thyrotropin-releasing hormone-1 (TRH) receptor or the beta2-adrenoreceptor (beta2-AR) were generated. Furthermore, C-terminally green fluorescent protein (GFP)-tagged forms of the receptors were built and stably expressed in HEK293 cells, thus enabling direct visualisation of receptor localisation and trafficking in intact cells. Pharmacological analysis of the receptor-GFP fusion proteins demonstrated that each bound [3H] iloprost with similar affinity and coupled to increased cAMP production. Sequestration studies revealed that iloprost-induced internalisation of the prostacyclin receptor was augmented by the addition of the TRH carboxyl tail. Conversely, the P2-tailed chimeras exhibited internalisation properties comparable to those of the full- length prostacyclin receptors. The receptors' internalisation kinetics were unaffected by the addition of the GFP moiety. Agonist-mediated sequestration of the constructs was abolished by treatments inducing clathrin depletion. In addition, sequestered receptors were found to colocalise in endosomes containing transferrin, as determined by confocal microscopy. Visual assessment of the dynamic interaction between P-arrestins and the receptor proteins demonstrated that sequestration of the full-length receptor proceeded primarily via an arrestin-independent mechanism. Switching of the receptor's carboxyl domain for the equivalent beta2-AR sequence did not confer beta-arrestin sensitivity to the receptor. In contrast, the TRH-tailed receptors exhibited an increased binding affinity for beta-arrestins, internalising in complexes with beta-arrestin 2. In a cellular milieu deficient of P- airestins and GRKs, the prostacyclin receptor and its chimeric forms retained the ability to undergo agonist-mediated sequestration. Analysis of receptor regulation revealed that the GFP-tagged IP receptor elicited rapid signal attenuation in response to iloprost challenge. A less striking desensitisation response was evident with receptors expressing the different carboxyl tails. During desensitisation of the receptor-GFP proteins, iloprost challenge induced rapid receptor phosphorylation which was, in part, mediated by the second messenger kinases PICA and PKC. PKA was demonstrated to be a major desensitising kinase of the receptors while PKC phosphorylation was identified as a possible determinant for receptor sequestration. Upon agonist withdrawal, the internalised GFP-tagged full-length receptor recycled rapidly back to the plasmalemmal surface, which was followed by the restoration in receptor responsiveness. By comparison, the agonist-activated chimeric receptors failed to recycle, and therefore resensitise, after agonist removal. Subsequently, the intracellularly retained chimeric receptors were sorted predominantly via a degradative pathway. Taken together, these data highlight the importance of the carboxyl terminal domain in prostacyclin receptor function.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Biochemistry
Date of Award: 2002
Depositing User: Enlighten Team
Unique ID: glathesis:2002-72575
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 11 Jun 2019 11:06
Last Modified: 11 Jun 2019 11:06

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