The stoichiometry of interaction between receptors, G-proteins and effector species

Kim, Gun-Do (1996) The stoichiometry of interaction between receptors, G-proteins and effector species. PhD thesis, University of Glasgow.

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G-proteins have been demonstrated to be necessary to allow transduction of information from hormone-activated cell surface receptors to a variety of effector systems. This study has focused on their expression and regulation in response to agonists at receptors which produce stimulation of adenylyl cyclase or stimulation of phospholipase C activity. To understand receptor regulation of cyclic AMP generation, it is important to know the relative or absolute levels of expression of each component (receptor, G- protein (Gs) and adenylyl cyclase) in individual cells, their cellular disposition and how alteration in levels of each component might alter the effectiveness of cellular signalling. In chapter 3, neuroblastoma x glioma hybrid, NG108-15, cells are shown to express Gsalpha in a substantial molar excess over its effector adenylyl cyclase. Sustained exposure of a cell to an agonist for a G-protein coupled receptor can lead to the down-regulation of levels of the receptor and the G-protein and this process can play a substantial part in the regulation of cellular sensitivity to the presence of agonist. Regulation of agonist access to the receptor population by pretreatment of NG108-15 cells transfected to express the human beta2-adrenoceptor with varying concentrations of an irreversible beta-adrenoceptor antagonist (BAAM) demonstrated that the extent of agonist-mediated Gsalpha down-regulation was dependent upon the availability of receptor to agonist and that the levels of receptor expression defines the intrinsic activity and potency of agonists. As noted above, however, the availability of sufficient Gsalpha to interact with the total cellular population of adenylyl cyclase suggests that the adenylyl cyclase is likely to be the limiting component for information transfer. As a means to examine quantitative aspects of the expression of adenylyl cyclase, human beta2-adrenoceptor expressing NG108-15 cells were further transfected to overexpress adenylyl cyclase type 2. The results demonstrated that receptor-mediated maximal output of the stimulatory arm of the adenylyl cyclase cascade can be increased by increasing total levels of adenylyl cyclase but this did not result in any significant alteration in the concentration-effect curves for stimulation of adenylyl cyclase activity produced by either the transfected human beta2-adrenoceptor or any of the endogenously expressed (IP prostanoid, A2 adenosine or secretin) Gsalpha-linked receptors. In chapter 4, human embryonic kidney (HEK-293) cells which express the long splice variant form of the rat thyrotropin-releasing hormone (TRH) receptor (clone E2) were examined to address whether both Gqa and/or G11alpha and Gsalpha are involved in TRH-stimulation of phospholipase C beta1 and/or adenylyl cyclase or whether different splice variants of the receptor selectively interact with different G- proteins to regulate different signal transduction cascades. Activation of this receptor with TRH caused only a large stimulation of production of inositol phosphates in a manner which was mediated by the pertussis-toxin-insensitive phospholipase C-linked Gq and G11 proteins despite the fact that TRH has been reported to stimulate adenylyl cyclase activity via activation of Gsalpha. In addition, with development of a 6 M urea containing SDS-PAGE (10% (w/v) acrylamide, 0.0625% (w/v) bisacrylamide) system which allowed resolution of Gqa and G11alpha I assessed whether a single receptor type could utilise species variants of the same G-protein equivalently. Treatment of clone E2M11 (which expresses the long isoform of the rat TRH receptor and both the endogenous human and exogenous introduced murine G11alpha) with TRH resulted in a qualitatively similar pattern of cellular redistribution and down-regulation of G11alpha isoforms. Quantitative analysis indicated, however, that the murine isoform of G11alpha was less effectively regulated by the long isoform of the rat TRH receptor than the endogenous human form of G11alpha.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Molecular biology.
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Milligan, Professor Graeme
Date of Award: 1996
Depositing User: Enlighten Team
Unique ID: glathesis:1996-71739
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 17 May 2019 09:31
Last Modified: 21 Jun 2022 12:58
Thesis DOI: 10.5525/gla.thesis.71739

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