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Regulation of signal transduction by RGS4

Brownlie, Zoe M. (2008) Regulation of signal transduction by RGS4. PhD thesis, University of Glasgow.

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Abstract

In the present study, the function and the mechanism of action of RGS4, a member of a family of proteins called Regulators of G protein Signalling (RGS) was investigated. A C-terminal fluorescent tag on RGS4 confirmed that transiently transfected RGS4 was predominantly cytosolic and underwent translocation to the plasma membrane of HEK293T cells following co-expression of Gi1, the 2A-adrenoceptor, or agonist activated 2A-adrenoceptor. This translocation of RGS4 to the plasma membrane was most pronounced with the co-expression of the constitutively active GTPase deficient Gi1 Q204L. High-affinity GTPase experiments indicated that RGS4S30C had enhanced GAP activity towards Go1 compared to wild type RGS4. This approach also demonstrated a simultaneous significant decrease in potency of both adrenaline and UK14304 to increase 2A-arenoceptor-activated high-affinity GTPase activity of Go1 in the presence of RGS4 and a further significant decrease in potency of both ligands in the presence of RGS4S30C. This enhanced GAP activity and observed decrease in agonist potency was also transferable to RGS16, an RGS protein closely related to RGS4. The selectivity of the G subunit was also investigated. The enhanced GAP activity and simultaneous significant decrease in potency of adrenaline and UK14304 to increase 2A-arenoceptor-activated high-affinity GTPase activity of RGS4S30C and RGS16S30C was selective for Go1 over Gi1. RGS4S30K and RGS4S30F also demonstrated higher GAP activity than wild type RGS4 but no consensus side chain could be identified that conferred a specific enhancement or loss of GAP activity. The ability to inhibit intracellular calcium release by an activated 1b-adrenoceptor-G11 fusion protein was used in order to investigate the GAP activity of RGS4N88S, RGS4N128A and RGS4N88S,N128A. All three mutants had ablated GAP activity towards G11 and therefore failed to inhibit intracellular calcium release. A novel role for the RGS insensitive mutation G188S was also observed when despite similar expression, G11 G188S significantly reduced agonist-stimulated [35S]GTPS binding compared to wild type G11. RGS4 represents a novel target for pharmaceutical drug development and the study of its regulation of signal transduction is an important area of investigation. These results highlight specific areas of RGS4 research with great pharmaceutical potential.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: RGS4
Subjects: Q Science > QH Natural history > QH301 Biology
R Medicine > RM Therapeutics. Pharmacology
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Supervisor's Name: Milligan, Dr Graeme
Date of Award: 2008
Depositing User: Miss Zoe M Brownlie
Unique ID: glathesis:2008-124
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Mar 2008
Last Modified: 10 Dec 2012 13:15
URI: http://theses.gla.ac.uk/id/eprint/124

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