Regulation of signal transduction by RGS4

QH301 Biology;RM Therapeutics. Pharmacology

Brownlie, Zoe M. ; Milligan, Graeme

University:University of Glasgow

Department:Institute of Molecular Cell and Systems Biology

关键词: RGS4;   

Others  :  http://theses.gla.ac.uk/124/1/2007Brownliephd.pdf

来源: University of Glasgow

【 摘 要 】

In the present study, the function and the mechanism of action of RGS4, a member of afamily of proteins called Regulators of G protein Signalling (RGS) was investigated.A C-terminal fluorescent tag on RGS4 confirmed that transiently transfected RGS4 waspredominantly cytosolic and underwent translocation to the plasma membrane ofHEK293T cells following co-expression of Gi1, the 2A-adrenoceptor, or agonist activated2A-adrenoceptor. This translocation of RGS4 to the plasma membrane was mostpronounced with the co-expression of the constitutively active GTPase deficient Gi1Q204L.High-affinity GTPase experiments indicated that RGS4S30C had enhanced GAP activitytowards Go1 compared to wild type RGS4. This approach also demonstrated asimultaneous significant decrease in potency of both adrenaline and UK14304 to increase2A-arenoceptor-activated high-affinity GTPase activity of Go1 in the presence of RGS4and 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 alsotransferable to RGS16, an RGS protein closely related to RGS4. The selectivity of the Gsubunit was also investigated. The enhanced GAP activity and simultaneous significantdecrease in potency of adrenaline and UK14304 to increase 2A-arenoceptor-activatedhigh-affinity GTPase activity of RGS4S30C and RGS16S30C was selective for Go1 overGi1. RGS4S30K and RGS4S30F also demonstrated higher GAP activity than wild typeRGS4 but no consensus side chain could be identified that conferred a specificenhancement or loss of GAP activity.The ability to inhibit intracellular calcium release by an activated 1b-adrenoceptor-G11fusion protein was used in order to investigate the GAP activity of RGS4N88S, RGS4N128Aand RGS4N88S,N128A. All three mutants had ablated GAP activity towards G11 andtherefore failed to inhibit intracellular calcium release.A novel role for the RGS insensitive mutation G188S was also observed when despitesimilar expression, G11G188S significantly reduced agonist-stimulated [35S]GTPS bindingcompared to wild type G11.RGS4 represents a novel target for pharmaceutical drug development and the study of itsregulation of signal transduction is an important area of investigation. These resultshighlight specific areas of RGS4 research with great pharmaceutical potential.