Proceedings of The Physiological Society

Life Sciences 2007 (2007) Proc Life Sciences, C81

Research Symposium

Investigating activation of human G protein-coupled receptor kinase 2 by c-SRC, using site-directed mutagenesis and real-time FRET measurements

S. Al-Sabah1, E. A. Ellis1, C. M. Moulton1, C. Krasel1

1. School of Pharmacy, University of Reading, Reading, Berkshire, United Kingdom.


Arrestin binding to G protein-coupled receptors requires phosphorylation of the agonist-stimulated receptor by GRK2. Once arrestin is bound to the receptor signaling via G proteins is arrested and the receptor is said to be desensitized. The receptor can then be targeted to clathrin-coated pits to be internalized and alternative signaling pathways may be activated. Using a previously established FRET-based assay to monitor arrestin3 binding to the β2-adrenergic receptor (β2AR) in single living cells, we show that GRK2 phosphorylation by c-Src accelerates arrestin3 binding to the β2AR, presumably by increasing GRK2 activity. HEK-293 cells were transfected with β2AR tagged with YFP, arrestin3 tagged with CFP and GRK2 with or without c-Src. A slow and fast phase of arrestin3 binding to β2AR was observed in agreement with previous work (J. Biol. Chem. 280:9528 (2005)). Cells that were not transfected with c-Src displayed a slow phase with a half-life of about 24 seconds, whereas for cells co-transfected with c-Src the half-life was reduced by approximately 50% to about 13 seconds. PP2, a src-family kinase inhibitor, was used to show that the observed acceleration of arrestin3 binding to the receptor was caused by the catalytic activity of c-Src. Treatment with 10µM PP2 for one hour had no effect on arestin3 binding to the receptor in cells not co-transfected with c-Src, but increased the half-life of arrestin3 binding to the receptor in cells co-transfected with c-Src to abot 25 seconds, identical to that of cells without c-Src. It has previously been reported that c-Src-mediated tyrosine phosphorylation of GRK2 occurs at residues 13, 86 and 92 (Cell Signal. 18(11):2004 (2006)). In order to show that phosphorylation of GRK2 by c-Src was required for the acceleration in arrestin3 binding to β2AR we tested a phosphorylation impaired GRK2 mutant (Y13,86,92F). There was no significant difference between the half-life of the slow phase of arrestin3 binding to β2AR in cells transfected with Y13,86,92F GRK2 with or without c-Src (a half-life of 54 and 59 seconds respectively), indicating that c-Src was having no effect on the mutated GRK2. However, cells expressing the mutated GRK2 without c-Src showed a half-life approximately 3 fold slower than that of wild-type GRK2. The half-life for the slow phase of arrestin3 binding to the β2AR in cells transfected with mutated GRK2 with c-Src was in the region of 5-fold slower than that of cells transfected with wild-type GRK2 with c-Src. We conclude that the Y13,86,93F GRK2 mutant is in some way catalytically impaired.

Where applicable, experiments conform with Society ethical requirements