G protein-coupled receptor kinase 2-mediated phosphorylation of ezrin is required for G protein-coupled receptor-dependent reorganisation of the actin cytoskeleton

• Main Author: Cant, Sarah Helen
• Published: University College London (University of London) 2005
• Subjects: 615.7
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.424329

G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitises activated G protein-coupled receptors (GPCRs). In this thesis, I identify ezrin, a member of the ezrin-radixin-moesin (ERM) family of actin/plasma membrane cross- linkers, as a novel non-GPCR substrate of GRK2. GRK2 phosphorylates ezrin within a 53 amino acid region that encompasses the phosphorylation site required for ezrin activation, threonine-567. Two lines of evidence indicate that GRK2 acts as an effector of activated GPCRs through phosphorylation of ERM proteins. Firstly, in Hep2 cells muscarinic Ml receptor (MlMR) activation promotes cytoskeletal reorganisation and membrane ruffling. This ruffling response is ERM-dependent and is accompanied by ERM protein phosphorylation. Inhibition of GRK2, but not Rho kinase, protein kinase C or cAMP-dependent protein kinase, prevents ERM protein phosphorylation and membrane ruffling. Secondly, agonist-induced internalisation of the (-adrenergic receptor (P2AR) and Ml MR is accompanied by ERM protein phosphorylation and localisation of phosphorylated ERM proteins to receptor-containing endosomal vesicles. The Na+/H+ exchanger regulatory factor, however, is not required for co-localisation of phosphorylated ERM proteins to the p2AR. Inhibition of ezrin function impedes P2AR internalisation, further linking GPCR activation, GRK activity and ezrin function. However, ERM protein phosphorylation does not occur following M2 muscarinic receptor (M2MR) activation and ERM proteins do not colocalise with internalised M2MR, suggesting that ERM proteins may be required for the internalisation of a subset of GPCRs. Overall, my results indicate that GRK2 serves not only to attenuate but also to transduce some GPCR-mediated signals.