A role for GRK2 in the aberrant de-differentiation of vascular smooth muscle cells.

Elizabeth Foxall, James Robinson, Julie Pitcher. MRC Laboratory for Molecular Cell Biology and Department of Neuroscience, Physiology and Pharmacology, University College London, MRC Laboratory for Molecular Cell Biology and Department of Neuroscience, Physiology and Pharmacology, University College London, Gower Street, London, WC1E 6BT, UK

The G protein-coupled receptor kinases (GRKs) are best known for their role in phosphorylating and desensitising G protein-coupled receptors (GPCRs). The GRKs also regulate signalling downstream of other families of receptors and have a number of non-receptor substrates and binding partners. Using co-immunoprecipitation and direct binding studies we have identified RhoAGTP and Raf1 as novel binding partners of GRK2, and report a previously unsuspected function for this kinase. GRK2 is a RhoA effector serving as a RhoA-activated scaffold protein for the ERK MAP kinase cascade. The ability of GRK2 to bind to Raf1, MEK1 and ERK2 is dependent on RhoAGTP binding to the catalytic domain of the kinase. Consistent with previously published results (Gao, J., Li, J., and Ma, L. (2005) Acta Biochim Biophys Sin (Shanghai) 37, 525-531, Wan, K. F., Sambi, B. S., Tate, R., Waters, C., and Pyne, N. J. (2003) J Biol Chem 278, 18658-18663), we find that overexpression of GRK2 in HEK-293 cells results in a ∼2-fold increase in EGF-induced ERK activation, as detected by western blotting for phospho-ERK. This increased ERK activity is associated with EGF-dependent, RhoAGTP binding and ERK scaffolding by GRK2, as assessed by the EGF-dependent co-immunoprecipitation of these proteins. Depletion of GRK2 expression by RNAi in vascular smooth muscle cells (VSMCs) reveals that GRK2 is required for EGF-induced, Rho- and ERK-dependent thymidine incorporation; thymidine incorporation in VSMCs depleted of GRK2 was reduced by 59 ± 12% relative to cells nucleofected with a scrambled control. Thus GRK2 is required for EGF-induced proliferation of VSMCs. Increased levels of GRK2 and RhoA have previously been associated with hypertension, in which a shift in the balance of differentiated versus proliferative VSMCs causes aberrant growth of vascular epithelia and restriction of blood flow. It is thought that EGF-induced phenotypic switching of VSMCs to the de-differentiated state may be mediated by GRK2’s ERK scaffolding function. Currently, we are looking to characterize the interactions between GRK2 and its ERK-scaffolding binding partners as direct or indirect, as well as investigating the role of GRK2 ERK-scaffolding in VSMCs downstream of EGF signalling.