Role of ssarrestins in bradykinin B2 receptor-mediated signalling

G protein-coupled receptors (GPCRs) can engage multiple pathways to activate ERK1/2 via both G proteins and/or ssarrestin. Receptor recruitment of ssarrestin is also important for GPCR desensitization, internalization and resensitization. Modulation of the receptor/ssarrestin interaction through modification of either component would presumably alter the output generated by receptor activation. Here we examined how ssarrestins regulate bradykinin (BK) B2 receptor (B2R) signalling and desensitization by either truncating ssarrestin1 or ssarrestin2 or by alanine substitution of a serine/threonine cluster in the C-terminal tail of B2R (B2R-4A), conditions which all affect the avidity of the B2R/ssarrestin complex. We first demonstrate that BK-mediated ERK1/2 activation is biphasic containing an early peak (between 2-5 min) followed by sustained activation for at least 60 min. The early but not the sustained phase was predictably affected by inhibition of either G alpha q/11 or G alpha i/o, whereas loss of ssarrestin2 but not ssarrestin1 resulted in diminished prolonged ERK1/2 activation. ssarrestin2's role was further examined using a truncation mutant with augmented avidity for the agonist-occupied receptor, revealing an increase in both immediate and extended ERK1/2 signalling. We also show that ERK1/2 is recruited to the B2R/ssarrestin complex on endosomes as well as the plasma membrane. Moreover, we investigated ssarrestin's role using the B2R-4A, which is deficient in ssarrestin binding and does not internalize. We show that ERK1/2 signalling downstream of the receptor is entirely G protein-dependent and receptor-mediated intracellular calcium mobilization studies revealed a lack of desensitization. Functionally, the lack of desensitization resulted in increased cell growth and migration compared to the wild-type receptor, which was sensitive to MEK inhibition. These results highlight ssarrestin's crucial role in the maintenance of proper B2R signalling. (C) 2010 Elsevier Inc. All rights reserved.