Role of endocytosis in the activation of the extracellular signal-regulated kinase cascade by sequestering and nonsequestering G protein-coupled receptors

Acting through a number of distinct pathways, many G protein-coupled recept ors (GPCRs) activate the extracellular signal-regulated kinase (ERK)/mitoge n-activated protein kinase (MAPK) cascade. Recently, it has been shown that in some cases, clathrin-mediated endocytosis is required for GPCR activati on of the ERK/MAPK cascade, whereas in others it is not. Accordingly, we co mpared ERK activation mediated by a GPCR that does not undergo agonist-stim ulated endocytosis, the alpha(2A) adrenergic receptor (alpha(2A) AR), with ERK activation mediated by the beta(2) adrenergic receptor (beta(2) AR), wh ich is endocytosed. Surprisingly, we found that in COS-7 cells, ERK activat ion by the alpha(2A) AR, like that mediated by both the beta(2) AR and the epidermal growth factor receptor (EGFR), is sensitive to mechanistically di stinct inhibitors of clathrin-mediated endocytosis, including monodansylcad averine. a mutant dynamin I, and a mutant beta-arrestin 1. Moreover, we det ermined that, as has been shown for many other GPCRs, both alpha(2A) and be ta(2) AR-mediated ERK activation involves transactivation of the EGFR, Usin g confocal immunofluorescence microscopy, we found that stimulation of the beta(2) AR, the alpha(2A) AR, or the EGFR each results in internalization o f a green fluorescent protein-tagged EGFR. Although beta(2) AR stimulation leads to redistribution of both the beta(2) AR and EGFR, activation of the alpha(2A) AR leads to redistribution of the EGFR but the alpha(2A) AR remai ns on the plasma membrane. These findings separate GPCR endocytosis from th e requirement for clathrin-mediated endocytosis in EGFR transactivation-med iated ERK activation and suggest that it is the receptor tyrosine kinase or another downstream effector that must engage the endocytic machinery.