SUN-479 Role of β-arrestin2 in ERK 1/2 Activation Mediated by Corticotropin-Releasing Factor Receptor Type 1

Abstract

Abstract The Corticotropin-Releasing Factor (CRF) is a neuropeptide released by the hypothalamus in response to stress. CRF activates the CRF1 and CRF2 receptors, which are members of the G protein-coupled receptor (GPCR) superfamily. Although it has been shown that the CRF1 receptor activates ERK 1/2 cascade in different cell models [1] however, to date is not well understood the roles of upstream pathways associated with this effects. β-arrestin2 is an adapter protein that participates in the regulation of GPCRs and has important functions in cell signaling by mediating activation of multiple transductional pathways, including Src and MAPK [2]. In the present work, we investigated the role of β-arrestin2 in the activation of MAPK by induction of 100 nM CRF. We used mouse embryonic fibroblast cells that lack β-arrestin2 (MEF KO-β-arrestin2), transfected with the CRF1 receptor that lack β-arrestin2 (MEF KO-β-arrestin2) as model. Our data revealed that β-arrestin2 is involved in the activation of ERK 1/2 since we detected lower (P<0.05) phosphorylation of ERK 1/2 in the KO-β-arrestin2 cells after 5 min CRF of stimulationCRF. Using MEF cells from double-knockout mice, lacking β-arrestin1 and β-arrestin2 (MEF 2KO-β-arrestin1/2), we found that β-arrestin1 is not involved in ERK 1/2 activation induced by CRF because there was no change on phosphorylation of ERK 1/2 between MEF KO-β-arrestin2 and 2KO-β-arrestin1/2 cells. The pretreatment with the EGFR inhibitor AG1478 promoted a decreased (P<0.05) ERK 1/2 phosphorylation in control cells. Interestingly, no EGFR activation was detected in the KO-β-arrestin2 cells. These results indicate that β-arrestin2 is necessary for the CRF1 receptor-mediated EGFR transactivation mechanism that induces ERK 1/2 phosphorylation. We also found that the transactivation of EGFR by CRF stimulation is independent of metalloproteinase activity through the pretreatment with the metalloproteinase inhibitor GM6001. To determine the role of Src in the CRF-mediated ERK activation, we pretreated the cells with the Src-specific inhibitor PP2. We observed that PP2 promoted a significant decrease (P<0.05) in CRF-mediated ERK activation. We also found a reduction (P<0.05) in Src phosphorylation at Y416 in MEF KO-β-arrestin2 cells stimulated with 5 min of CRF, suggesting that β-arrestin2 is involved in Src activation. Together, our data demonstrate that β-arrestin2 is an important player during the CRF1-dependent activation of the ERK 1/2 pathway. Reference: [1]Hauger et al., Ann. N. Y. Acad. Sci. (2009) 1179: 120-143.[2]Luttrell et al., Proc. Natl. Acad. Sci. (2001) 98: 2449-2454Sources of Research Support: UC MEXUS-CONACYT grant for collaborative projects to JAO-R and RLH; CONACYT grant to JAO-R; Merit Review grant; MIRECC of VISN22, and CESMH from the Department of VA; NIH grant to RLH. GKP-M was supported by CONACYT fellowship grant.