Corticotropin-releasing hormone (CRH) and its related peptides, the urocortins (UCNs) mediate their effects by binding to two types of GPCRs, CRH-R1 and CRH-R2. In most target tissues, the adenylyl cyclase/cAMP/PKA and the mitogen activated protein kinase (MAPK) are the two main pathways mediating the biological effects of CRH-Rs. For most multi-signal G-protein coupled receptors (GPCRs), there is considerable level of cross-talk between different signalling cascades, which is important for determining the overall biological response. In the present study we investigated potential signalling interactions between the cAMP and MAPK cascades following stimulation of HEK293 cells overexpressing recombinant CRH-R2β with UCN-II. Using specific kinase inhibitors we found that CRH-R2β mediated ERK1/2, but not p38MAPK activation was PI3-K dependent, and optimal ERK1/2, but not p38MAPK activation was dependent on an intact cAMP/PKA/AKAP pathway. Furthermore, inhibition of the ERK1/2 pathway by pre-treatment with the specific MEK1/2 inhibitor (UO126) attenuated UCN-II-induced ERK1/2 activation and cAMP responses. These observations may be directly related to CRH-R2β trafficking and endocytosis. Results from Indirect-confocal microscopy studies suggest that inhibition of either PKA (by myr-PKAi) or the ERK1/2 pathway accelerates CRH-R2β endocytosis, which was additionally confirmed by loss of CRH-R2 from the cell membrane. Indirect confocal analysis and immunoblotting using phospho-specific antibodies determined that UCN-II-activated ERK1/2 appeared to target β-arrestin1 (βarr1) and modulate, through phosphorylation at Ser412, its translocation to the plasma membrane, binary complex formation with CRH-R2β and receptor internalization kinetics. Depletion of endogenous βarr1 by siRNA reduced CRH-R2β internalization and enhanced UCN-II- induced ERK1/2 and p38 MAPK activation by 80% and 55% respectively. These findings reveal a complex interplay between the cAMP-ERK1/2 pathways that might allow fine-tuning of CRH-R2β functional responses.
03 - 07 May 2008
European Society of Endocrinology