Endocrine Abstracts

The family of urocortins (UCNs) exert important pathophysiological actions in the control of peripheral homeostatic mechanisms, through activation of the type 2-corticotropin releasing hormone receptor (CRH-R2). This G-protein coupled receptor preferentially binds urocortins (UCN, UCNII and UCNIII) than CRH. In most tissues, CRH-R2 activation leads to increased cAMP production. In this study we used HEK293 cells stably overexpressing recombinant CRH-R2β receptors to investigate intracellular events controlling receptor functional activity and their potential link to activation of distinct signalling cascades. Our results showed that agonist-induced CRH-R2β activation is followed by receptor endocytosis. Interestingly, we identified important agonist-specific temporal differences in receptor internalization kinetics; UCNII (a CRH-R2 specific agonist) induced CRH-R2β internalization within 15 min whereas the weaker agonist, CRH, induced CRH-R2β internalization only after 30–45 min of treatment. The role of intracellular molecules involved in GPCR internalization was also investigated. Confocal microscopy studies revealed that β-arrestin and clathrin were recruited to the plasma membrane as early as 2 min following UCNII treatment, and 5 min following CRH treatment. Furthermore, clathrin, but not β-arrestin, co-localize with the internalized receptor in the cytoplasm. We also investigated agonist induced ERK1/2 activation; both UCNII and CRH induced a transient ERK1/2 activation that returned to basal within 30 min. Confocal microscopy studies showed that activated ERK1/2 was uniformly distributed in the cytoplasm and nucleus. Receptor internalization inhibitors (conconavalin A and MDC) as well as expression of a dominant negative β-arrestin (319–418) markedly reduced UCNII and CRH induced ERK1/2 phosphorylation. In conclusion, we provide novel evidence of agonist-specific differences in the internalization characteristics of CRH-R2β which involve recruitment to clathrin coated-pits and β-arrestin to the plasma membrane. Receptor transport to the cytoplasm involves association with clathrin but not β-arrestin. This mechanism appears to be crucial for activation of distinct signaling cascades such as ERK1/2.