Searchable abstracts of presentations at key conferences in endocrinology
Endocrine Abstracts (2007) 14 OC9.4

ECE2007 Oral Communications Signal transduction (7 abstracts)

Signalling and internalisation properties of corticotrophin-releasing hormone (CRH) receptor type 2

Danijela Markovic , Hendrik Lehnert & Dimitris Grammatopoulos


Warwick Medical School, University of Warwick, Coventry, United Kingdom.


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.

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