Endocrine Abstracts

Corticotropin releasing hormone (CRH), and the urocortins (UCN) play key roles in mammalian pathophysiology. Their actions are mediated through two types of G-protein coupled CRH receptors, (R1 and R2) which exhibit distinct pharmacological characteristics and determine specificity of CRH and UCN actions. Several CRH-R1 mRNA splice variants have been identified in native tissues, encoding receptor isoforms with various aminoacid inserts or deletions, termed R1β, R1c, R1d etc. To obtain more information about the impact of these structural alterations on CRH-R1 functionality we expressed different CRH-R1 subtypes and CRH-R2β in HEK293 cells and we monitored their internalisation characteristics following agonist stimulation by using indirect confocal microscopy. Results showed that both CRH-R1α (the wild type receptor), and the signaling-impaired subtype CRH-R1β (which contains a 29 aa insert in the 1st IC loop), were expressed in plasma membrane and were internalised within 30–45 min of CRH activation in a β-arrestin dependent mechanism. Interestingly, the CRH-R1d that contains a 14 aa deletion in the 7th TMD which interferes with G-protein coupling, was distributed in the plasma membrane as well as the cytoplasm, suggesting that intact TMDs are essential for adequate receptor stability and localization to the plasma membrane. Nevertheless, this CRH-R1 subtype was also able to internalise normally in response to CRH binding. Studies on the CRH-R2β revealed important differences in receptor internalisation characteristics. In response to UCNII (a CRH-R2 specific agonist), the CRH-R2β internalised within 15 min of UCNII activation whereas the weaker agonist CRH, induced CRH-R2β internalization only after 30–45 min of treatment. These results suggest that intact CRH-R1 signaling potency is not essential for activating the intracellular machinery involved in receptor internalization endocytosis. In addition, CRH-R2 internalisation pathways might be directly related to individual agonists signalling potency and their ability to induce distinct receptor active conformations.