Agonist-induced internalization and desensitization of the human nociceptin receptor expressed in CHO cells

In this study, we examined agonist-induced internalization, recycling and signalling (measure of cAMP levels) of the cloned human nociceptin receptor (hNOP) expressed in CHO-K1 cells. Internalization was proven by a receptor-binding assay on viable cells. The agonist nociceptin/orphanin FQ (NC) promoted rapid internalization of the hNOP receptor (approximate to78% of cell surface receptors were lost after 2 min exposure to 1 muM NC) in a clathrin- and ATP-dependent manner. Internalization was more rapid and marked in CHO-K1 cells than, as we previously reported. in SK-N-BE cells. This difference may be related to higher levels of beta-arrestin isoforms detected in CHO-K1 than in SK-N-BE cells. hNOP receptor internalization was partially reversible and recycling occurred in the presence of the agonist; receptor recycling was de-pendent on okadaic acid-sensitive phosphatases and was blocked by monensin. Confocal microscopy analysis confirmed the internalization and the recycling back to the plasma membrane of an epitope-tagged hNOP receptor expressed in CHO-K1 cells. These receptors underwent rapid desensitization upon agonist challenge: NC efficacy in inhibiting forskolin-stimulated cAMP production was significantly reduced 10 min after exposure and correlated with the rate of receptor internalization, Moreover. we observed that blockade hNOP receptor recycling by monensin Would cause a more prolonged and relevant desensitization of this receptor. Thus, the dynamic cycle between hNOP receptor activation, internalization and recycling determines the activity of this receptor on the cell surface.