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

Abstract

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 (approximately 78% of cell surface receptors were lost after 2 min exposure to 1 microM 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 dependent 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 of 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.

Arrestin 3 Mediates Endocytosis of CCR7 following Ligation of CCL19 but Not CCL21

“…The CCR7 GPCR, like the nociceptin GPCR demonstrates ligand-dependent differences in receptor internalization and desensitization (57)(58)(59)(60). Both GPCRs differentially regulate receptor fate following ligand binding (45).…”

“…2), the nociceptin receptor required different ligand-dependent incubation times to mediate receptor internalization. This may reflect the differential functions of the described ligands, since agonists and antagonists were internalized at different rates (57)(58)(59)(60).…”

Nociceptin/Orphanin FQ Receptor Structure, Signaling, Ligands, Functions, and Interactions with Opioid Systems

“…Numerous groups have investigated the many stages of NOP receptor trafficking (for a thorough review, see Donica et al, 2013). Early work in the NOP receptor field had difficulty in finding agonistinduced internalization (Dautzenberg et al, 2001); however, later reports showed that NOP receptors indeed internalized in response to N/OFQ treatment (Spampinato et al, 2001(Spampinato et al, , 2002. Similar to the kappa opioid receptor disparities in internalization conditions , it is likely that differences reported in the internalization of NOP receptors are due to expression variability and model system used.…”

“…NOP receptor recycling has not been extensively examined, although some groups have shown that, in transfected cells, once internalized receptors remain internalized after washout for up to 90 minutes to 2 hours in some reports (Spampinato et al, 2001;Spampinato et al, 2002). Long-term treatment of GPCRs with agonists generally causes them to either become recycled after some critical time window or to become transported to proteasomes and lysosomes.…”

Cellular Mechanisms of Nociceptin/Orphanin FQ (N/OFQ) Peptide (NOP) Receptor Regulation and Heterologous Regulation by N/OFQ

“…Clathrin-coated vesicles are trafficked to early endosomal compartments, where the receptor is completely internalized. Agonist-induced receptor internalization is (Cannarsa et al, 2012;Connor et al, 1996b;Donica et al, 2011;Mandyam et al, 2003;Thakker et al, 2007;Thakker and Standifer, 2002a,b) (Barnes et al, 2007;Bevan et al, 1998;Fukuda et al, 1998;Fukuda et al, 1997;Hashimoto et al, 2002;Hawes et al, 1998;Lou et al, 1997;McDonald et al, 2003;Okawa et al, 1999;Pan et al, 2002;Peluso et al, 2001;Spampinato and Baiula, 2006;Spampinato et al, 2002;Waits et al, 2004) COS-7 African green monkey kidney fibroblast-like cells transformed with SV40 ++++ G-protein coupling and NOP receptor expression (Chan and Wong, 2000;Chan et al, 1998;Ho et al, 2002 (Altier et al, 2006;Beedle et al, 2004;Evans et al, 2010) the intermediate phase of GPCR desensitization, where the receptor is now inaccessible for activation by the extracellular agonist, thereby inhibiting further agonist-mediated cellular responses. However, it is also an essential process for resensitization of receptors (Gainetdinov et al, 2004), as explained below.…”