Characterization of a naturally occurring variant of the mu-Opioid Receptor (MOPr): L83I

Alexandra E Cooke, Graeme Henderson, Eamonn Kelly, Stuart J Mundell. University of Bristol, School of Physiology and Pharmacology,Medical Sciences Building, Bristol, BS8 1TD, UK

DAMGO-induced desensitisation of the mu-opioid receptor (MOPr) is mediated primarily by G-protein-coupled receptor kinase 2 (GRK2) and results in rapid MOPr internalization, whereas morphine-induced desensitization is largely PKC-dependent and results in little MOPr internalization (Bailey et al., 2003; Johnson et al., 2006). A recently identified naturally occurring variant (L83I) has been observed to undergo significant internalization in response to morphine (Ravindranathan et al., 2009). The current study examined the trafficking and signalling of the wild-type (WT) MOPr in comparison with that of the L83I variant.

All experiments were carried out on HEK 293 cells expressing either HA-MOPr or the HA-MOPr-L83I variant. Internalization of HA-tagged receptors was assessed by ELISA and immunofluorescence microscopy as previously described (Mundell et al., 2006). Phosphorylation of serine 375 was assessed by western blot following immunoprecipitation of HA-tagged receptors from whole cell lysates. cAMP measurements were performed using a cAMP enzyme immunoassay (EIA).

DAMGO stimulation of the receptors resulted in significant internalization of both the WT-MOPr and the L83I variant (33.9 ± 3.0% and 36.6 ± 4.3% respectively; n = 6). In contrast, morphine induced significant internalization of the L83I variant but had little effect on the WT-MOPr (27.2 ± 4.1% and 1.6 ± 7.8% respectively; n = 6). Marked internalization of the L83I variant in response to morphine was also seen by immunofluorescence confocal microscopy. Inhibition of dynamin by pre-treatment of cells with dynasore (40μM; 15 min) effectively inhibited the DAMGO-induced internalization of both the WT-MOPr and the L83I variant. Furthermore, dynasore also inhibited the morphine-induced internalization of the L83I variant (58.0 ± 6.6 % v. -8.6 ± 7.7%; P<0.001; n = 3).

Antagonism of GRK2 with a dominant negative mutant (DNM) GRK2 (K220R) attenuated the internalization of the L83I variant in response to both DAMGO (50.2 ± 5.7% and 7.9 ± 4.6% in the absence and presence of GRK2-DNM ; P<0.01; n = 4) and morphine (41.4 ± 5.3% and 3.6 ± 9.6% in the absence and presence of GRK2-DNM; P<0.01) in addition to inhibiting the internalization of the WT-MOPr in response to DAMGO (52.3 ± 2.4% and 3.6 ± 4.7% in the absence and presence of GRK2-DNM; P<0.001; n = 4). Of the numerous potential phosphorylation sites in the MOPr, serine 375 in the C-terminal tail has been the most extensively studied and identified as a probable GRK phosphorylation site. Following immunoprecipitation of the HA-tagged receptors, DAMGO induced substantial phosphorylation of this residue in both the WT-MOPr and the L83I variant, whereas, morphine induced far less phosphorylation of this residue but which was the same in the WT-MOPr and L83I (n.s.; n = 5).

Investigations into the G-protein signalling of the L83I variant by measurement of cAMP inhibition revealed no significant change in the EC50 of either DAMGO or morphine when compared with values obtained for the WT-MOPr (n.s; n = 4).

In conclusion these results show that unlike the WT-MOPr, the L83I variant rapidly internalizes in response to morphine in a GRK- and dynamin-dependent manner. The enhanced internalization of L83I in response to morphine is not due to increased phosphorylation of Serine 375, or an increased ability to signal via G-proteins.

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