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. 2004 Jul 22;23(16):3282–3289. doi: 10.1038/sj.emboj.7600334

Figure 2.

Figure 2

Relationship between Ser375 phosphorylation and internalization of MOR. (A) HEK 293 cells expressing the wild-type MOR or the S375A mutant were transiently transfected with GRK2 (+GRK2) or empty vector (−GRK2). After 2 days, cells were either not treated (−) or treated with 10 μM DAMGO (D) or 10 μM morphine (M) for 30 min, lysed and immunoblotted with an antibody specific for the Ser375-phosphorylated MOR (pMOR, upper panel). Expression of HA-tagged MOR and HA-tagged S375A receptors was confirmed by immunoblotting with an anti-HA antibody (MOR, lower panel). Note, (I) Ser375 phosphorylation was not detectable in untreated cells, (II) the morphine-induced Ser375 phosphorylation was ∼35% of DAMGO-induced phosphorylation, (III) overexpression of GRK2 strongly enhanced the morphine-induced Ser375 phosphorylation and (IV) phospho-Ser375 immunoreactivity was not detectable in cells expressing the S375A mutant. The positions of molecular mass markers are indicated on the left (in kDa). Three additional experiments gave similar results. (B) HEK 293 cells expressing the wild-type MOR or the S375A mutant were transiently transfected with GRK2 (+GRK2) or empty vector (−GRK2). After 2 days, cells were either not treated (−) or treated with 10 μM DAMGO (D) or 10 μM morphine (M) for 30 min. Cell surface receptors were labeled with rabbit anti-HA antibodies, followed by a peroxidase-conjugated secondary antibody. Receptor sequestration, quantified as the percent loss of cell-surface receptors in agonist-treated cells, was measured by ELISA. Data are presented as mean±s.e. of five independent experiments performed in quadruplicate. The results were analyzed by two-tailed Student's paired t-test (*P<0.05 versus DAMGO (−GRK2); #P<0.05 versus morphine (−GRK2)). Note, (I) in contrast to DAMGO, morphine failed to cause substantial receptor endocytosis, (II) overexpression of GRK2 strongly enhanced the level of morphine-induced internalization and (III) mutation of Ser375 to Ala (S375A) strongly reduced agonist-driven MOR endocytosis.