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Vol. 61, Issue 1, 73-84, January 2002
)(trans)-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidiny)-cyclohexyl]benzeneacetamide-Induced
Desensitization and Phosphorylation between Human and Rat 
-Opioid
Receptors Expressed in Chinese Hamster Ovary Cells
Department of Pharmacology and Center for Substance Abuse Research,
Temple University School of Medicine, Philadelphia, Pennsylvania
The agonist
(
)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)-cyclohexyl]benzeneacetamide
[()U50,488H] caused desensitization of the human 
-opioid
receptor (hkor) and Flag-tagged hkor (Flag-hkor) but not the rat
-opioid receptor (rkor) and Flag-tagged rkor (Flag-rkor) stably
expressed in CHO cells as assessed by guanosine 5'-O-(3-[35S]thiotriphosphate) binding. In
addition, ()U50,488H stimulation enhanced phosphorylation of the
Flag-hkor, but not Flag-rkor. ()U50,488H-induced phosphorylation of
the Flag-hkor was reduced by expression of the dominant negative mutant
GRK2-K220R, demonstrating the involvement of G protein-coupled receptor
kinases (GRKs). However, expression of GRK2 and arrestin-2 or GRK3 and
arrestin-3 did not result in desensitization or phosphorylation of the
Flag-rkor after ()U50,488H pretreatment. To understand the molecular
basis of the species differences, we constructed two Flag-tagged
chimeric receptors, Flag-h/rkor and Flag-r/hkor, in which the
C-terminal domains of Flag-hkor and Flag-rkor were switched. When
stably expressed in CHO cells, Flag-r/hkor, but not Flag-h/rkor, was desensitized and phosphorylated after exposure to ()U50,488H, indicating that the C-terminal domain plays a critical role in the
differences. We then generated a Flag-hkor mutant, in which S358 was
mutated to N (Flag-hkorS358N) and a Flag-rkor mutant, in which N358 was
substituted with S (Flag-rkorN358S). Although Flag-hkorS358N was not
phosphorylated or desensitized by ()U50,488H stimulation,
Flag-rkorN358S underwent ()U50,488H-induced desensitization with
slightly increased phosphorylation. These results indicate that there
are differences in ()U50,488H-induced desensitization and
phosphorylation between the hkor and the rkor. In addition, the
C-terminal domain plays a crucial role in these differences and the 358 locus contributes to the differences. Our findings suggest caution in
extrapolating studies on -opioid receptor regulation from rats to humans.
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