Phosphorylation state of mu-opioid receptor determines the alternative recycling of receptor via Rab4 or Rab11 pathway.
Agonist-induced phosphorylation, internalization, and intracellular trafficking of G protein-coupled receptors are critical in regulating both cellular responsiveness and signal transduction. The current study investigated the role of receptor phosphorylation state in regulation of agonist-induced internalization and intracellular trafficking of mu-opioid receptor (MOR). Our results showed that after agonist stimulation, the recycle of a mutant MOR that lacks the C-terminal residues after Asn(362) (MOR362T) was greatly decreased, whereas a C-terminal phosphorylation sites-mutated MOR (MOR3A), which is deficient in agonist-induced phosphorylation recycled back to the membrane at a level comparable to that of the wild-type receptor, however, interestingly at a slower rate. Inhibition of functions of either Rab4 or Rab11 by dominant-negative mutants and small interfering RNA both significantly impaired the recycling of the wild-type MOR, whereas the recycling of the phosphorylation-deficient mutant was only inhibited by the dominant-negative mutant and small interfering RNA of Rab11, suggesting that the recycling of nonphosphorylated MOR is exclusively via Rab11-mediated pathway. Furthermore, phosphorylated MOR was observed accumulated in Rab5- and Rab4-, but not Rab11-positive vesicles. Our data indicate that both phosphorylated and nonphosphorylated MOR internalize via Rab5-dependent pathway after agonist stimulation, and the phosphorylated and nonphosphorylated MORs recycle through distinct vesicular trafficking pathways mediated by Rab4 and Rab11, respectively, which may ultimately lead to differential cellular responsiveness or downstream signaling.
Pharmacology Research Center, Fudan University, 138 Yi Xue Yuan Road, Shanghai 200032, People's Republic of China.
Feifei
F
Wang
Xiaoqing
X
Chen
Xiaoqing
X
Zhang
Lan
L
Ma
D000818:Animals; D001710:Biotin; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D011957:Receptors, Opioid / Q000819:agonists / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D020695:rab4 GTP-Binding Proteins / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins
D001710:Biotin; D049452:Green Fluorescent Proteins; D050505:Mutant Proteins; D034741:RNA, Small Interfering; D011957:Receptors, Opioid; D011993:Recombinant Fusion Proteins; D020691:rab GTP-Binding Proteins; D020695:rab4 GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins