RT Journal Article
SR Electronic
T1 Agonist-dependent and -independent kappa opioid receptor phosphorylation showed distinct phosphorylation patterns and resulted in different cellular outcomes
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 137570
DO 10.1101/137570
A1 Yi-Ting Chiu
A1 Chongguang Chen
A1 Stefan Schulz
A1 Lee-Yuan Liu-Chen
YR 2017
UL http://biorxiv.org/content/early/2017/05/13/137570.abstract
AB We reported previously that the selective agonist U50,488H promoted phosphorylation of the mouse kappa opioid receptor (KOPR) at residues S356, T357, T363 and S369. Here, we found that agonist (U50,488H)-dependent KOPR phosphorylation at all the residues were mediated by Gi/oα proteins and multiple protein kinases [GRKs2, 3, 5 and 6 and protein kinase C (PKC)]. In addition, PKC activation by phorbol ester induced agonist-independent KOPR phosphorylation. Compared with U50,488H, PKC activation promoted much higher S356/T357 phosphorylation, much lower T363 phosphorylation and similar levels of S369 phosphorylation. Following U50,488H, GRKs, but not PKC, were involved in agonist-induced KOPR internalization. In contrast, PKC activation caused a lower level of agonist-independent KOPR internalization, compared to U50,488H. U50,488H-induced activation of extracellular signal regulated kinase 1/2 (ERK1/2) was G protein-, but not β-arrestin-, dependent. After U50,488H, GRK- mediated, but not PKC-mediated, KOPR phosphorylation followed by β-arrestin recruitment desensitized U50,488H-induced ERK1/2 response. Therefore, agonist-dependent (GRK- and PKC-mediated) and agonist-independent (PKC-promoted) KOPR phosphorylations show distinct phosphorylation patterns, leading to diverse cellular outcomes.CHO cellsChinese hamster ovary cellsCHLChelerythrine chlorideDDMDodecyl-β-DmaltosideERK1/2extracellular signal regulated kinase 1/2ECLenhanced chemiluminescenceGRKG protein-coupled receptor kinaseGPCRsG protein-coupled receptorsHEK293 cellshuman embryonic kidney 293 cellsHRPHorseradish peroxidaseKOPRkappa opioid receptorMEMMinimal essential mediumN2A cellsneuro2A mouse neuroblastoma cellsNi-NTANickel-nitrilotriacetateNTFno transfectionPKCprotein kinase CPMAphorbol-12-myristate-13-acetatePTXpertussis toxinCHO cellsChinese hamster ovary cellsCHLChelerythrine chlorideDDMDodecyl-β-DmaltosideERK1/2extracellular signal regulated kinase 1/2ECLenhanced chemiluminescenceGRKG protein-coupled receptor kinaseGPCRsG protein-coupled receptorsHEK293 cellshuman embryonic kidney 293 cellsHRPHorseradish peroxidaseKOPRkappa opioid receptorMEMMinimal essential mediumN2A cellsneuro2A mouse neuroblastoma cellsNi-NTANickel-nitrilotriacetateNTFno transfectionPKCprotein kinase CPMAphorbol-12-myristate-13-acetatePTXpertussis toxin