Serine 331 is the major site of receptor phosphorylation induced by agents that activate protein kinase G in HEK 293 cells overexpressing thromboxane receptor alpha.
Human embryonic kidney (HEK)293 cells stably transfected with the His-tagged thromboxane receptor alpha (TPalpha) was used to study the phosphorylation and desensitization of the receptor induced by 8-bromo-cyclic GMP (8-Br-cGMP), sodium nitroprusside (SNP), or S-nitroso-glutathione (SNG). These agents are known to activate cGMP-dependent protein kinase (PKG). Pretreatment of cells with these agents attenuated significantly agonist I-BOP induced Ca(2+) release. These agents also induced dose-dependent phosphorylation of the TPalpha as demonstrated by increased (32)P-labeling of the receptor from cells prelabeled with (32)Pi. To facilitate the identification of the intracellular domains involved in phosphorylation, glutathione S-transferase (GST)-intracellular domain fusion proteins were used as substrates for the purified PKG. It was found that only the GST-C-terminal tail fusion protein could serve as a substrate for the PKG. To identify the specific serine/threonine residues in the C-terminal tail being phosphorylated, various alanine mutants of these serine/threonine residues were checked for their ability to serve as substrates. It was found that the Ser-331 of the C-terminal tail was primarily involved in the PKG-mediated phosphorylation. That Ser-331 is a predominant site of phosphorylation was supported by in vivo studies in which HEK293 cells expressing the S331A mutant receptor showed little phosphorylation induced by any of the above three agents. Furthermore, HEK293 cells expressing the S331A mutant receptor pretreated with any of the above three agents became responsive to the agonist I-BOP-induced Ca(2+) release. These results indicate that Ser-331 of the TPalpha is the primary site responsible for the phosphorylation and the desensitization of the receptor induced by agents that activate the PKG.
Yamamoto
S
S
Division of Pharmaceutical Sciences, University of Kentucky, Lexington, Kentucky 40536-0082, USA.
Yan
F
F
Zhou
H
H
Tai
H H
HH
D001483:Base Sequence; D001665:Binding Sites; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002460:Cell Line; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D016297:Mutagenesis, Site-Directed; D009599:Nitroprusside / Q000494:pharmacology; D009603:Nitroso Compounds / Q000494:pharmacology; D010766:Phosphorylation; D017482:Receptors, Thromboxane / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D026422:S-Nitrosoglutathione; D012694:Serine / Q000737:chemistry
D019086:Bridged Bicyclo Compounds, Heterocyclic; D017931:DNA Primers; D005231:Fatty Acids, Unsaturated; D009603:Nitroso Compounds; D017482:Receptors, Thromboxane; D011993:Recombinant Fusion Proteins; C061750:7-(3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo(2.2.1)heptan-2-yl)-5-heptenoic acid; D009599:Nitroprusside; C016276:8-bromocyclic GMP; D012694:Serine; D026422:S-Nitrosoglutathione; D017869:Cyclic GMP-Dependent Protein Kinases; D005978:Glutathione; D006152:Cyclic GMP