Desensitization and internalization of endothelin receptor A: impact of G protein-coupled receptor kinase 2 (GRK2)-mediated phosphorylation.
Endothelin receptor A (ETA), a G protein-coupled receptor, mediates endothelin signaling, which is regulated by GRK2. Three Ser and seven Thr residues recently proven to be phosphoacceptor sites are located in the C-terminal extremity (CTE) of the receptor following its palmitoylation site. We created various phosphorylation-deficient ETA mutants. The phospholipase C activity of mutant receptors in HEK-293 cells was analyzed during continuous endothelin stimulation to investigate the impact of phosphorylation sites on ETA desensitization. Total deletion of phosphoacceptor sites in the CTE affected proper receptor regulation. However, proximal and distal phosphoacceptor sites both turned out to be sufficient to induce WT-like desensitization. Overexpression of the Gαq coupling-deficient mutant GRK2-D110A suppressed ETA-WT signaling but failed to decrease phospholipase C activity mediated by the phosphorylation-deficient mutant ETA-6PD. In contrast, GRK2-WT acted on both receptors, whereas the kinase-inactive mutant GRK2-D110A/K220R failed to inhibit signaling of ETA-WT and ETA-6PD. This demonstrates that ETA desensitization involves at least two autonomous GRK2-mediated components: 1) a phosphorylation-independent signal decrease mediated by blocking of Gαq and 2) a mechanism involving phosphorylation of Ser and Thr residues in the CTE of the receptor in a redundant fashion, able to incorporate either proximal or distal phosphoacceptor sites. High level transfection of GRK2 variants influenced signaling of ETA-WT and ETA-6PD and hints at an additional phosphorylation-independent regulatory mechanism. Furthermore, internalization of mRuby-tagged receptors was observed with ETA-WT and the phosphorylation-deficient mutant ETA-14PD (lacking 14 phosphoacceptor sites) and turned out to be based on a phosphorylation-independent mechanism.
From the E. & H. Klessmann Institute for Cardiovascular Research & Development, Clinic of Thoracic and Cardiovascular Surgery, Heart and Diabetes Center NRW, Ruhr University Bochum, D-32545 Bad Oeynhausen.
Florian
F
Gärtner
Dynamic Cell Imaging, Faculty of Biology, Bielefeld University, D-33501 Bielefeld, Germany.
Thorsten
T
Seidel
From the E. & H. Klessmann Institute for Cardiovascular Research & Development, Clinic of Thoracic and Cardiovascular Surgery, Heart and Diabetes Center NRW, Ruhr University Bochum, D-32545 Bad Oeynhausen.
Uwe
U
Schulz
From the E. & H. Klessmann Institute for Cardiovascular Research & Development, Clinic of Thoracic and Cardiovascular Surgery, Heart and Diabetes Center NRW, Ruhr University Bochum, D-32545 Bad Oeynhausen.
Jan
J
Gummert
From the E. & H. Klessmann Institute for Cardiovascular Research & Development, Clinic of Thoracic and Cardiovascular Surgery, Heart and Diabetes Center NRW, Ruhr University Bochum, D-32545 Bad Oeynhausen. Electronic address: hmilting@hdz-nrw.de.
Hendrik
H
Milting
D019943:Amino Acid Substitution; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000235:genetics / Q000378:metabolism*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D020125:Mutation, Missense; D010766:Phosphorylation / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D017466:Receptors, Endothelin / Q000235:genetics / Q000378:metabolism*; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism
D017466:Receptors, Endothelin; C496567:GRK2 protein, human; D054769:G-Protein-Coupled Receptor Kinase 2; D010738:Type C Phospholipases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11
Endothelin; G Protein-coupled Receptor (GPCR); Phosphorylation; Receptor Desensitization; Receptor Endocytosis; Receptor Regulation