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Research ArticleArticle

Role of Helix 8 of the Thyrotropin-Releasing Hormone Receptor in Phosphorylation by G Protein-Coupled Receptor Kinase

Austin U. Gehret, Brian W. Jones, Phuong N. Tran, Laurie B. Cook, Emileigh K. Greuber and Patricia M. Hinkle
Molecular Pharmacology February 2010, 77 (2) 288-297; DOI: https://doi.org/10.1124/mol.109.059733
Austin U. Gehret
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Brian W. Jones
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Phuong N. Tran
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Laurie B. Cook
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Emileigh K. Greuber
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Patricia M. Hinkle
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Abstract

The thyrotropin-releasing hormone (TRH) receptor undergoes rapid and extensive agonist-dependent phosphorylation attributable to G protein-coupled receptor (GPCR) kinases (GRKs), particularly GRK2. Like many GPCRs, the TRH receptor is predicted to form an amphipathic helix, helix 8, between the NPXXY motif at the cytoplasmic end of the seventh transmembrane domain and palmitoylation sites at Cys335 and Cys337. Mutation of all six lysine and arginine residues between the NPXXY and residue 340 to glutamine (6Q receptor) did not prevent the receptor from stimulating inositol phosphate turnover but almost completely prevented receptor phosphorylation in response to TRH. Phosphorylation at all sites in the cytoplasmic tail was inhibited. The phosphorylation defect was not reversed by long incubation times or high TRH concentrations. As expected for a phosphorylation-defective receptor, the 6Q-TRH receptor did not recruit arrestin, undergo the typical arrestin-dependent increase in agonist affinity, or internalize well. Lys326, directly before phenylalanine in the common GPCR motif NPXXY(X)5–6F(R/K), was critical for phosphorylation. The 6Q-TRH receptor was not phosphorylated effectively in cells overexpressing GRK2 or in in vitro kinase assays containing purified GRK2. Phosphorylation of the 6Q receptor was partially restored by coexpression of a receptor with an intact helix 8 but without phosphorylation sites. Phosphorylation was inhibited but not completely prevented by alanine substitution for cysteine palmitoylation sites. Positively charged amino acids in the proximal tail of the β2-adrenergic receptor were also important for GRK-dependent phosphorylation. The results indicate that positive residues in helix 8 of GPCRs are important for GRK-dependent phosphorylation.

Footnotes

  • This work was supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant DK19974].

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

    doi:10.1124/mol.109.059733

  • ABBREVIATIONS:

    TRH
    thyrotropin-releasing hormone
    GPCR
    G protein-coupled receptor
    GFP
    green fluorescent protein
    GRK
    G protein-coupled receptor kinase
    HEK
    human embryonic kidney
    WT
    wild-type
    ELISA
    enzyme-linked immunosorbent assay
    PBS
    phosphate-buffered saline
    RIPA
    radioimmunoprecipitation assay
    HBSS
    Hanks' balanced salt solution
    HA
    hemagglutinin
    GF109203X
    3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride.

  • Received July 23, 2009.
  • Accepted November 11, 2009.
  • Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 77 (2)
Molecular Pharmacology
Vol. 77, Issue 2
1 Feb 2010
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Research ArticleArticle

Role of Helix 8 of the Thyrotropin-Releasing Hormone Receptor in Phosphorylation by G Protein-Coupled Receptor Kinase

Austin U. Gehret, Brian W. Jones, Phuong N. Tran, Laurie B. Cook, Emileigh K. Greuber and Patricia M. Hinkle
Molecular Pharmacology February 1, 2010, 77 (2) 288-297; DOI: https://doi.org/10.1124/mol.109.059733

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Research ArticleArticle

Role of Helix 8 of the Thyrotropin-Releasing Hormone Receptor in Phosphorylation by G Protein-Coupled Receptor Kinase

Austin U. Gehret, Brian W. Jones, Phuong N. Tran, Laurie B. Cook, Emileigh K. Greuber and Patricia M. Hinkle
Molecular Pharmacology February 1, 2010, 77 (2) 288-297; DOI: https://doi.org/10.1124/mol.109.059733
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