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Research ArticleCELLULAR AND MOLECULAR

Two Amino Acids in the Sixth Transmembrane Segment of the Mouse Gastrin-Releasing Peptide Receptor Are Important for Receptor Activation

Yuhmei Lin, Xiaoying Jian, Zolan Lin, Glenn S. Kroog, Samuel Mantey, Robert T. Jensen, James Battey and John Northup
Journal of Pharmacology and Experimental Therapeutics September 2000, 294 (3) 1053-1062;
Yuhmei Lin
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Xiaoying Jian
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Zolan Lin
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Glenn S. Kroog
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Samuel Mantey
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Robert T. Jensen
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James Battey
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John Northup
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Abstract

The gastrin-releasing peptide receptor (GRP-R) is a G protein-coupled receptor that mediates a variety of cellular responses, including cell growth and modulation of neuronal activity by activation of heterotrimeric GTP-binding proteins in the Gq family. To understand the regulation of GRP-R signaling we have substituted alanine for each of 10 amino acid residues within the transmembrane (TM) helices of the GRP-R predicted to project into the binding pocket of the receptor and analyzed the importance of each of these residues for receptor function. Two mutations showed selective loss of either agonist (Y285A) or antagonist (F313A) affinity for the GRP-R. In addition, we identified two amino acid residues, Phe270 and Asn281, in the sixth TM segment, which are important for receptor-G protein interaction. In a competition-binding assay with an antagonist radioligand, bombesin showed a 20- to 100-fold decreased affinity for the N281A and F270A mutant GRP-R compared with wild-type GRP-R. The saturation-binding isotherms are best fit by a two-state model, indicating that the receptors are in either a low-affinity (KD2) or a high-affinity (KD1) state. The ratio of the two affinities (KD2/KD1) was significantly increased for both mutants compared with wild-type GRP-R, whereas the fraction of mutant receptors in the high-affinity state (R1) was decreased. GDP/guanosine-5′-O-(3-thio)triphosphate exchange catalyzed by the N281A mutant was lower than that observed for the wild-type GRP-R. However, for both mutants, bombesin was still able to stimulate 1,4,5-inositol triphosphate in transfected cells albeit with reduced activity. We conclude that these two TM residues are important for receptor-G protein coupling, and postulate that each mutation may affect GRP-R conformational change to the high-affinity, G protein-coupled state.

Footnotes

  • Send reprint requests to: Dr. James Battey, National Institute on Deafness and Other Communication Disorders/National Institutes of Health, Bldg. 31, Room 3C02, 31 Center Dr., MSC 2320, Bethesda, MD 20892-2320. E-mail:batteyj{at}nidcd.nih.gov

  • Abbreviations:
    BN
    bombesin
    GRP
    gastrin-releasing peptide
    GRP-R
    GRP receptor (bb2)
    NMB-R
    neuromedin B-preferring peptide receptor (bb1)
    BRS-3
    bombesin receptor subtype 3 (bb3)
    GPCR
    G protein-coupled receptor
    TM
    transmembrane
    DMEM
    Dulbecco's modified Eagle's medium
    125I-ME
    125I-[d-Tyr6]BN(6-13)methyl ester
    GTPγS
    guanosine-5′-O-(3-thio)triphosphate
    125I-BN
    125I-[Tyr4]bombesin
    1,4,5-IP3
    1,4,5-inositol triphosphate
    ic3
    intracellular loop 3
    EC
    extracellular
    5HT2A
    5-hydroxytryptamine2A
    • Received February 18, 2000.
    • Accepted May 9, 2000.
  • U.S. Government
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Journal of Pharmacology and Experimental Therapeutics: 294 (3)
Journal of Pharmacology and Experimental Therapeutics
Vol. 294, Issue 3
1 Sep 2000
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Research ArticleCELLULAR AND MOLECULAR

Two Amino Acids in the Sixth Transmembrane Segment of the Mouse Gastrin-Releasing Peptide Receptor Are Important for Receptor Activation

Yuhmei Lin, Xiaoying Jian, Zolan Lin, Glenn S. Kroog, Samuel Mantey, Robert T. Jensen, James Battey and John Northup
Journal of Pharmacology and Experimental Therapeutics September 1, 2000, 294 (3) 1053-1062;

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Research ArticleCELLULAR AND MOLECULAR

Two Amino Acids in the Sixth Transmembrane Segment of the Mouse Gastrin-Releasing Peptide Receptor Are Important for Receptor Activation

Yuhmei Lin, Xiaoying Jian, Zolan Lin, Glenn S. Kroog, Samuel Mantey, Robert T. Jensen, James Battey and John Northup
Journal of Pharmacology and Experimental Therapeutics September 1, 2000, 294 (3) 1053-1062;
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