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Research ArticleArticle

Identification and Characterization of Distinct C-Terminal Domains of the Human Hydroxycarboxylic Acid Receptor-2 That Are Essential for Receptor Export, Constitutive Activity, Desensitization, and Internalization

Guo Li, Qi Zhou, Yena Yu, Linjie Chen, Ying Shi, Jiansong Luo, Jeffrey Benovic, Jianxin Lu and Naiming Zhou
Molecular Pharmacology December 2012, 82 (6) 1150-1161; DOI: https://doi.org/10.1124/mol.112.081307
Guo Li
Zhejiang Provincial Key Laboratory of Medical Genetics, School of Laboratory Medicine and Life Science, Wenzhou Medical College, Wenzhou, Zhejiang, China (Q.Z., Y.Y., J.Lu., N.Z.); College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou Zhejiang, China (G.L., L.C., Y.S., N.Z.); Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania (J.Luo, J.B.); and Institute of Aging Research, Hangzhou Normal University, Hangzhou, Zhejian, China (G.L.)
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Qi Zhou
Zhejiang Provincial Key Laboratory of Medical Genetics, School of Laboratory Medicine and Life Science, Wenzhou Medical College, Wenzhou, Zhejiang, China (Q.Z., Y.Y., J.Lu., N.Z.); College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou Zhejiang, China (G.L., L.C., Y.S., N.Z.); Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania (J.Luo, J.B.); and Institute of Aging Research, Hangzhou Normal University, Hangzhou, Zhejian, China (G.L.)
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Yena Yu
Zhejiang Provincial Key Laboratory of Medical Genetics, School of Laboratory Medicine and Life Science, Wenzhou Medical College, Wenzhou, Zhejiang, China (Q.Z., Y.Y., J.Lu., N.Z.); College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou Zhejiang, China (G.L., L.C., Y.S., N.Z.); Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania (J.Luo, J.B.); and Institute of Aging Research, Hangzhou Normal University, Hangzhou, Zhejian, China (G.L.)
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Linjie Chen
Zhejiang Provincial Key Laboratory of Medical Genetics, School of Laboratory Medicine and Life Science, Wenzhou Medical College, Wenzhou, Zhejiang, China (Q.Z., Y.Y., J.Lu., N.Z.); College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou Zhejiang, China (G.L., L.C., Y.S., N.Z.); Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania (J.Luo, J.B.); and Institute of Aging Research, Hangzhou Normal University, Hangzhou, Zhejian, China (G.L.)
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Ying Shi
Zhejiang Provincial Key Laboratory of Medical Genetics, School of Laboratory Medicine and Life Science, Wenzhou Medical College, Wenzhou, Zhejiang, China (Q.Z., Y.Y., J.Lu., N.Z.); College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou Zhejiang, China (G.L., L.C., Y.S., N.Z.); Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania (J.Luo, J.B.); and Institute of Aging Research, Hangzhou Normal University, Hangzhou, Zhejian, China (G.L.)
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Jiansong Luo
Zhejiang Provincial Key Laboratory of Medical Genetics, School of Laboratory Medicine and Life Science, Wenzhou Medical College, Wenzhou, Zhejiang, China (Q.Z., Y.Y., J.Lu., N.Z.); College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou Zhejiang, China (G.L., L.C., Y.S., N.Z.); Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania (J.Luo, J.B.); and Institute of Aging Research, Hangzhou Normal University, Hangzhou, Zhejian, China (G.L.)
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Jeffrey Benovic
Zhejiang Provincial Key Laboratory of Medical Genetics, School of Laboratory Medicine and Life Science, Wenzhou Medical College, Wenzhou, Zhejiang, China (Q.Z., Y.Y., J.Lu., N.Z.); College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou Zhejiang, China (G.L., L.C., Y.S., N.Z.); Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania (J.Luo, J.B.); and Institute of Aging Research, Hangzhou Normal University, Hangzhou, Zhejian, China (G.L.)
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Jianxin Lu
Zhejiang Provincial Key Laboratory of Medical Genetics, School of Laboratory Medicine and Life Science, Wenzhou Medical College, Wenzhou, Zhejiang, China (Q.Z., Y.Y., J.Lu., N.Z.); College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou Zhejiang, China (G.L., L.C., Y.S., N.Z.); Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania (J.Luo, J.B.); and Institute of Aging Research, Hangzhou Normal University, Hangzhou, Zhejian, China (G.L.)
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Naiming Zhou
Zhejiang Provincial Key Laboratory of Medical Genetics, School of Laboratory Medicine and Life Science, Wenzhou Medical College, Wenzhou, Zhejiang, China (Q.Z., Y.Y., J.Lu., N.Z.); College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou Zhejiang, China (G.L., L.C., Y.S., N.Z.); Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania (J.Luo, J.B.); and Institute of Aging Research, Hangzhou Normal University, Hangzhou, Zhejian, China (G.L.)
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Abstract

The human hydroxycarboxylic acid receptor 2 (HCA2), also known as GPR109A and HM74a, was first identified as a niacin receptor and has recently received significant attention because of its potential to clinically modify plasma lipids in a favorable manner. Our recent studies have demonstrated that the niacin-induced internalization of HCA2 receptors is regulated by G protein-coupled receptor kinase (GRK) 2 and arrestin3 and that internalized receptors rapidly recycle back to the cell surface. The investigation presented here used a combination of amino acid deletion and site-directed mutagenesis to identify structural and functional domains within the HCA2 C terminus and explore their potential roles in receptor phosphorylation, desensitization, and internalization. We first constructed four mutants with deletions of 10 to 15 amino acids each that were distinct from truncated mutants. We successfully identified different domains responsible for receptor export, constitutive activity, desensitization, phosphorylation, and internalization. We also generated a comprehensive series of alanine substitution mutants, replacing conserved serine and threonine residues in the C terminus with alanine residues to pinpoint the key residues that are essential for GRK2-mediated phosphorylation and arrestin3 association. Moreover, we found that a sequence from residues 329 to 343 in the C-terminal tail of HCA2 plays a crucial role in keeping HCA2 in an inactive conformation. These data demonstrate the importance of distinct domains within the C terminus of HCA2 for receptor cell surface expression, desensitization, and internalization and phosphorylation and stabilization of an inactive receptor conformation.

Footnotes

  • ↵Embedded Image The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

  • This work was supported by the Ministry of Science and Technology [Grants 2012CB910402, 2012AA020303-05]; National Natural Science Foundation of China [Grant 81173106]; Zhejiang Natural Science Foundation [Grant Z2080207]; and National Institutes of Health National Institute of General Medical Sciences [Grant GM44944].

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

    http://dx.doi.org/10.1124/mol.112.081307.

  • ABBREVIATIONS:

    GRK
    G protein-coupled receptor kinase
    ERK1/2
    extracellular signal-regulated kinase1/2
    siRNA
    small interfering RNA
    GPCR
    G protein-coupled receptor
    DiI
    1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate
    FITC
    fluorescein isothiocyanate
    HRP
    horseradish peroxidase
    HEK
    human embryonic kidney
    EGFP
    enhanced green fluorescent protein
    ELISA
    enzyme-linked immunosorbent assay
    WT
    wild-type
    TBS
    Tris-buffered saline
    BSA
    bovine serum albumin
    DMEM
    Dulbecco's modified Eagle's medium
    PBS
    phosphate-buffered saline
    CRE
    cAMP response element
    ER
    endoplasmic reticulum
    MβCD
    methyl-β-cyclodextrin
    PKA
    protein kinase A
    PKC
    protein kinase C
    5-HT
    5-hydroxytryptamine.

  • Received July 18, 2012.
  • Accepted September 7, 2012.
  • Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 82 (6)
Molecular Pharmacology
Vol. 82, Issue 6
1 Dec 2012
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Research ArticleArticle

Role of C Terminus in Hydroxycarboxylic Acid Receptor-2

Guo Li, Qi Zhou, Yena Yu, Linjie Chen, Ying Shi, Jiansong Luo, Jeffrey Benovic, Jianxin Lu and Naiming Zhou
Molecular Pharmacology December 1, 2012, 82 (6) 1150-1161; DOI: https://doi.org/10.1124/mol.112.081307

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Research ArticleArticle

Role of C Terminus in Hydroxycarboxylic Acid Receptor-2

Guo Li, Qi Zhou, Yena Yu, Linjie Chen, Ying Shi, Jiansong Luo, Jeffrey Benovic, Jianxin Lu and Naiming Zhou
Molecular Pharmacology December 1, 2012, 82 (6) 1150-1161; DOI: https://doi.org/10.1124/mol.112.081307
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