RIC-3 Exclusively Enhances the Surface Expression of Human Homomeric 5-Hydroxytryptamine Type 3A (5-HT(3)A) Receptors Despite Direct Interactions with 5-HT3A, -C, -D, and -E Subunits

Jutta Walstab, Christian Hammer, Felix Lasitschka, Dorothee Möller, Christopher N. Connolly, Gudrun Rappold, Michael Bruess, Heinz Boenish, Beate Niesler

    Research output: Contribution to journalArticle

    17 Citations (Scopus)

    Abstract

    Although five 5-hydroxytryptamine type 3 (5-HT3) subunits (A-E) have been cloned, knowledge on the regulation of their assembly is limited. RIC-3 has been identified as a chaperone specific for the pentameric ligand-gated nicotinic acetylcholine and 5-HT3 receptors. Therefore, we examined the impact of RIC-3 on differently composed 5-HT3 receptors with the focus on 5-HT3C, -D, and -E subunits. The influence of RIC-3 on these receptor subtypes is supported by the presence of RIC3 mRNA in tissues expressing at least one of the subunits 5-HT3C, -D, and -E. Furthermore, immunocytochemical studies on transfected mammalian cells revealed co-localization in the endoplasmic reticulum and direct interaction of RIC-3 with 5-HT3A, -C, -D, and -E. Functional and pharmacological characterization was performed using HEK293 cells expressing 5-HT3A or 5-HT3A + 5-HT3B (or -C, -D, or -E) in the presence or absence of RIC-3. Ca2+ influx analyses revealed that RIC-3 does not influence the 5-HT concentration-response relationship on 5-HT(3)A receptors but leads to differential increases of 5-HT-induced maximum response (E-max) on cells expressing different subunits. Increases of E-max were due to analogously enhanced B-max values for binding of the 5-HT3 receptor antagonist [H-3]GR65630. The observed enhanced cell surface expression of the tested 5-HT3 subunit combinations correlated with the increased surface expression of 5-HT3A as determined by flow cytometry. In conclusion, we showed that RIC-3 can interact with 5-HT3A, -C, -D, and -E subunits and predominantly enhances the surface expression of homomeric 5-HT(3)A receptors in HEK293 cells. These data implicate a possible role of RIC-3 in determining 5-HT3 receptor composition in vivo.

    Original languageEnglish
    Pages (from-to)26956-26965
    Number of pages10
    JournalJournal of Biological Chemistry
    Volume285
    Issue number35
    DOIs
    Publication statusPublished - 27 Aug 2010

    Keywords

    • NICOTINIC ACETYLCHOLINE-RECEPTORS
    • ENDOPLASMIC-RETICULUM CHAPERONE
    • FUNCTIONAL EXPRESSION
    • MOLECULAR-CLONING
    • SEROTONIN
    • PROTEIN
    • GENE
    • TRAFFICKING
    • MATURATION

    Cite this

    Walstab, Jutta ; Hammer, Christian ; Lasitschka, Felix ; Möller, Dorothee ; Connolly, Christopher N. ; Rappold, Gudrun ; Bruess, Michael ; Boenish, Heinz ; Niesler, Beate. / RIC-3 Exclusively Enhances the Surface Expression of Human Homomeric 5-Hydroxytryptamine Type 3A (5-HT(3)A) Receptors Despite Direct Interactions with 5-HT3A, -C, -D, and -E Subunits. In: Journal of Biological Chemistry. 2010 ; Vol. 285, No. 35. pp. 26956-26965.
    @article{78f73a5f129849d78a7312239d7b84d5,
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    abstract = "Although five 5-hydroxytryptamine type 3 (5-HT3) subunits (A-E) have been cloned, knowledge on the regulation of their assembly is limited. RIC-3 has been identified as a chaperone specific for the pentameric ligand-gated nicotinic acetylcholine and 5-HT3 receptors. Therefore, we examined the impact of RIC-3 on differently composed 5-HT3 receptors with the focus on 5-HT3C, -D, and -E subunits. The influence of RIC-3 on these receptor subtypes is supported by the presence of RIC3 mRNA in tissues expressing at least one of the subunits 5-HT3C, -D, and -E. Furthermore, immunocytochemical studies on transfected mammalian cells revealed co-localization in the endoplasmic reticulum and direct interaction of RIC-3 with 5-HT3A, -C, -D, and -E. Functional and pharmacological characterization was performed using HEK293 cells expressing 5-HT3A or 5-HT3A + 5-HT3B (or -C, -D, or -E) in the presence or absence of RIC-3. Ca2+ influx analyses revealed that RIC-3 does not influence the 5-HT concentration-response relationship on 5-HT(3)A receptors but leads to differential increases of 5-HT-induced maximum response (E-max) on cells expressing different subunits. Increases of E-max were due to analogously enhanced B-max values for binding of the 5-HT3 receptor antagonist [H-3]GR65630. The observed enhanced cell surface expression of the tested 5-HT3 subunit combinations correlated with the increased surface expression of 5-HT3A as determined by flow cytometry. In conclusion, we showed that RIC-3 can interact with 5-HT3A, -C, -D, and -E subunits and predominantly enhances the surface expression of homomeric 5-HT(3)A receptors in HEK293 cells. These data implicate a possible role of RIC-3 in determining 5-HT3 receptor composition in vivo.",
    keywords = "NICOTINIC ACETYLCHOLINE-RECEPTORS, ENDOPLASMIC-RETICULUM CHAPERONE, FUNCTIONAL EXPRESSION, MOLECULAR-CLONING, SEROTONIN, PROTEIN, GENE, TRAFFICKING, MATURATION",
    author = "Jutta Walstab and Christian Hammer and Felix Lasitschka and Dorothee M{\"o}ller and Connolly, {Christopher N.} and Gudrun Rappold and Michael Bruess and Heinz Boenish and Beate Niesler",
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    RIC-3 Exclusively Enhances the Surface Expression of Human Homomeric 5-Hydroxytryptamine Type 3A (5-HT(3)A) Receptors Despite Direct Interactions with 5-HT3A, -C, -D, and -E Subunits. / Walstab, Jutta; Hammer, Christian; Lasitschka, Felix; Möller, Dorothee; Connolly, Christopher N.; Rappold, Gudrun; Bruess, Michael; Boenish, Heinz; Niesler, Beate.

    In: Journal of Biological Chemistry, Vol. 285, No. 35, 27.08.2010, p. 26956-26965.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - RIC-3 Exclusively Enhances the Surface Expression of Human Homomeric 5-Hydroxytryptamine Type 3A (5-HT(3)A) Receptors Despite Direct Interactions with 5-HT3A, -C, -D, and -E Subunits

    AU - Walstab, Jutta

    AU - Hammer, Christian

    AU - Lasitschka, Felix

    AU - Möller, Dorothee

    AU - Connolly, Christopher N.

    AU - Rappold, Gudrun

    AU - Bruess, Michael

    AU - Boenish, Heinz

    AU - Niesler, Beate

    PY - 2010/8/27

    Y1 - 2010/8/27

    N2 - Although five 5-hydroxytryptamine type 3 (5-HT3) subunits (A-E) have been cloned, knowledge on the regulation of their assembly is limited. RIC-3 has been identified as a chaperone specific for the pentameric ligand-gated nicotinic acetylcholine and 5-HT3 receptors. Therefore, we examined the impact of RIC-3 on differently composed 5-HT3 receptors with the focus on 5-HT3C, -D, and -E subunits. The influence of RIC-3 on these receptor subtypes is supported by the presence of RIC3 mRNA in tissues expressing at least one of the subunits 5-HT3C, -D, and -E. Furthermore, immunocytochemical studies on transfected mammalian cells revealed co-localization in the endoplasmic reticulum and direct interaction of RIC-3 with 5-HT3A, -C, -D, and -E. Functional and pharmacological characterization was performed using HEK293 cells expressing 5-HT3A or 5-HT3A + 5-HT3B (or -C, -D, or -E) in the presence or absence of RIC-3. Ca2+ influx analyses revealed that RIC-3 does not influence the 5-HT concentration-response relationship on 5-HT(3)A receptors but leads to differential increases of 5-HT-induced maximum response (E-max) on cells expressing different subunits. Increases of E-max were due to analogously enhanced B-max values for binding of the 5-HT3 receptor antagonist [H-3]GR65630. The observed enhanced cell surface expression of the tested 5-HT3 subunit combinations correlated with the increased surface expression of 5-HT3A as determined by flow cytometry. In conclusion, we showed that RIC-3 can interact with 5-HT3A, -C, -D, and -E subunits and predominantly enhances the surface expression of homomeric 5-HT(3)A receptors in HEK293 cells. These data implicate a possible role of RIC-3 in determining 5-HT3 receptor composition in vivo.

    AB - Although five 5-hydroxytryptamine type 3 (5-HT3) subunits (A-E) have been cloned, knowledge on the regulation of their assembly is limited. RIC-3 has been identified as a chaperone specific for the pentameric ligand-gated nicotinic acetylcholine and 5-HT3 receptors. Therefore, we examined the impact of RIC-3 on differently composed 5-HT3 receptors with the focus on 5-HT3C, -D, and -E subunits. The influence of RIC-3 on these receptor subtypes is supported by the presence of RIC3 mRNA in tissues expressing at least one of the subunits 5-HT3C, -D, and -E. Furthermore, immunocytochemical studies on transfected mammalian cells revealed co-localization in the endoplasmic reticulum and direct interaction of RIC-3 with 5-HT3A, -C, -D, and -E. Functional and pharmacological characterization was performed using HEK293 cells expressing 5-HT3A or 5-HT3A + 5-HT3B (or -C, -D, or -E) in the presence or absence of RIC-3. Ca2+ influx analyses revealed that RIC-3 does not influence the 5-HT concentration-response relationship on 5-HT(3)A receptors but leads to differential increases of 5-HT-induced maximum response (E-max) on cells expressing different subunits. Increases of E-max were due to analogously enhanced B-max values for binding of the 5-HT3 receptor antagonist [H-3]GR65630. The observed enhanced cell surface expression of the tested 5-HT3 subunit combinations correlated with the increased surface expression of 5-HT3A as determined by flow cytometry. In conclusion, we showed that RIC-3 can interact with 5-HT3A, -C, -D, and -E subunits and predominantly enhances the surface expression of homomeric 5-HT(3)A receptors in HEK293 cells. These data implicate a possible role of RIC-3 in determining 5-HT3 receptor composition in vivo.

    KW - NICOTINIC ACETYLCHOLINE-RECEPTORS

    KW - ENDOPLASMIC-RETICULUM CHAPERONE

    KW - FUNCTIONAL EXPRESSION

    KW - MOLECULAR-CLONING

    KW - SEROTONIN

    KW - PROTEIN

    KW - GENE

    KW - TRAFFICKING

    KW - MATURATION

    U2 - 10.1074/jbc.M110.122838

    DO - 10.1074/jbc.M110.122838

    M3 - Article

    VL - 285

    SP - 26956

    EP - 26965

    JO - Journal of Biological Chemistry

    JF - Journal of Biological Chemistry

    SN - 0021-9258

    IS - 35

    ER -