Gβ5-RGS7 inhibits Gαq-mediated signaling via a direct protein-protein interaction

D. Scott Witherow, Steven C. Tovey, Qiang Wang, Gary B. Willars, Vladlen Z Slepak

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

A subfamily of regulators of G protein signaling (RGS) proteins consisting of RGS6, -7, -9, and -11 is characterized by the presence of a unique Gγ-like domain through which they form obligatory dimers with the G protein subunit Gβ5 in vivo. In Caenorhabditis elegans, orthologs of Gβ5-RGS dimers are implicated in regulating both Gαi and Gαq signaling, and in cell-based assays these dimers regulate Gαi/o- and Gαq/11-mediated pathways. However, initial studies with purified Gβ5-RGS6 or Gβ5-RGS7 showed that they only serve as GTPase activating proteins for Gαo. Pull-down assays and co-immunoprecipitation with these dimers failed to detect their binding to either Gαo or Gαq, indicating that the interaction might require additional factors present in vivo. Here, we asked if the RGS7-Gβ5 complex binds to Gαq using fluorescence resonance energy transfer (FRET) in transiently transfected mammalian cells. RGS7, Gβ5, and Gα subunits were tagged with yellow variants of green fluorescent protein. First we confirmed the functional activity of the fusion proteins by co-immunoprecipitation and also their effect on signaling. Second, we again demonstrate the interaction between RGS7 and Gβ5 using FRET. Finally, using both FRET spectroscopy on cell suspensions and microscopy of individual cells, we showed FRET between the yellow fluorescence protein-tagged RGS7-Gβ5 complex and cyan fluorescence protein-tagged Gαq, indicating a direct interaction between these molecules.

Original languageEnglish
Pages (from-to)21307-21313
Number of pages7
JournalJournal of Biological Chemistry
Volume278
Issue number23
DOIs
StatePublished - Jun 6 2003

Fingerprint

Fluorescence Resonance Energy Transfer
Dimers
Immunoprecipitation
Assays
Proteins
Gq-G11 GTP-Binding Protein alpha Subunits
Fluorescence
RGS Proteins
GTP-Binding Protein Regulators
GTPase-Activating Proteins
Caenorhabditis elegans
Protein Subunits
Green Fluorescent Proteins
GTP-Binding Proteins
Microscopy
Spectrum Analysis
Suspensions
Microscopic examination
Fusion reactions
Cells

ASJC Scopus subject areas

  • Biochemistry

Cite this

Gβ5-RGS7 inhibits Gαq-mediated signaling via a direct protein-protein interaction. / Witherow, D. Scott; Tovey, Steven C.; Wang, Qiang; Willars, Gary B.; Slepak, Vladlen Z.

In: Journal of Biological Chemistry, Vol. 278, No. 23, 06.06.2003, p. 21307-21313.

Research output: Contribution to journalArticle

Witherow, D. Scott ; Tovey, Steven C. ; Wang, Qiang ; Willars, Gary B. ; Slepak, Vladlen Z. / Gβ5-RGS7 inhibits Gαq-mediated signaling via a direct protein-protein interaction. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 23. pp. 21307-21313.
@article{28a6d5950f674824a7b76a615537a000,
title = "Gβ5-RGS7 inhibits Gαq-mediated signaling via a direct protein-protein interaction",
abstract = "A subfamily of regulators of G protein signaling (RGS) proteins consisting of RGS6, -7, -9, and -11 is characterized by the presence of a unique Gγ-like domain through which they form obligatory dimers with the G protein subunit Gβ5 in vivo. In Caenorhabditis elegans, orthologs of Gβ5-RGS dimers are implicated in regulating both Gαi and Gαq signaling, and in cell-based assays these dimers regulate Gαi/o- and Gαq/11-mediated pathways. However, initial studies with purified Gβ5-RGS6 or Gβ5-RGS7 showed that they only serve as GTPase activating proteins for Gαo. Pull-down assays and co-immunoprecipitation with these dimers failed to detect their binding to either Gαo or Gαq, indicating that the interaction might require additional factors present in vivo. Here, we asked if the RGS7-Gβ5 complex binds to Gαq using fluorescence resonance energy transfer (FRET) in transiently transfected mammalian cells. RGS7, Gβ5, and Gα subunits were tagged with yellow variants of green fluorescent protein. First we confirmed the functional activity of the fusion proteins by co-immunoprecipitation and also their effect on signaling. Second, we again demonstrate the interaction between RGS7 and Gβ5 using FRET. Finally, using both FRET spectroscopy on cell suspensions and microscopy of individual cells, we showed FRET between the yellow fluorescence protein-tagged RGS7-Gβ5 complex and cyan fluorescence protein-tagged Gαq, indicating a direct interaction between these molecules.",
author = "Witherow, {D. Scott} and Tovey, {Steven C.} and Qiang Wang and Willars, {Gary B.} and Slepak, {Vladlen Z}",
year = "2003",
month = "6",
day = "6",
doi = "10.1074/jbc.M212884200",
language = "English",
volume = "278",
pages = "21307--21313",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "23",

}

TY - JOUR

T1 - Gβ5-RGS7 inhibits Gαq-mediated signaling via a direct protein-protein interaction

AU - Witherow, D. Scott

AU - Tovey, Steven C.

AU - Wang, Qiang

AU - Willars, Gary B.

AU - Slepak, Vladlen Z

PY - 2003/6/6

Y1 - 2003/6/6

N2 - A subfamily of regulators of G protein signaling (RGS) proteins consisting of RGS6, -7, -9, and -11 is characterized by the presence of a unique Gγ-like domain through which they form obligatory dimers with the G protein subunit Gβ5 in vivo. In Caenorhabditis elegans, orthologs of Gβ5-RGS dimers are implicated in regulating both Gαi and Gαq signaling, and in cell-based assays these dimers regulate Gαi/o- and Gαq/11-mediated pathways. However, initial studies with purified Gβ5-RGS6 or Gβ5-RGS7 showed that they only serve as GTPase activating proteins for Gαo. Pull-down assays and co-immunoprecipitation with these dimers failed to detect their binding to either Gαo or Gαq, indicating that the interaction might require additional factors present in vivo. Here, we asked if the RGS7-Gβ5 complex binds to Gαq using fluorescence resonance energy transfer (FRET) in transiently transfected mammalian cells. RGS7, Gβ5, and Gα subunits were tagged with yellow variants of green fluorescent protein. First we confirmed the functional activity of the fusion proteins by co-immunoprecipitation and also their effect on signaling. Second, we again demonstrate the interaction between RGS7 and Gβ5 using FRET. Finally, using both FRET spectroscopy on cell suspensions and microscopy of individual cells, we showed FRET between the yellow fluorescence protein-tagged RGS7-Gβ5 complex and cyan fluorescence protein-tagged Gαq, indicating a direct interaction between these molecules.

AB - A subfamily of regulators of G protein signaling (RGS) proteins consisting of RGS6, -7, -9, and -11 is characterized by the presence of a unique Gγ-like domain through which they form obligatory dimers with the G protein subunit Gβ5 in vivo. In Caenorhabditis elegans, orthologs of Gβ5-RGS dimers are implicated in regulating both Gαi and Gαq signaling, and in cell-based assays these dimers regulate Gαi/o- and Gαq/11-mediated pathways. However, initial studies with purified Gβ5-RGS6 or Gβ5-RGS7 showed that they only serve as GTPase activating proteins for Gαo. Pull-down assays and co-immunoprecipitation with these dimers failed to detect their binding to either Gαo or Gαq, indicating that the interaction might require additional factors present in vivo. Here, we asked if the RGS7-Gβ5 complex binds to Gαq using fluorescence resonance energy transfer (FRET) in transiently transfected mammalian cells. RGS7, Gβ5, and Gα subunits were tagged with yellow variants of green fluorescent protein. First we confirmed the functional activity of the fusion proteins by co-immunoprecipitation and also their effect on signaling. Second, we again demonstrate the interaction between RGS7 and Gβ5 using FRET. Finally, using both FRET spectroscopy on cell suspensions and microscopy of individual cells, we showed FRET between the yellow fluorescence protein-tagged RGS7-Gβ5 complex and cyan fluorescence protein-tagged Gαq, indicating a direct interaction between these molecules.

UR - http://www.scopus.com/inward/record.url?scp=0038079771&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0038079771&partnerID=8YFLogxK

U2 - 10.1074/jbc.M212884200

DO - 10.1074/jbc.M212884200

M3 - Article

C2 - 12670932

AN - SCOPUS:0038079771

VL - 278

SP - 21307

EP - 21313

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 23

ER -