Elsevier

Methods in Enzymology

Volume 522, 2013, Pages 153-167
Methods in Enzymology

Chapter Nine - Group II Activators of G-protein Signaling: Monitoring the Interaction of Gα with the G-protein Regulatory Motif in the Intact Cell

https://doi.org/10.1016/B978-0-12-407865-9.00009-1Get rights and content

Abstract

The G-protein regulatory (GPR) motif serves as a docking site for Gαi–GDP free of Gβγ. The GPR–Gα complex may function at the cell cortex and/or at intracellular sites. GPR proteins include the Group II Activators of G-protein signaling identified in a functional screen for receptor-independent activators of G-protein signaling (GPSM1-3, RGS12) each of which contain 1–4 GPR motifs. GPR motifs are also found in PCP2/L7(GPSM4), Rap1-Gap1 Transcript Variant 1, and RGS14. While the biochemistry of the interaction of GPR proteins with purified Gα is generally understood, the dynamics of this signaling complex and its regulation within the cell remains undefined. Major questions in the field revolve around the factors that regulate the subcellular location of GPR proteins and their interaction with Gαi and other binding partners in the cell. As an initial approach to this question, we established a platform to monitor the GPR–Gαi complex in intact cells using bioluminescence resonance energy transfer.

Introduction

Activators of G-protein signaling refer to a group of proteins identified in a yeast-based functional screen of mammalian cDNAs that activated G-protein signaling in the absence of a receptor (Blumer et al., 2007, Cismowski et al., 1999, Sato et al., 2006, Takesono et al., 1999). There are three groups of AGS proteins distinguished by their mechanism of action and/or engagement of G-protein subunits. Group II AGS proteins (AGS3–GPSM1, AGS4–GPSM3, AGS5–GPSM2, AGS6–RGS12) contain 1–4 G-protein regulatory (GPR) or GoLoco motifs. GPR motifs are also found in PCP2/L7(GPSM4), Rap1-Gap1 Transcript Variant 1, and RGS14 (Fig. 9.1A). The GPR motif is a 20–25 amino acid region that binds to and stabilizes the GDP-bound conformation of Gαi, Gαt, and/or Gαo. The GPR–Gα complex is a target for nonreceptor guanine nucleotide exchange factors such as Ric-8A and GIV1/Girdin (Garcia-Marcos et al., 2011, Tall and Gilman, 2005, Thomas et al., 2008, Oner et al., 2012) and can function as a discrete signaling module. GPR proteins may also influence the dynamics of the activation–deactivation cycle for the classical heterotrimeric G-protein.

A number of biochemical- and structural-based approaches have been utilized to define the GPR–G-protein interaction including yeast two-hybrid assays, nucleotide binding assays, GST pull-down assays, coimmunoprecipitation, and surface plasmon resonance (Adhikari and Sprang, 2003, Bernard et al., 2001, Bosch et al., 2011, Cao et al., 2004, Giguere et al., 2011, Kimple et al., 2001, Kimple et al., 2004, Natochin et al., 2000, Peterson et al., 2000, Peterson et al., 2002, Tall and Gilman, 2005, Willard et al., 2007, Willard et al., 2006). While many basic aspects of the biochemistry of the interaction of GPR proteins with purified Gα are generally understood, the dynamics of this signaling complex and its regulation within the cell remain undefined.

Resonance energy transfer, in particular bioluminescence resonance energy transfer (BRET), allows analysis of protein–protein interactions in live cells in real time. The enzymatic oxidation of substrates such as coelentrazine by Renilla luciferase and subsequent nonradiative emission can excite suitable acceptors such as yellow fluorescent protein (YFP) if the donor and acceptor are in close proximity (< 100 Å). There are many excellent detailed descriptions of the BRET platform for measuring protein interaction dynamics (Bacart et al., 2008, Hamdan et al., 2006, Pfleger et al., 2006). This chapter focuses specifically on the use of this platform to study the interaction of GPR proteins with Gα in intact cells and the regulation of this interaction by a G-protein-coupled receptor. We initially focused on AGS3 and AGS4 as two subtypes of Group II AGS proteins (Oner et al., 2010a, Oner et al., 2010b) and we recently reported a subsequent study on the GPR protein RGS14 (Vellano, Maher, Hepler, & Blumer, 2011). AGS3 (650 amino acids) has four GPR motifs and a clearly defined series of tetratricopeptide repeats in the amino terminal half of the protein. AGS4 (160 amino acids) has three GPR motifs without any defined regulatory domains in the region upstream of the GPR repeats.

Section snippets

Generation of Probes for Measurement of GPR–Gαi Interaction in Cells by BRET

  • 1.

    AGS3 (650 aa), AGS3 short cDNA (166 aa), and AGS4 (160 aa) were tagged at the carboxyl terminus by insertion of the cDNAs into humanized phRluc-N vectors (PerkinElmer #6310220) and pVenus-N or pEYFPN1 vectors (Fig. 9.1B).

  • 2.

    AGS3-Q/A and AGS4-Q/A were inserted into humanized phRluc-N vectors (PerkinElmer #6310220) and pVenus-N or pEYFPN1 vectors. Each construct contains a single amino acid substitution in each GPR motif (AGS3: GPR1-Q488A, GPR2-Q541A, GPR3-Q589A, GPR4-Q623A; AGS4: GPR1-Q80A,

Expression of Donor and Acceptor in Cells

  • 1.

    The human embryonic kidney 293 cell line (HEK-293) (ATCC #CRL-1573) was maintained in Dulbecco's minimal essential medium (high glucose, without phenol red) supplemented with 5% fetal bovine serum, 2 mM glutamine, 100 units/ml of penicillin, and 100 mg/ml of streptomycin. Cells were grown in a humidified incubator in the presence of 5% CO2 at 37 °C.

  • 2.

    HEK-293 cells were seeded at a density of 8 × 105 cells/well on 6-well plates in 2 ml medium and cultured overnight at 37 °C.

  • 3.

    Cells were then transiently

Analysis of GPR–Gαi1 Interaction by BRET

  • 1.

    48 h after transfection, the culture medium was removed and cells were washed once and harvested with 800 μl/well of Tyrode's solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 0.37 mM NaH2PO4, 24 mM NaHCO3, 10 mM HEPES, pH 7.4, and 0.1% glucose, w/v).

  • 2.

    Cells were counted and the volume adjusted such that 100 μl contained 1 × 105 cells.

  • 3.

    Cells (1 × 105 in 100 μl) were distributed in triplicate into gray 96 HS OptiPlates (PerkinElmer Life Sciences, #6005330). The cell number and volume can be adjusted as

Regulation of the GPR–Gαi Complex by a G-protein-Coupled Receptor

  • 1.

    Cells are transfected as above with phRluc::AGS4 or phRluc::AGS3 and pcDNA3::Gαi1-YFP with and without pcDNA3::α2A/D-AR (200–1000 μg). The amounts of receptor plasmid used for transfection are dependent upon the desired levels of receptor expression. Receptor expression should be determined in each experiment by radioligand binding.

  • 2.

    48 h after transfection, cells are harvested and aliquoted into gray 96-well plates as described above.

  • 3.

    Total fluorescence is measured (excitation 485 nm, emission 535 

Analysis of GPR–Gαi1 Interaction in Subcellular Fractions by BRET

BRET can also be used to define the subcellular location of the GPR–Gαi1 interaction. In these experiments, cells are lysed in hypotonic buffer and fractionated by centrifugation followed by fluorescence and luminescence measurements to determine BRET. These measurements also allow for the determination of the relative distribution of donor and acceptor as well as the relative BRET signals in the two subcellular fractions (Fig. 9.5). Coulon and colleagues also reported the visualization of

Summary

The BRET system described herein provides a strong platform to define the regulation of GPR–Gαi complexes in intact cells. The BRET signal is robust and regulated by a G-protein-coupled receptor. It is not clear if the G-protein-coupled receptor is directly coupling to the AGS-GPR–Gαi in a manner analogous to receptor coupling to Gαβγ or whether it exists as part of a larger signaling complex. AGS3 and AGS4 are both stabilized at the cell cortex upon coexpression with Gαi (Oner et al., 2010b,

Acknowledgments

This work was supported by grants from the National Institutes of Health [NS24821 (SML), DA025896 (SML), and GM086510 (JBB)]. We thank Heather Bainbridge and Ellen Maher for technical assistance. The authors thank Dr. Atsushi Miyawaki (Laboratory for Cell Function and Dynamics, Saitama, Japan) for pNPY-Venus-N1 and Dr. Michel Bouvier, Department of Biochemistry, Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, Québec, Canada for the Rluc plasmids. The authors

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