Identification of G Protein α_i Signaling Partners by Proximity Labeling Reveals a Network of Interactions that Includes PDZ-RhoGEF

Abstract

G protein-coupled receptors (GPCRs) that couple to the Gi family of G proteins are key regulators of cell and tissue physiology. Our recent work has discovered novel roles for Gαi in migration of neutrophils and fibrosarcoma cells downstream of activated chemoattractant receptors, but the molecular target(s) of Gαi in these processes remain to be identified. We adopted an intact cell proximity-based labeling approach using BioID2 coupled to tandem mass tag (TMT)-based quantitative proteomics to identify proteins that selectively interact with the GTP-bound form of Gαi1. Multiple targets were identified and validated for selective biotinylation by active BioID2-Gαi1(Q204L), suggesting a previously unappreciated network of interactions for activated Gαi proteins in intact cells. Extensive characterization of one candidate protein, PDZ-RhoGEF (PRG), revealed that active-Gαi1 strongly activates PRG. Strikingly, large differences in the ability of Gαi1, Gαi2, and Gαi3 isoforms to activate PRG were observed despite over 85% sequence identity. We also demonstrate the functional relevance of the interaction between active Gαi and PRG ex vivo in primary human neutrophils. Identification and characterization of new targets regulated by Gαi both individually and in networks provide insights that will aid not only in investigation of diverse functional roles of Gi-coupled GPCRs in biology but also in the development of novel therapeutic approaches.

Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs

“…As shown in Figure 2—figure supplement 2 , only over-expression of the Gα family members corresponding to their selective effectors (Rap1GAP for G i/o , p63-RhoGEF for G q/11 , and PDZ-RhoGEF for G 12/13 ) significantly increased the recruitment of the effector-RlucII to the plasma membrane. A recent study ( Chandan et al, 2021 ) showed that G i/o can also activate full length PDZ-RhoGEF. Although the domain of PDZ-RhoGEF required for this activation has not been identified yet, the selectivity of our PDZ-RhoGEF sensor for G 12/13 vs .…”

“…#: H-5645 Chemical compound, drug Ghrelin Tocris Cat. #: 1,463 Chemical compound, drug Glucagon ( Aittaleb et al, 2010 ; Aittaleb et al, 2011 ; Armando et al, 2014 ; Atwood et al, 2011 ; Avet et al, 2020 ; Azzi et al, 2003 ; Bowes et al, 2012 ; Brabet et al, 1998 ; Breton et al, 2010 ; Bünemann et al, 2003 ; Carr et al, 2014 ; Casey et al, 1990 ; Chandan et al, 2021 ; De Haan and Hirst, 2004 ; Devost et al, 2017 ; Fukuhara et al, 2001 ; Galandrin et al, 2007 ; Galés et al, 2005 ; Galés et al, 2006 ; Goupil et al, 2015 ; Hauser et al, 2017 ; Hauser et al, 2022 ; Hoffmann et al, 2005 ; Inoue et al, 2019 ; Jordan et al, 1999 ; Kawamata et al, 2003 ; Kenakin, 2019 ; Kim et al, 2002 ) Bachem Cat. #: H-6790 Chemical compound, drug Glucagon-like peptide-1 GLP-1 ( Bowes et al, 2012 ; Brabet et al, 1998 ; Breton et al, 2010 ; Bünemann et al, 2003 ; Carr et al, 2014 ; Casey et al, 1990 ; Chandan et al, 2021 ; De Haan and Hirst, 2004 ; …”

“…#: H-6790 Chemical compound, drug Glucagon-like peptide-1 GLP-1 ( Bowes et al, 2012 ; Brabet et al, 1998 ; Breton et al, 2010 ; Bünemann et al, 2003 ; Carr et al, 2014 ; Casey et al, 1990 ; Chandan et al, 2021 ; De Haan and Hirst, 2004 ; Devost et al, 2017 ; Fukuhara et al, 2001 ; Galandrin et al, 2007 ; Galés et al, 2005 ; Galés et al, 2006 ; Goupil et al, 2015 ; Hauser et al, 2017 ; Hauser et al, 2022 ; Hoffmann et al, 2005 ; Inoue et al, 2019 ; Jordan et al, 1999 ; Kawamata et al, 2003 ; Kenakin, 2019 ; Kim et al, 2002 ; Kobayashi et al, 2019 ; Laschet et al, 2019 ; Lavoie et al, 2002 ; Leduc et al, 2009 ; Leguay et al, 2021 ; Lu et al, 2014 ; Lutz et al, 2007 ) Bachem Cat. #: H-6795 Chemical compound, drug Glucagon-like peptide-2 GLP-2 ( Aittaleb et al, 2010 ; Aittaleb et al, 2011 ; Armando et al, 2014 ; Atwood et al, 2011 ; Avet et al, 2020 ; Azzi et al, 2003 ; Bowes et al, 2012 ; Brabet et al, 1998 ; Breton et al, 2010 ; Bünemann et al, 2003 ; Carr et al, 2014 ; Casey et al, 1990 …”

Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs

“…As shown in Figure S2, only over-expression of the Gα family members corresponding to their selective effectors (Rap1GAP for Gi/o, p63-RhoGEF for Gq/11 and PDZ-RhoGEF for G12/13) significantly increased the recruitment of the effector-RlucII to the plasma membrane. A recent study (Chandan N.R., 2021) showed that Gi/o can also activate full length PDZ-RhoGEF. Although the domain of PDZ-RhoGEF required for this activation has not been identified yet, the selectivity of our PDZ-RhoGEF sensor for G12/13 vs. all other G protein families most likely results from the fact that we used a truncated version of PDZ-RhoGEF that only contains the G12/13 binding domain and lacks the PDZ domain involved in protein-protein interaction, the actin-binding domain and the DH/PH domains involved in GEF activity and RhoA activation (Aittaleb, Boguth, & Tesmer, 2010).…”

Tandem Mass Tag Analysis of the Effect of the Anterior Cingulate Cortex in Nonerosive Reflux Disease Rats with Shugan Jiangni Hewei Granules Treatment

“…Moreover, adhesion receptors on adjacent cells can adapt to changes in the microenvironment because of the information conveyed by adhesion receptors on their cells. Adhesion receptors use their cytoplasmic domains (including FERM, proline-rich motifs, PDZ domains, and phosphorylation sites) to bind to appropriate specific interaction motifs in cytoplasmic proteins and transmit intercellular signals [30][31][32]. SJHG's therapy of VH may be based on the hypothesis that JAM-A expression is being increased.…”