Gγ identity dictates efficacy of Gßγ signaling and macrophage migration.

G protein ßγ subunit (Gßγ) is a major signal transducer and controls processes ranging from cell migration to gene transcription. Despite having significant subtype heterogeneity and exhibiting diverse cell- and tissue-specific expression levels, Gßγ is often considered a unified signaling entity with a defined functionality. However, the molecular and mechanistic basis of Gßγ's signaling specificity is unknown. Here, we demonstrate that Gγ subunits, bearing the sole plasma membrane (PM)-anchoring motif, control the PM affinity of Gßγ and thereby differentially modulate Gßγ effector signaling in a Gγ-specific manner. Both Gßγ signaling activity and the migration rate of macrophages are strongly dependent on the PM affinity of Gγ. We also found that the type of C-terminal prenylation and five to six pre-CaaX motif residues at the PM-interacting region of Gγ control the PM affinity of Gßγ. We further show that the overall PM affinity of the Gßγ pool of a cell type is a strong predictor of its Gßγ signaling-activation efficacy. A kinetic model encompassing multiple Gγ types and parameterized for empirical Gßγ behaviors not only recapitulated experimentally observed signaling of Gßγ, but also suggested a Gγ-dependent, active-inactive conformational switch for the PM-bound Gßγ, regulating effector signaling. Overall, our results unveil crucial aspects of signaling and cell migration regulation by Gγ type-specific PM affinities of Gßγ.

Measurement of GPCR-G protein activity in living cells.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors in eukaryotic genomes. They control a variety of cellular and physiological processes such as hormone secretion and heart rate, and therefore are associated with a majority of pathological conditions including cancer and heart diseases. Currently established assays to measure ligand-induced activation of GPCRs and G proteins possess limitations such as being time consuming, indirect, and expensive. Thus, an efficient method to measure GPCR-G protein activation is required to identify novel pharmacological modulators to control them and gain insights about molecular underpinnings of the associated pathways. Activation of GPCRs induces dissociation of G protein heterotrimers to form GαGTP and free Gßγ. Free Gßγ subunits have been shown to translocate reversibly from the plasma membrane to internal membranes. Gßγ translocation therefore represents the GPCR-G protein activation, and thus, imaging of this process can be used to quantify the kinetics and magnitude of the pathway activation-deactivation in real time in living cells. The objective of this chapter is to elaborate the protocols of (i) generation and optimization of the required sensor constructs; (ii) development of cell culture, transient transfection, imaging, and optogenetic procedures; (iii) imaging and data analysis methods; and (iv) stable cell line generation, pertaining to this assay to measure GPCR-G protein activation.

Two independent but synchronized Gßγ subunit-controlled pathways are essential for trailing-edge retraction during macrophage migration.

Chemokine-induced directional cell migration is a universal cellular mechanism and plays crucial roles in numerous biological processes, including embryonic development, immune system function, and tissue remodeling and regeneration. During the migration of a stationary cell, the cell polarizes, forms lamellipodia at the leading edge (LE), and triggers the concurrent retraction of the trailing edge (TE). During cell migration governed by inhibitory G protein (Gi)-coupled receptors (GPCRs), G protein ßγ (Gßγ) subunits control the LE signaling. Interestingly, TE retraction has been linked to the activation of the small GTPase Ras homolog family member A (RhoA) by the Gα12/13 pathway. However, it is not clear how the activation of Gi-coupled GPCRs at the LE orchestrates the TE retraction in RAW264.7 macrophages. Here, using an optogenetic approach involving an opsin to activate the Gi pathway in defined subcellular regions of RAW cells, we show that in addition to their LE activities, free Gßγ subunits also govern TE retraction by operating two independent, yet synchronized, pathways. The first pathway involves RhoA activation, which prevents dephosphorylation of the myosin light chain, allowing actomyosin contractility to proceed. The second pathway activates phospholipase Cß and induces myosin light chain phosphorylation to enhance actomyosin contractility through increasing cytosolic calcium. We further show that both of these pathways are essential, and inhibition of either one is sufficient to abolish the Gi-coupled GPCR-governed TE retraction and subsequent migration of RAW cells.

Reversible G Protein ßγ9 Distribution-Based Assay Reveals Molecular Underpinnings in Subcellular, Single-Cell, and Multicellular GPCR and G Protein Activity.

Current assays to measure the activation of G protein coupled receptors (GPCRs) and G proteins are time-consuming, indirect, and expensive. Therefore, an efficient method which directly measures the ability of a ligand to govern GPCR-G protein interactions can help to understand the molecular underpinnings of the associated signaling. A live cell imaging-based approach is presented here to directly measure ligand-induced GPCR and G protein activity in real time. The number of active GPCRs governs G protein heterotrimer (αßγ) dissociation, thereby controlling the concentration of free ßγ subunits. The described γ9 assay measures the GPCR activation-induced extent of the reversible ßγ9 subunit exchange between the plasma membrane (PM) and internal membranes (IMs). Confocal microscopy-based γ9 assay quantitatively determines the concentration dependency of ligands on GPCR activation. Demonstrating the high-throughput screening (HTS) adaptability, the γ9 assay performed using an imaging plate reader measures the ligand-induced GPCR activation. This suggests that the γ9 assay can be employed to screen libraries of compounds for their ability to activate GPCRs. Together with subcellular optogenetics, the spatiotemporal sensitivity of the γ9 assay permits experimental determination of the limits of spatially restricted activation of GPCRs and G proteins in subcellular regions of single cells. This assay works effectively for GPCRs coupled to αi/o and αs heterotrimers, including light-sensitive GPCRs. In addition, computational modeling of experimental data from the assay is used to decipher intricate molecular details of the GPCR-G protein activation process. Overall, the γ9 assay provides a robust strategy for quantitative as well as qualitative determination of GPCR and G protein function on a single-cell, multicell, and subcellular level. This assay not only provides information about the inner workings of the signaling pathway, but it also strengthens GPCR deorphanization as well as drug discovery efforts.