Molecular Mechanisms of G?i Signaling Selectivity

Abstract

Nearly all of human physiology is under the control of G protein-coupled receptors (GPCRs), which transmit signals from extracellular stimuli to affect intracellular processes. The heterotrimeric G proteins that these receptors are coupled to transduce signals from the GPCR and pass it on to intracellular effector proteins, which have diverse functions. The α subunit of heterotrimeric G proteins acts as a molecular switch, binding different guanine nucleotides which control its functional state. Sixteen human Gα subunits form four distinct families: Gαs, Gαi/o, Gαq/11, and Gα12/13. Each of these families are highly similar in sequence and function, resulting in highly unique signaling patterns between families. While seemingly functionally redundant, members of these families differ in tissue distribution and cellular function. The Gαi/o family consists of Gαi1-3, Gαo, GαT1-3, and Gαz. These subunits vary widely in tissue expression, but Gαi1-3 are expressed relatively ubiquitously. They are nearly identical in their canonical function: Gαi1, Gαi2, and Gαi3 equipotently inhibit the membrane enzyme adenylyl cyclase. They also have similar binding and hydrolysis rates of guanosine-5’-diphosphate (GDP) and guanosine-5’-triphosphate (GTP), respectively. Without downstream signaling partners which display specificity for interaction with Gαi subtypes, investigators have turned to studies in vivo to parse their functional differences. These studies have revealed important, non-overlapping roles for Gαi subtypes in different tissues and systems, but have not revealed any molecular details of the interactions responsible for such effects. Some differences in Gαi subtype interactions with other proteins have been demonstrated at the molecular level, but the mechanism for this selectivity is not well understood. Recently, our laboratory used proximity labeling proteomics to discover a novel effector of Gαi: PDZ-RhoGEF (PRG), a guanine nucleotide exchange factor for the monomeric G protein Rho. Remarkably, this downstream effector is activated strongly by Gαi1 and Gαi3, but activation by Gαi2 in cells is significantly weaker. Here, I outline our investigation into the molecular basis for this stark difference in selectivity of PRG for Gαi subtypes using Gαi1 and Gαi2. Using cell-based functional assays and molecular dynamics simulations, we demonstrated that nucleotide-dependent activation of PRG by Gαi1 is controlled by interactions at the interface of the two domains of Gα, the Ras-like domain (RLD) and the helical domain (HD). In particular, one amino acid in the Switch III loop of Gαi1, D229, makes an interaction with R144 in the helical domain, permitting an array of other interdomain interactions and stabilizing the Switch III loop. The corresponding residue of Gαi1 D229 is Gαi2 A230, which does not interact with the cognate arginine in the HD, and does not support these additional interdomain interactions. Substitution of the whole Gαi1 HD into Gαi2 also confers the ability to activate PRG in this manner. Finally, using unbiased proximity labeling in cells coupled to tandem mass spectrometry proteomics, we show that this mechanism of Switch III stabilization, which confers Gαi nucleotide-dependent interaction with and activation of PRG, also increases interaction of Gαi subtypes with other novel effector proteins. These results describe a novel mechanism which may extend to other Gα protein families, modulating their selectivity for effector proteins and determining their signaling. Elucidating such molecular processes is key to our understanding of the nature of G protein-effector interactions. This has clear implications for signaling downstream of all GPCRs, the most prevalent protein target for the treatment of human disease.