Sharon L. Campbell

Heterotrimeric G proteins (Gα, Gβ and Gγ) act downstream of G-protein-coupled receptors (GPCRs) to mediate signaling pathways that regulate various physiological processes and human disease conditions. While human Gαi and its yeast homolog Gpa1 were previously postulated to function as intracellular pH sensors, the pH–sensing capabilities of Gαi and the underlying mechanism remain to be established. Our research shows that variations in pH significantly affect the structure and stability of Gαi-GDP. Specifically, at the lower end of the physiological pH range, the protein undergoes an order-to-disorder transition due to the loss of electrostatic interactions within the Gαi Switch regions, resulting in a reduction in agonist-mediated Gαi-Gβγ release. Further, we identified key residues within the Gαi Switch regions that form the pH–sensing network. Mutation of these residues in Gαi gives rise to ‘low pH mimetics’ that abolish pH-dependent thermostability changes and reduce Gαi-Gβγ release. Overall, our findings suggest that pH-sensitive structural changes in Gαi impact the agonist-mediated dissociation of Gβγ, which is essential for proper signaling.

C-di-GAMP was first identified in bacteria to promote colonization, while mammalian 2′3′-cGAMP is synthesized by cGAS to activate STING for innate immune stimulation. However, 2′3′-cGAMP function beyond innate immunity remains elusive. Here, we report that 2′3′-cGAMP promotes cell migration independent of innate immunity. 2′3′-cGAMP interactome analysis identifies the small GTPase Rab18 as a 2′3′-cGAMP binding partner and effector in cell migration control. Mechanistically, 2′3′-cGAMP binds Rab18 to facilitate GTP loading and subsequent Rab18 activation, which further promotes FosB transcription in facilitating cell migration. Induced synthesis of endogenous 2′3′-cGAMP by intrabreast tumor bacterium S. aureus infection or low-dose doxorubicin treatment facilitates cell migration depending on the cGAS/cGAMP/Rab18/FosB signaling. We find that lovastatin induces Rab18 deprenylation that abolishes 2′3′-cGAMP recognition therefore suppressing cell migration. Together, our study reveals a previously unidentified 2′3′-cGAMP function in cell migration control via the 2′3′-cGAMP/Rab18/FosB signaling that provides additional insights into clinical applications of 2′3′-cGAMP.

Heterotrimeric G proteins (Gα, Gβ and Gγ) act downstream of G-protein-coupled receptors (GPCRs) to mediate signaling pathways that regulate various physiological processes and human disease conditions. Previously, human Gαi and its yeast homolog Gpa1 have been reported to function as intracellular pH sensors, yet the pH sensing capabilities of Gαi and the underlying mechanism remain to be established. Herein, we identify a pH sensing network within Gαi, and evaluate the consequences of pH modulation on the structure and stability of the G-protein. We find that changes over the physiological pH range significantly alter the structure and stability of Gαi-GDP, with the protein undergoing a disorder-to-order transition as the pH is raised from 6.8 to 7.5. Further, we find that modulation of intracellular pH in HEK293 cells regulates Gαi-Gβγ release. Identification of key residues in the pH-sensing network allowed the generation of low pH mimetics that attenuate Gαi-Gβγ release. Our findings, taken together, indicate that pH-dependent structural changes in Gαi alter the agonist-mediated Gβγ dissociation necessary for proper signaling.

The ability of cells and tissues to respond differentially to mechanical forces applied in distinct directions is mediated by the ability of load-bearing proteins to preferentially maintain physical linkages in certain directions. However, the molecular basis and biological consequences of directional force-sensitive binding remain unclear. Vinculin (Vcn) is a load-bearing linker protein that exhibits directional catch bonding due to interactions between the Vcn tail domain (Vt) and filamentous (F)-actin. We developed a computational approach to predict Vcn residues involved in directional catch bonding and produced a set of associated Vcn variants with unaltered Vt structure, actin binding, or phospholipid interactions. Incorporation of the variants did not affect Vcn activation but reduced Vcn loading and altered exchange dynamics, consistent with the loss of directional catch bonding. Expression of Vcn variants perturbed the coordination of subcellular structures and cell migration, establishing key cellular functions for Vcn directional catch bonding.

Structure factors and crystal information

Heterotrimeric guanine nucleotide–binding proteins (G proteins) that function as molecular switches for cellular growth and metabolism are activated by GTP and inactivated by GTP hydrolysis. In uveal melanoma, a conserved glutamine residue critical for GTP hydrolysis in the G protein α subunit is often mutated in Gα _q or Gα ₁₁ to either leucine or proline. In contrast, other glutamine mutations or mutations in other Gα subtypes are rare. To uncover the mechanism of the genetic selection and the functional role of this glutamine residue, we analyzed all possible substitutions of this residue in multiple Gα isoforms. Through cell-based measurements of activity, we showed that some mutants were further activated and inactivated by G protein–coupled receptors. Through biochemical, molecular dynamics, and nuclear magnetic resonance–based structural studies, we showed that the Gα mutants were functionally distinct and conformationally diverse, despite their shared inability to hydrolyze GTP. Thus, the catalytic glutamine residue contributes to functions beyond GTP hydrolysis, and these functions include subtype-specific, allosteric modulation of receptor-mediated subunit dissociation. We conclude that G proteins do not function as simple on-off switches. Rather, signaling emerges from an ensemble of active states, a subset of which are favored in disease and may be uniquely responsive to receptor-directed ligands.

The ability of cells and tissues to differentially resist or adapt to mechanical forces applied in distinct directions is mediated by the ability of load-bearing proteins to preferentially maintain physical linkages in certain directions. However, the molecular basis and biological consequences of directional force-sensitive binding are unclear. Vinculin (Vcn) is a load-bearing linker protein that exhibits directional catch bonding due to interactions between the Vcn tail domain (Vt) and filamentous (F)-actin. We developed a computational approach to predict Vcn residues involved in directional catch bonding and produced a set of associated Vcn variants with unaltered Vt structure, actin binding, or phospholipid interactions. Incorporation of these variants into Vcn biosensors did not perturb Vcn conformation, but reduced Vcn loading consistent with loss of directional catch bonding. Expression of Vcn variants perturbed the coalignment of FAs and F-actin and directed cell migration, establishing key cellular functions for Vcn directional catch bonding.