G protein-coupled receptor (GPCR) signaling is the primary method eukaryotes use to respond to specific cues in their environment. However, the relationship between stimulus and response for each GPCR is difficult to predict due to diversity in natural signal transduction architecture and expression. Using genome engineering in yeast, we constructed an insulated, modular GPCR signal transduction system to study how the response to stimuli can be predictably tuned using synthetic tools. We delineated the contributions of a minimal set of key components via computational and experimental refactoring, identifying simple design principles for rationally tuning the dose response. Using five different GPCRs, we demonstrate how this enables cells and consortia to be engineered to respond to desired concentrations of peptides, metabolites, and hormones relevant to human health. This work enables rational tuning of cell sensing while providing a framework to guide reprogramming of GPCR-based signaling in other systems.

Strategies aimed at mimicking or enhancing the action of the incretin hormone glucagon-like peptide 1 (GLP-1) therapeutically improve glucose-stimulated insulin secretion (GSIS); however, it is not clear whether GLP-1 directly drives insulin secretion in pancreatic islets. Here, we examined the mechanisms by which GLP-1 stimulates insulin secretion in mouse and human islets. We found that GLP-1 enhances GSIS at a half-maximal effective concentration of 0.4 pM. Moreover, we determined that GLP-1 activates PLC, which increases submembrane diacylglycerol and thereby activates PKC, resulting in membrane depolarization and increased action potential firing and subsequent stimulation of insulin secretion. The depolarizing effect of GLP-1 on electrical activity was mimicked by the PKC activator PMA, occurred without activation of PKA, and persisted in the presence of PKA inhibitors, the K_ATP channel blocker tolbutamide, and the L-type Ca²⁺ channel blocker isradipine; however, depolarization was abolished by lowering extracellular Na⁺. The PKC-dependent effect of GLP-1 on membrane potential and electrical activity was mediated by activation of Na⁺-permeable TRPM4 and TRPM5 channels by mobilization of intracellular Ca²⁺ from thapsigargin-sensitive Ca²⁺ stores. Concordantly, GLP-1 effects were negligible in Trpm4 or Trpm5 KO islets. These data provide important insight into the therapeutic action of GLP-1 and suggest that circulating levels of this hormone directly stimulate insulin secretion by β cells.

Ras is a prototypical small G-protein and a central regulator of growth, proliferation and differentiation processes in virtually every nucleated cell. As such, Ras becomes engaged and activated by multiple growth factors, mitogens, cytokines or adhesion receptors. Ras activation comes about by changes in the steady-state equilibrium between the inactive guanosine diphosphate (GDP)-bound and active guanosine triphosphate (GTP)-bound states of Ras, resulting in the mostly transient accumulation of Ras-GTP. Three decades of intense Ras research have disclosed various families of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) as the two principal regulatory elements of the Ras-GDP/GTP loading status. However, with the possible exception of the GEF Sos, we still have only a rudimentary knowledge of the precise role played by many GEF and GAP members in the signalling network upstream of Ras. As for GAPs, we even lack the fundamental understanding of whether they function as genuine signal transducers in the context of growth factor-elicited Ras activation or rather act as passive modulators of the Ras-GDP/GTP cycle. Here we sift through the large body of Ras literature and review the relevant data for understanding the participation and precise role played by GEFs and GAPs in the process of Ras activation.

The calcitonin gene-related peptide (CGRP) family of G protein-coupled receptors (GPCRs) is formed through the association of the calcitonin receptor-like receptor (CLR) and one of three receptor activity-modifying proteins (RAMPs). Binding of one of the three peptide ligands, CGRP, adrenomedullin (AM), and intermedin/adrenomedullin 2 (AM2), is well known to result in a Gα_s-mediated increase in cAMP. Here we used modified yeast strains that couple receptor activation to cell growth, via chimeric yeast/Gα subunits, and HEK-293 cells to characterize the effect of different RAMP and ligand combinations on this pathway. We not only demonstrate functional couplings to both Gα_s and Gα_q but also identify a Gα_i component to CLR signaling in both yeast and HEK-293 cells, which is absent in HEK-293S cells. We show that the CGRP family of receptors displays both ligand- and RAMP-dependent signaling bias among the Gα_s, Gα_i, and Gα_q/11 pathways. The results are discussed in the context of RAMP interactions probed through molecular modeling and molecular dynamics simulations of the RAMP-GPCR-G protein complexes. This study further highlights the importance of RAMPs to CLR pharmacology and to bias in general, as well as identifying the importance of choosing an appropriate model system for the study of GPCR pharmacology.

IP₃ receptor (IP₃R)-mediated Ca²⁺ transfer at the mitochondria-endoplasmic reticulum (ER) contact sites (MERCS) drives mitochondrial Ca²⁺ uptake and oxidative metabolism and is linked to different pathologies, including Parkinson’s disease (PD). The dependence of Ca²⁺ transfer efficiency on the ER-mitochondria distance remains unexplored. Employing molecular rulers that stabilize ER-mitochondrial distances at 5 nm resolution, and using genetically encoded Ca²⁺ indicators targeting the ER lumen and the sub-mitochondrial compartments, we now show that a distance of ~20 nm is optimal for Ca²⁺ transfer and mitochondrial oxidative metabolism due to enrichment of IP₃R at MERCS. In human iPSC-derived astrocytes from PD patients, 20 nm MERCS were specifically reduced, which correlated with a reduction of mitochondrial Ca²⁺ uptake. Stabilization of the ER-mitochondrial interaction at 20 nm, but not at 10 nm, fully rescued mitochondrial Ca²⁺ uptake in PD astrocytes. Our work determines with precision the optimal distance for Ca²⁺ flux between ER and mitochondria and suggests a new paradigm for fine control over mitochondrial function.