Heterogeneity of tethered agonist signaling in adhesion G protein-coupled receptors.

Adhesion G protein-coupled receptor (aGPCR) signaling influences development and homeostasis in a wide range of tissues. In the current model for aGPCR signaling, ligand binding liberates a conserved sequence that acts as an intramolecular, tethered agonist (TA), yet this model has not been evaluated systematically for all aGPCRs. Here, we assessed the TA-dependent activities of all 33 aGPCRs in a suite of transcriptional reporter, G protein activation, and ß-arrestin recruitment assays using a new fusion protein platform. Strikingly, only ∼50% of aGPCRs exhibited robust TA-dependent activation, and unlike other GPCR families, aGPCRs showed a notable preference for G12/13 signaling. AlphaFold2 predictions assessing TA engagement in the predicted intramolecular binding pocket aligned with the TA dependence of the cellular responses. This dataset provides a comprehensive resource to inform the investigation of all human aGPCRs and for targeting aGPCRs therapeutically.

The M3 Muscarinic Acetylcholine Receptor Can Signal through Multiple G Protein Families.

The M3 muscarinic acetylcholine receptor (M3R) is a G protein-coupled receptor (GPCR) that regulates important physiologic processes, including vascular tone, bronchoconstriction, and insulin secretion. It is expressed on a wide variety of cell types, including pancreatic beta, smooth muscle, neuronal, and immune cells. Agonist binding to the M3R is thought to initiate intracellular signaling events primarily through the heterotrimeric G protein Gq. However, reports differ on the ability of M3R to couple to other G proteins beyond Gq. Using members from the four primary G protein families (Gq, Gi, Gs, and G13) in radioligand binding, GTP turnover experiments, and cellular signaling assays, including live cell G protein dissociation and second messenger assessment of cAMP and inositol trisphosphate, we show that other G protein families, particularly Gi and Gs, can also interact with the human M3R. We further show that these interactions are productive as assessed by amplification of classic second messenger signaling events. Our findings demonstrate that the M3R is more promiscuous with respect to G protein interactions than previously appreciated. SIGNIFICANCE STATEMENT: The study reveals that the human M3 muscarinic acetylcholine receptor (M3R), known for its pivotal roles in diverse physiological processes, not only activates intracellular signaling via Gq as previously known but also functionally interacts with other G protein families such as Gi and Gs, expanding our understanding of its versatility in mediating cellular responses. These findings signify a broader and more complex regulatory network governed by M3R and have implications for therapeutic targeting.

A spatiotemporal Notch interaction map from plasma membrane to nucleus.

Notch signaling relies on ligand-induced proteolysis of the transmembrane receptor Notch to liberate a nuclear effector that drives cell fate decisions. Upon ligand binding, sequential cleavage of Notch by the transmembrane protease ADAM10 and the intracellular protease γ-secretase releases the Notch intracellular domain (NICD), which translocates to the nucleus and forms a complex that induces target gene transcription. To map the location and timing of the individual steps required for the proteolysis and movement of Notch from the plasma membrane to the nucleus, we used proximity labeling with quantitative, multiplexed mass spectrometry to monitor the interaction partners of endogenous NOTCH2 after ligand stimulation in the presence of a γ-secretase inhibitor and as a function of time after inhibitor removal. Our studies showed that γ-secretase-mediated cleavage of NOTCH2 occurred in an intracellular compartment and that formation of nuclear complexes and recruitment of chromatin-modifying enzymes occurred within 45 min of inhibitor washout. These findings provide a detailed spatiotemporal map tracking the path of Notch from the plasma membrane to the nucleus and identify signaling events that are potential targets for modulating Notch activity.

Tethered agonist activated ADGRF1 structure and signalling analysis reveal basis for G protein coupling.

Adhesion G Protein Coupled Receptors (aGPCRs) have evolved an activation mechanism to translate extracellular force into liberation of a tethered agonist (TA) to effect cell signalling. We report here that ADGRF1 can signal through all major G protein classes and identify the structural basis for a previously reported Gαq preference by cryo-EM. Our structure shows that Gαq preference in ADGRF1 may derive from tighter packing at the conserved F569 of the TA, altering contacts between TM helix I and VII, with a concurrent rearrangement of TM helix VII and helix VIII at the site of Gα recruitment. Mutational studies of the interface and of contact residues within the 7TM domain identify residues critical for signalling, and suggest that Gαs signalling is more sensitive to mutation of TA or binding site residues than Gαq. Our work advances the detailed molecular understanding of aGPCR TA activation, identifying features that potentially explain preferential signal modulation.

The structure of the yeast Ctf3 complex.

Kinetochores are the chromosomal attachment points for spindle microtubules. They are also signaling hubs that control major cell cycle transitions and coordinate chromosome folding. Most well-studied eukaryotes rely on a conserved set of factors, which are divided among two loosely-defined groups, for these functions. Outer kinetochore proteins contact microtubules or regulate this contact directly. Inner kinetochore proteins designate the kinetochore assembly site by recognizing a specialized nucleosome containing the H3 variant Cse4/CENP-A. We previously determined the structure, resolved by cryo-electron microscopy (cryo-EM), of the yeast Ctf19 complex (Ctf19c, homologous to the vertebrate CCAN), providing a high-resolution view of inner kinetochore architecture (Hinshaw and Harrison, 2019). We now extend these observations by reporting a near-atomic model of the Ctf3 complex, the outermost Ctf19c sub-assembly seen in our original cryo-EM density. The model is sufficiently well-determined by the new data to enable molecular interpretation of Ctf3 recruitment and function.