Background: G protein-coupled receptors (GPCRs) adopt multiple structural conformations whose functional significance remains unclear. Results: Novel FRET-based sensors were developed to detect the stabilization of G protein-specific GPCR conformations in live cells. Conclusion: FRET measurements delineate distinct structural mechanisms for three 2-adrenergic receptor ligands. Significance: This study extensively validates a new technology that links GPCR conformation and function in live cells.
We achieved optofluidic protein lasing using genetically encoded fluorescent protein FRET pairs linked by length-tunable peptides. Up to 25-fold reduction in the donor laser emission was observed when the donor and the acceptor were brought to close proximity, as compared to only 17% reduction in the donor emission using the conventional FRET detection. Our work opens a door to a broad range of applications in studying protein-protein interactions and protein-drug interactions.
We achieve optofluidic lasers with a single molecular layer of gain, in which green fluorescent protein, dye-labeled bovine serum albumin, and dye-labeled DNA are respectively used as the gain medium and attached to the surface of a ring resonator via surface immobilization biochemical methods. It is estimated that the surface density of the gain molecules is on the order of 1012/cm2, sufficient for lasing under pulsed optical excitation. It is further shown that the optofluidic laser can be tuned by energy transfer mechanisms through biomolecular interactions. This work not only opens a door to novel photonic devices that can be controlled at the level of a single molecular layer, but also provides a promising sensing platform to analyze biochemical processes at the solid-liquid interface.
Natural killer (NK) cells safeguard against early tumor formation by destroying transformed target cells in a process referred to as NK immune surveillance. However, the immune escape mechanisms used by malignant brain tumors to subvert this innate type of immune surveillance remain unclear. Here we show that malignant glioma cells suppress NK immune surveillance by overexpressing the β-galactoside-binding lectin galectin-1. Conversely, galectin-1 deficient glioma cells could be eradicated by host NK cells prior to the initiation of an anti-tumor T-cell response. In vitro experiments demonstrated that galectin-1 deficient GL26-Cit glioma cells are ~3-fold more sensitive to NK-mediated tumor lysis that galectin-1 expressing cells. Our findings suggest that galectin-1 suppression in human glioma could improve patient survival by restoring NK immune surveillance that can eradicate glioma cells.
Although individual G-protein-coupled receptors (GPCRs) are known to activate one or more G proteins, the GPCR-G-protein interaction is viewed as a bimolecular event involving the formation of a ternary ligand-GPCR-G-protein complex. Here, we present evidence that individual GPCR-G-protein interactions can reinforce each other to enhance signaling through canonical downstream second messengers, a phenomenon we term "GPCR priming." Specifically, we find that the presence of noncognate Gq protein enhances cAMP stimulated by two Gs-coupled receptors, β2-adrenergic receptor (β2-AR) and D dopamine receptor (D-R). Reciprocally, Gs enhances IP through vasopressin receptor (V-R) but not α1 adrenergic receptor (α1-AR), suggesting that GPCR priming is a receptor-specific phenomenon. The C terminus of either the Gαs or Gαq subunit is sufficient to enhance Gα subunit activation and cAMP levels. Interaction of Gαs or Gαq C termini with the GPCR increases signaling potency, suggesting an altered GPCR conformation as the underlying basis for GPCR priming. We propose three parallel mechanisms involving () sequential G-protein interactions at the cognate site, () G-protein interactions at distinct allosteric and cognate sites on the GPCR, and () asymmetric GPCR dimers. GPCR priming suggests another layer of regulation in the classic GPCR ternary-complex model, with broad implications for the multiplicity inherent in signaling networks.
Despite the crowded nature of the cellular milieu, ligand–GPCR–G protein interactions are traditionally viewed as spatially and temporally isolated events. In contrast, recent reports suggest the spatial and temporal coupling of receptor–effector interactions, with the potential to diversify downstream responses. In this study, we combine protein engineering of GPCR–G protein interactions with affinity sequestration and photo-manipulation of the crucial Gα C terminus, to demonstrate the temporal coupling of cognate and non-cognate G protein interactions through priming of the GPCR conformation. We find that interactions of the Gαs and Gαq C termini with the β2-adrenergic receptor (β2-AR), targeted at the G-protein-binding site, enhance Gs activation and cyclic AMP levels. β2-AR–Gα C termini interactions alter receptor conformation, which persists for ~90 s following Gα C terminus dissociation. Non-cognate G-protein expression levels impact cognate signaling in cells. Our study demonstrates temporal allostery in GPCRs, with implications for the modulation of downstream responses through the canonical G-protein-binding interface.
Background: Protein kinase C (PKC) is a nodal regulator of cell signaling. Results: Multiple interactions between conserved regulatory domains in PKC synergistically stabilize a nanomolar affinity homodimer critical for cellular function. Conclusion: Homodimerization regulates the equilibrium between the auto-inhibited and active states of PKC␣. Significance: Multiplexed interactions between modular domains dictate PKC function.
FRET and BRET approaches are well established for detecting ligand induced GPCR-G protein interactions in cells. Currently, FRET/BRET assays rely on co-expression of GPCR and G protein, and hence depend on the stoichiometry and expression levels of the donor and acceptor probes. On the other hand, GPCR-G protein fusions have been used extensively to understand the selectivity of GPCR signaling pathways. However, the signaling properties of fusion proteins are not consistent across GPCRs. In this study, we describe and characterize novel sensors based on the Systematic Protein Affinity Strength Modulation (SPASM) technique. Sensors consist of a GPCR and G protein tethered by an ER/K linker flanked by FRET probes. SPASM sensors are tested for the β2-, α1-, and α2- adrenergic receptors, and adenosine type 1 receptor (A1R), tethered to Gαs-XL, Gαi2, or Gαq subunits. Agonist stimulation of β2-AR and α2-AR increases FRET signal comparable to co-expressed FRET/BRET sensors. SPASM sensors also retain signaling through the endogenous G protein milieu. Importantly, ER/K linker length systematically tunes the GPCR-G protein interaction, with consequent modulation of second messenger signaling for cognate interactions. SPASM GPCR sensors serve the dual purpose of detecting agonist-induced changes in GPCR-G protein interactions, and linking these changes to downstream signaling.
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