Heterogeneity and dynamics of ERK and Akt activation by G protein-coupled receptors depend on the activated heterotrimeric G proteins

Kinases are fundamental regulators of cellular functions and play key roles in GPCR-mediated signaling pathways. Kinase activities are generally inferred from cell lysates, hiding the heterogeneity of the individual cellular responses to extracellular stimuli. Here, we study the dynamics and heterogeneity of ERK and Akt in genetically identical cells in response to activation of endogenously expressed GPCRs. We use kinase translocation reporters, highcontent imaging, automated segmentation and clustering methods to assess cell-to-cell signaling heterogeneity. We observed ligand-concentration dependent response kinetics to histamine, a2-adrenergic, and S1P receptor stimulation that varied between cells. By using G protein inhibitors, we observed that Gq mediated the ERK and Akt responses to histamine. In contrast, Gi was necessary for ERK and Akt activation in response to α2-adrenergic receptor activation. ERK and Akt were also strongly activated by S1P, showing high heterogeneity at the single cell level, especially for ERK. In all cases, the cellular heterogeneity was not explained by distinct pre-stimulation levels or saturation of the measured response. Cluster analysis of time-series derived from 68,000 cells obtained under the different conditions revealed several distinct populations of cells that display similar response dynamics. The single-cell ERK responses to histamine and brimonidine showed remarkably similar dynamics, despite the activation of different heterotrimeric G proteins. In contrast, the ERK response dynamics to S1P showed high heterogeneity, which was reduced by the inhibition of Gi. To conclude, we have set up an imaging and analysis strategy that reveals substantial cell-to-cell heterogeneity in kinase activity driven by GPCRs

Single-cell imaging of ERK and Akt activation dynamics and heterogeneity induced by G-protein-coupled receptors

Kinases play key roles in signaling networks that are activated by G-protein-coupled receptors (GPCRs). Kinase activities are generally inferred from cell lysates, hiding cell-to-cell variability. To study the dynamics and heterogeneity of ERK and Akt proteins, we employed high-content biosensor imaging with kinase translocation reporters.ThekinaseswereactivatedwithGPCRligands.Weobserved ligand concentration-dependent response kinetics to histamine, α2-adrenergic and S1P receptor stimulation. By using G-protein inhibitors, we observed that Gq mediated the ERK and Akt responses to histamine. In contrast,Giwas necessary forERKand Akt activationin response to α2-adrenergic receptor activation. ERK and Akt were also strongly activated by S1P, showing high heterogeneity at the single-cell level, especially for ERK. Cluster analysis of time series derived from 68,000 cells obtained under the different conditions revealed several distinct populations of cells that display similar response dynamics. ERKresponse dynamicstoS1Pshowedhighheterogeneity,whichwas reduced by the inhibition of Gi. To conclude, we have set up an imaging and analysis strategy that reveals substantial cell-to-cell heterogeneity in kinase activity driven by GPCRs

Mutational analysis and modeling of negative allosteric modulator binding sites in AMPA receptors

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) constitute a subclass of the ionotropic glutamate receptor superfamily, which functions as glutamate-gated cation channels to mediate the majority of excitatory neurotransmission in the central nervous system. AMPARs are therapeutic targets in a range of brain disorders associated with abnormal glutamate hyperactivity. Multiple classes of AMPAR inhibitors have been developed during the past decades, including competitive antagonists, ion channel blockers, and negative allosteric modulators (NAMs). At present, the NAM is the only class of AMPAR ligands that have been developed into safe and useful drugs in humans in the form of perampanel (Fycompa), which was recently approved for treatment of epilepsy. Compared with the detailed understanding of other AMPAR ligand classes, surprisingly little information has been available regarding the molecular mechanism of perampanel and other classes of NAMs at AMPARs; including the location and structure of NAM binding pockets in the receptor complex. However, structures of the AMPAR GluA2 in complex with NAMs were recently reported that unambiguously identified the NAM binding sites. In parallel with this work, our aim with the present study was to identify specific residues involved in the formation of the NAM binding site for three prototypical AMPAR NAMs. Hence, we have performed a mutational analysis of the AMPAR region that links the four extracellular ligand-binding domains to the central ion channel in the transmembrane domain region. Furthermore, we perform computational ligand docking of the NAMs into structural models of the homomeric GluA2 receptor and optimize side chain conformations around the NAMs to model how NAMs bind in this specific site. The new insights provide potentially valuable input for structure-based drug design of new NAMs. SIGNIFICANCE STATEMENT: The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are glutamate-gated ion channels that mediate the majority of excitatory neurotransmission in the brain. Negative allosteric modulators of AMPA receptors are considered to have significant therapeutic potential in diseases linked to glutamate hyperactivity. The present work employs mutational analysis and molecular modeling of the binding site for prototypical NAMs to provide new molecular insight into how NAMs interact with the AMPA receptor, which is of potential use for future design of new types of NAMs

Mutational Analysis and Modeling of Negative Allosteric Modulator Binding Sites in AMPA Receptors

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) constitute a subclass of the ionotropic glutamate receptor superfamily, which functions as glutamate-gated cation channels to mediate the majority of excitatory neurotransmission in the central nervous system. AMPARs are therapeutic targets in a range of brain disorders associated with abnormal glutamate hyperactivity. Multiple classes of AMPAR inhibitors have been developed during the past decades, including competitive antagonists, ion channel blockers, and negative allosteric modulators (NAMs). At present, the NAM is the only class of AMPAR ligands that have been developed into safe and useful drugs in humans in the form of perampanel (Fycompa), which was recently approved for treatment of epilepsy. Compared with the detailed understanding of other AMPAR ligand classes, surprisingly little information has been available regarding the molecular mechanism of perampanel and other classes of NAMs at AMPARs; including the location and structure of NAM binding pockets in the receptor complex. However, structures of the AMPAR GluA2 in complex with NAMs were recently reported that unambiguously identified the NAM binding sites. In parallel with this work, our aim with the present study was to identify specific residues involved in the formation of the NAM binding site for three prototypical AMPAR NAMs. Hence, we have performed a mutational analysis of the AMPAR region that links the four extracellular ligand-binding domains to the central ion channel in the transmembrane domain region. Furthermore, we perform computational ligand docking of the NAMs into structural models of the homomeric GluA2 receptor and optimize side chain conformations around the NAMs to model how NAMs bind in this specific site. The new insights provide potentially valuable input for structure-based drug design of new NAMs. SIGNIFICANCE STATEMENT: The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are glutamate-gated ion channels that mediate the majority of excitatory neurotransmission in the brain. Negative allosteric modulators of AMPA receptors are considered to have significant therapeutic potential in diseases linked to glutamate hyperactivity. The present work employs mutational analysis and molecular modeling of the binding site for prototypical NAMs to provide new molecular insight into how NAMs interact with the AMPA receptor, which is of potential use for future design of new types of NAMs