John R. Hepler

Cellular Regulation of RGS Proteins: Modulators and Integrators of G Protein Signaling

Regulators of G protein signaling (RGS) and RGS-like proteins are a family (>30 members) of highly diverse, multifunctional signaling proteins that bind directly to activated Gα subunits. Family members are defined by a shared RGS domain, which is responsible for Gα binding and markedly stimulates the GTPase activity of Gα subunits leading to their deactivation and termination of downstream signals. Although much has been learned in recent years about the biochemistry of RGS/Gα interactions, considerably less is known about the broader cellular roles and regulation of RGS proteins. Recent findings indicate that cellular mechanisms such as covalent modification, alternative gene splicing, and protein processing can dictate the activity and subcellular localization of RGS proteins. Many family members also directly link G proteins to a growing list of signaling proteins with diverse cellular roles. New findings indicate that RGS proteins act not as dedicated inhibitors but, rather, as tightly regulated modulators and integrators of G protein signaling. In some cases, RGS proteins modulate the lifetime and kinetics of both slow-acting (e.g., Ca2+ oscillations) and fast-acting (e.g., ion conductances, phototransduction) signaling responses. In other cases, RGS proteins integrate G proteins with signaling pathways linked to such diverse cellular responses as cell growth and differentiation, cell motility, and intracellular trafficking. These and other recent studies with animal model systems indicate that RGS proteins play important roles in both physiology and disease. Recognition of the central functions these proteins play in vital cellular processes has focused our attention on RGS proteins as exciting new candidates for therapeutic intervention and drug development.

Emerging roles for RGS proteins in cell signalling

Regulators of G-protein signalling (RGS proteins) are a family of highly diverse, multifunctional signalling proteins that share a conserved 120 amino acid domain (RGS domain). RGS domains bind directly to activated Galpha subunits and act as GTPase-activating proteins (GAPs) to attenuate and/or modulate hormone and neurotransmitter receptor-initiated signalling by both Galpha-GTP and Gbetagamma. Apart from this structural domain, which is shared by all known RGS proteins, these proteins differ widely in their overall size and amino acid identity and possess a remarkable variety of structural domains and motifs. These biochemical features impart signalling functions and/or enable RGS proteins to interact with a growing list of unexpected protein-binding partners with diverse cellular roles. New appreciation for the broader cellular functions of RGS proteins challenges established models of G-protein signalling and serves to identify these proteins as central participants in receptor signalling and cell physiology.

G Protein Selectivity Is a Determinant of RGS2 Function

RGS (regulator of Gprotein signaling) proteins are GTPase-activating proteins that attenuate signaling by heterotrimeric G proteins. Whether the biological functions of RGS proteins are governed by quantitative differences in GTPase-activating protein activity toward various classes of Gα subunits and how G protein selectivity is achieved by differences in RGS protein structure are largely unknown. Here we provide evidence indicating that the function of RGS2 is determined in part by differences in potency toward Gq versus Gi family members. RGS2 was 5-fold more potent than RGS4 as an inhibitor of Gq-stimulated phosphoinositide hydrolysis in vivo. In contrast, RGS4 was 8-fold more potent than RGS2 as an inhibitor of Gi-mediated signaling. RGS2 mutants were identified that display increased potency toward Gi family members without affecting potency toward Gq. These mutations and the structure of RGS4-Giα1 complexes suggest that RGS2-Giα interaction is unfavorable in part because of the geometry of the switch I binding pocket of RGS2 and a potential interaction between the α8-α9 loop of RGS2 and αA of Gi class α subunits. The results suggest that the function of RGS2 relative to other RGS family members is governed in part by quantitative differences in activity toward different classes of Gα subunits.

RGS4 Inhibits Signaling by Group I Metabotropic Glutamate Receptors

Metabotropic glutamate receptors (mGluRs) couple to heterotrimeric G-proteins and regulate cell excitability and synaptic transmission in the CNS. Considerable effort has been focused on understanding the cellular and biochemical mechanisms that underlie regulation of signaling by G-proteins and their linked receptors, including the mGluRs. Recent findings demonstrate that regulators of G-protein signaling (RGS) proteins act as effector antagonists and GTPase-activating proteins for Gα subunits to inhibit cellular responses by G-protein-coupled receptors. RGS4 blocks Gq activation of phospholipase Cβ and is expressed broadly in rat brain. The group I mGluRs (mGluRs 1 and 5) couple to Gq pathways to regulate several effectors in the CNS. We examined the capacity of RGS4 to regulate group I mGluR responses. InXenopus oocytes, purified RGS4 virtually abolishes the mGluR1a- and mGluR5a-mediated but not the inositol trisphospate-mediated activation of a calcium-dependent chloride current. Additionally, RGS4 markedly attenuates the mGluR5-mediated inhibition of potassium currents in hippocampal CA1 neurons. This inhibition is dose-dependent and occurs at concentrations that are virtually identical to those required for inhibition of phospholipase C activity in NG108–15 membranes and reconstituted systems using purified proteins. These findings demonstrate that RGS4 can modulate mGluR responses in neurons, and they highlight a previously unknown mechanism for regulation of G-protein-coupled receptor signaling in the CNS.

Activation of Protease-Activated Receptor-1 Triggers Astrogliosis after Brain Injury

We have studied the involvement of the thrombin receptor [protease-activated receptor-1 (PAR-1)] in astrogliosis, because extravasation of PAR-1 activators, such as thrombin, into brain parenchyma can occur after blood-brain barrier breakdown in a number of CNS disorders. PAR1-/- animals show a reduced astrocytic response to cortical stab wound, suggesting that PAR-1 activation plays a key role in astrogliosis associated with glial scar formation after brain injury. This interpretation is supported by the finding that the selective activation of PAR-1 in vivo induces astrogliosis. The mechanisms by which PAR-1 stimulates glial proliferation appear to be related to the ability of PAR-1 receptor signaling to induce sustained extracellular receptor kinase (ERK) activation. In contrast to the transient activation of ERK by cytokines and growth factors, PAR-1 stimulation induces a sustained ERK activation through its coupling to multiple G-protein-linked signaling pathways, including Rho kinase. This sustained ERK activation appears to regulate astrocytic cyclin D1 levels and astrocyte proliferation in vitro and in vivo. We propose that this PAR-1-mediated mechanism underlying astrocyte proliferation will operate whenever there is sufficient injury-induced blood-brain barrier breakdown to allow extravasation of PAR-1 activators.

Protease-activated receptor-1 in human brain: Localization and functional expression in astrocytes

Protease-activated receptor-1 (PAR1) is a G-protein coupled receptor that is proteolytically activated by blood-derived serine proteases. Although PAR1 is best known for its role in coagulation and hemostasis, recent findings demonstrate that PAR1 activation has actions in the central nervous system (CNS) apart from its role in the vasculature. Rodent studies have demonstrated that PAR1 is expressed throughout the brain on neurons and astrocytes. PAR1 activation in vitro and in vivo appears to influence neurodegeneration and neuroprotection in animal models of stroke and brain injury. Because of increasing evidence that PAR1 has important and diverse roles in the CNS, we explored the protein localization and function of PAR1 in human brain. PAR1 is most intensely expressed in astrocytes of white and gray matter and moderately expressed in neurons. PAR1 and GFAP co-localization demonstrates that PAR1 is expressed on the cell body and on astrocytic endfeet that invest capillaries. PAR1 activation in the U178MG human glioblastoma cell line increased PI hydrolysis and intracellular Ca(2+), indicating that PAR1 is functional in human glial-derived tumor cells. Primary cultures of human astrocytes and human glioblastoma cells respond to PAR1 activation by increasing intracellular Ca(2+). Together, these results demonstrate that PAR1 is expressed in human brain and functional in glial tumors and cultures derived from it. Because serine proteases may enter brain tissue and activate PAR1 when the blood brain barrier (BBB) breaks down, pharmacological manipulation of PAR1 signaling may provide a potential therapeutic target for neuroprotection in human neurological disorders.

RGS14 is a natural suppressor of both synaptic plasticity in CA2 neurons and hippocampal-based learning and memory

Learning and memory have been closely linked to strengthening of synaptic connections between neurons (i.e., synaptic plasticity) within the dentate gyrus (DG)–CA3–CA1 trisynaptic circuit of the hippocampus. Conspicuously absent from this circuit is area CA2, an intervening hippocampal region that is poorly understood. Schaffer collateral synapses on CA2 neurons are distinct from those on other hippocampal neurons in that they exhibit a perplexing lack of synaptic long-term potentiation (LTP). Here we demonstrate that the signaling protein RGS14 is highly enriched in CA2 pyramidal neurons and plays a role in suppression of both synaptic plasticity at these synapses and hippocampal-based learning and memory. RGS14 is a scaffolding protein that integrates G protein and H-Ras/ERK/MAP kinase signaling pathways, thereby making it well positioned to suppress plasticity in CA2 neurons. Supporting this idea, deletion of exons 2–7 of the RGS14 gene yields mice that lack RGS14 (RGS14-KO) and now express robust LTP at glutamatergic synapses in CA2 neurons with no impact on synaptic plasticity in CA1 neurons. Treatment of RGS14-deficient CA2 neurons with a specific MEK inhibitor blocked this LTP, suggesting a role for ERK/MAP kinase signaling pathways in this process. When tested behaviorally, RGS14-KO mice exhibited marked enhancement in spatial learning and in object recognition memory compared with their wild-type littermates, but showed no differences in their performance on tests of nonhippocampal-dependent behaviors. These results demonstrate that RGS14 is a key regulator of signaling pathways linking synaptic plasticity in CA2 pyramidal neurons to hippocampal-based learning and memory but distinct from the canonical DG–CA3–CA1 circuit.

RGS7 Is Palmitoylated and Exists as Biochemically Distinct Forms

Abstract: Regulator of G protein signaling (RGS) proteins are GTPase‐activating proteins that modulate neurotransmitter and G protein signaling. RGS7 and its binding partners Gα and Gβ5 are enriched in brain, but biochemical mechanisms governing RGS7/Gα/Gβ5 interactions and membrane association are poorly defined. We report that RGS7 exists as one cytosolic and three biochemically distinct membrane‐bound fractions (salt‐extractable, detergent‐extractable, and detergent‐insensitive) in brain. To define factors that determine RGS7 membrane attachment, we examined the biochemical properties of recombinant RGS7 and Gβ5 synthesized in Spodoptera frugiperda insect cells. We have found that membrane‐bound but not cytosolic RGS7 is covalently modified by the fatty acid palmitate. Gβ5 is not palmitoylated. Both unmodified (cytosolic) and palmitoylated (membrane‐derived) forms of RGS7, when complexed with Gβ5, are equally effective stimulators of Gαo GTPase activity, suggesting that palmitoylation does not prevent RGS7/Gαo interactions. The isolated core RGS domain of RGS7 selectively binds activated Gαi/o in brain extracts and is an effective stimulator of both Gαo and Gαi1 GTPase activities in vitro. In contrast, the RGS7/Gβ5 complex selectively interacts with Gαo only, suggesting that features outside the RGS domain and/or Gβ5 association dictate RGS7‐Gα interactions. These findings define previously unrecognized biochemical properties of RGS7, including the first demonstration that RGS7 is palmitoylated.

RGS14 is a bifunctional regulator of Gαi/o activity that exists in multiple populations in brain

Members of the regulators of G protein signaling (RGS) family modulate Gα‐directed signals as a result of the GTPase‐activating protein (GAP) activity of their conserved RGS domain. In addition to its RGS domain, RGS14 contains a Rap binding domain (RBD) and a GoLoco motif. To define the cellular and biochemical properties of RGS14 we utilized two different affinity purified antisera that specifically recognize recombinant and native RGS14. In brain, we observed two RGS14‐like immunoreactive bands of distinct size (60 kDa and 55 kDa). Both forms are present in brain cytosol and in two, biochemically distinct, membrane subpopulations: one detergent‐extractable and the other detergent‐insensitive. Recombinant RGS14 binds specifically to activated Gαi/o, but not Gαq/11, Gα12/13, or Gαs in brain membranes. In reconstitution studies, we found that RGS14 is a non‐selective GAP for Gαi1 and Gαo and that full‐length RGS14 is an approximately 10‐fold more potent stimulator of Gα GTPase activity than the RGS domain alone. In contrast, neither full‐length RGS14 nor the isolated RBD domain is a GAP for Rap1. RGS14 is also a highly selective guanine nucleotide dissociation inhibitor (GDI) for Gαi but not Gαo, and this activity is restricted to the C‐terminus containing the GoLoco domain. These findings highlight previously unknown biochemical properties of RGS14 in brain, and provide one of the first examples of an RGS protein that is a bifunctional regulator of Gα actions.

Plasmin potentiates synaptic N-methyl-D-aspartate receptor function in hippocampal neurons through activation of protease-activated receptor-1.

Protease-activated receptor-1 (PAR1) is activated by a number of serine proteases, including plasmin. Both PAR1 and plasminogen, the precursor of plasmin, are expressed in the central nervous system. In this study we examined the effects of plasmin in astrocyte and neuronal cultures as well as in hippocampal slices. We find that plasmin evokes an increase in both phosphoinositide hydrolysis (EC50 64 nm) and Fura-2/AM fluorescence (195 ± 6.7% above base line, EC50 65 nm) in cortical cultured murine astrocytes. Plasmin also activates extracellular signal-regulated kinase (ERK1/2) within cultured astrocytes. The plasmin-induced rise in intracellular Ca2+ concentration ([Ca2+]i) and the increase in phospho-ERK1/2 levels were diminished in PAR1-/- astrocytes and were blocked by 1 μm BMS-200261, a selective PAR1 antagonist. However, plasmin had no detectable effect on ERK1/2 or [Ca2+]i signaling in primary cultured hippocampal neurons or in CA1 pyramidal cells in hippocampal slices. Plasmin (100-200 nm) application potentiated the N-methyl-d-aspartate (NMDA) receptor-dependent component of miniature excitatory postsynaptic currents recorded from CA1 pyramidal neurons but had no effect on α-amino-3-hydroxy-5-methyl-4-isoxazole propionate- or γ-aminobutyric acid receptor-mediated synaptic currents. Plasmin also increased NMDA-induced whole cell receptor currents recorded from CA1 pyramidal cells (2.5 ± 0.3-fold potentiation over control). This effect was blocked by BMS-200261 (1 μm; 1.02 ± 0.09-fold potentiation over control). These data suggest that plasmin may serve as an endogenous PAR1 activator that can increase [Ca2+]i in astrocytes and potentiate NMDA receptor synaptic currents in CA1 pyramidal neurons.