Ching-Kang Chen

Slowed recovery of rod photoresponse in mice lacking the GTPase accelerating protein RGS9-1.

Timely deactivation of the α-subunit of the rod G-protein transducin (Gαt) is essential for the temporal resolution of rod vision. Regulators of G-protein signalling (RGS) proteins accelerate hydrolysis of GTP by the α-subunits of heterotrimeric G proteins in vitro. Several retinal RGS proteins can act in vitro as GTPase accelerating proteins (GAP) for Gαt. Recent reconstitution experiments indicate that one of these, RGS9-1, may account for much of the Gαt GAP activity in rod outer segments (ROS). Here we report that ROS membranes from mice lacking RGS9-1 hydrolyse GTP more slowly than ROS membranes from control mice. The Gβ5-L protein that forms a complex with RGS9-1 (ref. 10) was absent from RGS9-/- retinas, although Gβ5-L messenger RNA was still present. The flash responses of RGS9-/- rods rose normally, but recovered much more slowly than normal. We conclude that RGS9-1, probably in a complex with Gβ5-L, is essential for acceleration of hydrolysis of GTP by Gαt and for normal recovery of the photoresponse.

Retinal remodeling triggered by photoreceptor degenerations

Many photoreceptor degenerations initially affect rods, secondarily leading to cone death. It has long been assumed that the surviving neural retina is largely resistant to this sensory deafferentation. New evidence from fast retinal degenerations reveals that subtle plasticities in neuronal form and connectivity emerge early in disease. By screening mature natural, transgenic, and knockout retinal degeneration models with computational molecular phenotyping, we have found an extended late phase of negative remodeling that radically changes retinal structure. Three major transformations emerge: 1) Müller cell hypertrophy and elaboration of a distal glial seal between retina and the choroid/retinal pigmented epithelium; 2) apparent neuronal migration along glial surfaces to ectopic sites; and 3) rewiring through evolution of complex neurite fascicles, new synaptic foci in the remnant inner nuclear layer, and new connections throughout the retina. Although some neurons die, survivors express molecular signatures characteristic of normal bipolar, amacrine, and ganglion cells. Remodeling in human and rodent retinas is independent of the initial molecular targets of retinal degenerations, including defects in the retinal pigmented epithelium, rhodopsin, or downstream phototransduction elements. Although remodeling may constrain therapeutic intervals for molecular, cellular, or bionic rescue, it suggests that the neural retina may be more plastic than previously believed. J. Comp. Neurol. 464:1–16, 2003.

RGS9 modulates dopamine signaling in the basal ganglia.

Regulators of G protein signaling (RGS) modulate heterotrimeric G proteins in part by serving as GTPase-activating proteins for Galpha subunits. We examined a role for RGS9-2, an RGS subtype highly enriched in striatum, in modulating dopamine D2 receptor function. Viral-mediated overexpression of RGS9-2 in rat nucleus accumbens (ventral striatum) reduced locomotor responses to cocaine (an indirect dopamine agonist) and to D2 but not to D1 receptor agonists. Conversely, RGS9 knockout mice showed heightened locomotor and rewarding responses to cocaine and related psychostimulants. In vitro expression of RGS9-2 in Xenopus oocytes accelerated the off-kinetics of D2 receptor-induced GIRK currents, consistent with the in vivo data. Finally, chronic cocaine exposure increased RGS9-2 levels in nucleus accumbens. Together, these data demonstrate a functional interaction between RGS9-2 and D2 receptor signaling and the behavioral actions of psychostimulants and suggest that psychostimulant induction of RGS9-2 represents a compensatory adaptation that diminishes drug responsiveness.

RGS expression rate-limits recovery of rod photoresponses.

Signaling through G protein-coupled receptors (GPCRs) underlies many cellular processes, yet it is not known which molecules determine the duration of signaling in intact cells. Two candidates are G protein-coupled receptor kinases (GRKs) and Regulators of G protein signaling (RGSs), deactivation enzymes for GPCRs and G proteins, respectively. Here we investigate whether GRK or RGS governs the overall rate of recovery of the light response in mammalian rod photoreceptors, a model system for studying GPCR signaling. We show that overexpression of rhodopsin kinase (GRK1) increases phosphorylation of the GPCR rhodopsin but has no effect on photoresponse recovery. In contrast, overexpression of the photoreceptor RGS complex (RGS9-1.Gbeta5L.R9AP) dramatically accelerates response recovery. Our results show that G protein deactivation is normally at least 2.5 times slower than rhodopsin deactivation, resolving a long-standing controversy concerning the mechanism underlying the recovery of rod visual transduction.

Evidence for two apoptotic pathways in light-induced retinal degeneration

Excessive phototransduction signaling is thought to be involved in light-induced and inherited retinal degeneration. Using knockout mice with defects in rhodopsin shut-off and transducin signaling, we show that two different pathways of photoreceptor-cell apoptosis are induced by light. Bright light induces apoptosis that is independent of transducin and accompanied by induction of the transcription factor AP-1. By contrast, low light induces an apoptotic pathway that requires transducin. We also provide evidence that additional genetic factors regulate sensitivity to light-induced damage. Our use of defined mouse mutants resolves some of the complexity underlying the mechanisms that regulate susceptibility to retinal degeneration.

Instability of GGL domain-containing RGS proteins in mice lacking the G protein β-subunit Gβ5

RGS (regulator of G protein signaling) proteins containing the G protein γ-like (GGL) domain (RGS6, RGS7, RGS9, and RGS11) interact with the fifth member of the G protein β-subunit family, Gβ5. This interaction is necessary for the stability of both the RGS protein and for Gβ5. Consistent with this notion, we have found that elevation of RGS9-1 mRNA levels by transgene expression does not increase RGS9-1 protein level in the retina, suggesting that Gβ5 levels may be limiting. To examine further the interactions of Gβ5 and the GGL domain-containing RGS proteins, we inactivated the Gβ5 gene. We found that the levels of GGL domain-containing RGS proteins in retinas and in striatum are eliminated or reduced drastically, whereas the levels of Gγ2 and RGS4 proteins remain normal in the absence of Gβ5. The homozygous Gβ5 knockout (Gβ5–/–) mice derived from heterozygous knockout mating are runty and exhibit a high preweaning mortality rate. We concluded that complex formation between GGL domain-containing RGS proteins and the Gβ5 protein is necessary to maintain their mutual stability in vivo. Furthermore, in the absence of Gβ5 and all four RGS proteins that form protein complexes with Gβ5, the animals that survive into adulthood are viable and have no gross defects in brain or retinal morphology.

D2 Dopamine Receptors Colocalize Regulator of G-Protein Signaling 9-2 (RGS9-2) via the RGS9 DEP Domain, and RGS9 Knock-Out Mice Develop Dyskinesias Associated with Dopamine Pathways

Regulator of G-protein signaling 9-2 (RGS9-2), a member of the RGS family of Gα GTPase accelerating proteins, is expressed specifically in the striatum, which participates in antipsychotic-induced tardive dyskinesia and in levodopa-induced dyskinesia. We report that RGS9 knock-out mice develop abnormal involuntary movements when inhibition of dopaminergic transmission is followed by activation of D2-like dopamine receptors (DRs). These abnormal movements resemble drug-induced dyskinesia more closely than other rodent models. Recordings from striatal neurons of these mice establish that activation of D2-like DRs abnormally inhibits glutamate-elicited currents. We show that RGS9-2, via its DEP domain (for Disheveled, EGL-10, Pleckstrin homology), colocalizes with D2DRs when coexpressed in mammalian cells. Recordings from oocytes coexpressing D2DR or the m2 muscarinic receptor and G-protein-gated inward rectifier potassium channels show that RGS9-2, via its DEP domain, preferentially accelerates the termination of D2DR signals. Thus, alterations in RGS9-2 may be a key factor in the pathway leading from D2DRs to the side effects associated with the treatment both of psychoses and Parkinsons disease.

Photoreceptor cGMP Phosphodiesterase δ Subunit (PDEδ) Functions as a Prenyl-binding Protein

Bovine PDEδ was originally copurified with rod cGMP phosphodiesterase (PDE) and shown to interact with prenylated, carboxymethylated C-terminal Cys residues. Other studies showed that PDEδ can interact with several small GTPases including Rab13, Ras, Rap, and Rho6, all of which are prenylated, as well as the N-terminal portion of retinitis pigmentosa GTPase regulator and Arl2/Arl3, which are not prenylated. We show by immunocytochemistry with a PDEδ-specific antibody that PDEδ is present in rods and cones. We find by yeast two-hybrid screening with a PDEδ bait that it can interact with farnesylated rhodopsin kinase (GRK1) and that prenylation is essential for this interaction. In vitro binding assays indicate that both recombinant farnesylated GRK1 and geranylgeranylated GRK7 co-precipitate with a glutathione S-transferase-PDEδ fusion protein. Using fluorescence resonance energy transfer techniques exploiting the intrinsic tryptophan fluorescence of PDEδ and dansylated prenyl cysteines as fluorescent ligands, we show that PDEδ specifically binds geranylgeranyl and farnesyl moieties with a Kd of 19.06 and 0.70 μm, respectively. Our experiments establish that PDEδ functions as a prenyl-binding protein interacting with multiple prenylated proteins.

Amino-terminal Cysteine Residues of RGS16 Are Required for Palmitoylation and Modulation of Gi- and Gq-mediated Signaling

RGS proteins (Regulators ofG protein Signaling) are a recently discovered family of proteins that accelerate the GTPase activity of heterotrimeric G protein α subunits of the i, q, and 12 classes. The proteins share a homologous core domain but have divergent amino-terminal sequences that are the site of palmitoylation for RGS-GAIP and RGS4. We investigated the function of palmitoylation for RGS16, which shares conserved amino-terminal cysteines with RGS4 and RGS5. Mutation of cysteine residues at residues 2 and 12 blocked the incorporation of [3H]palmitate into RGS16 in metabolic labeling studies of transfected cells or into purified RGS proteins in a cell-free palmitoylation assay. The purified RGS16 proteins with the cysteine mutations were still able to act as GTPase-activating protein for Giα. Inhibition or a decrease in palmitoylation did not significantly change the amount of protein that was membrane-associated. However, palmitoylation-defective RGS16 mutants demonstrated impaired ability to inhibit both Gi- and Gq-linked signaling pathways when expressed in HEK293T cells. These findings suggest that the amino-terminal region of RGS16 may affect the affinity of these proteins for Gα subunits in vivo or that palmitoylation localizes the RGS protein in close proximity to Gα subunits on cellular membranes.

Modules in the photoreceptor RGS9-1•Gβ5L GTPase-accelerating protein complex control effector coupling, GTPase acceleration, protein folding, and stability

RGS (regulators of Gprotein signaling) proteins regulate G protein signaling by accelerating GTP hydrolysis, but little is known about regulation of GTPase-accelerating protein (GAP) activities or roles of domains and subunits outside the catalytic cores. RGS9-1 is the GAP required for rapid recovery of light responses in vertebrate photoreceptors and the only mammalian RGS protein with a defined physiological function. It belongs to an RGS subfamily whose members have multiple domains, including Gγ-like domains that bind Gβ 5 proteins. Members of this subfamily play important roles in neuronal signaling. Within the GAP complex organized around the RGS domain of RGS9-1, we have identified a functional role for the Gγ-like-Gβ 5L complex in regulation of GAP activity by an effector subunit, cGMP phosphodiesterase γ and in protein folding and stability of RGS9-1. The C-terminal domain of RGS9-1 also plays a major role in conferring effector stimulation. The sequence of the RGS domain determines whether the sign of the effector effect will be positive or negative. These roles were observed in vitro using full-length proteins or fragments for RGS9-1, RGS7, Gβ 5S, and Gβ 5L. The dependence of RGS9-1 on Gβ 5 co-expression for folding, stability, and function has been confirmed in vivo using transgenicXenopus laevis. These results reveal how multiple domains and regulatory polypeptides work together to fine tune Gtα inactivation.