The effector enzyme regulates the duration of G protein signaling in vertebrate photoreceptors by increasing the affinity between transducin and RGS protein

The photoreceptor-specific G protein transducin acts as a molecular switch, stimulating the activity of its downstream effector in its GTP-bound form and inactivating the effector upon GTP hydrolysis. This activity makes the rate of transducin GTPase an essential factor in determining the duration of photoresponse in vertebrate rods and cones. In photoreceptors, the slow intrinsic rate of transducin GTPase is accelerated by the complex of the ninth member of the regulators of G protein signaling family with the long splice variant of type 5 G protein beta subunit (RGS9.Gbeta5L). However, physiologically rapid GTPase is observed only when transducin forms a complex with its effector, the gamma subunit of cGMP phosphodiesterase (PDEgamma). In this study, we addressed the mechanism by which PDEgamma regulates the rate of transducin GTPase. We found that RGS9.Gbeta5L alone has a significant ability to activate transducin GTPase, but its affinity for transducin is low. PDEgamma acts by enhancing the affinity between activated transducin and RGS9.Gbeta5L by more than 15-fold, which is evident both from kinetic measurements of transducin GTPase rate and from protein binding assays with immobilized transducin. Furthermore, our data indicate that a single RGS9.Gbeta5L molecule is capable of accelerating the GTPase activity of approximately 100 transducin molecules/s. This rate is faster than the rates reported previously for any RGS protein and is sufficient for timely photoreceptor recovery in both rod and cone photoreceptors.     

Kinetic mechanism of RGS9-1 potentiation by R9AP

The duration of the photoreceptor's response to a light stimulus determines the speed at which an animal adjusts to ever-changing conditions of the visual environment. One critical component which regulates the photoresponse duration on the molecular level is the complex between the ninth member of the regulators of G protein signaling family (RGS9-1) and its partner, type 5 G protein beta-subunit (Gbeta5L). RGS9-1.Gbeta5L is responsible for the activation of the GTPase activity of the photoreceptor-specific G protein, transducin. Importantly, this function of RGS9-1.Gbeta5L is regulated by its membrane anchor, R9AP, which drastically potentiates the ability of RGS9-1.Gbeta5L to activate transducin GTPase. In this study, we address the kinetic mechanism of R9AP action and find that it consists primarily of a direct increase in the RGS9-1.Gbeta5L activity. We further showed that the binding site for RGS9-1.Gbeta5L is located within the N-terminal putative trihelical domain of R9AP, and even though this domain is sufficient for binding, it takes the entire R9AP molecule to potentiate the activity of RGS9-1.Gbeta5L. The mechanism revealed in this study is different from and complements another well-established mechanism of regulation of RGS9-1.Gbeta5L by the effector enzyme, cGMP phosphodiesterase, which is based entirely on the enhancement in the affinity between RGS9-1.Gbeta5L and transducin. Together, these mechanisms ensure timely transducin inactivation in the course of the photoresponse, a requisite for normal vision.     

Specific binding of RGS9-Gbeta 5L to protein anchor in photoreceptor membranes greatly enhances its catalytic activity

The complex between the short splice variant of the ninth member of the RGS protein family and the long splice variant of type 5 G protein beta subunit (RGS9-Gbeta5L) plays a critical role in regulating the duration of the light response in vertebrate photoreceptors by activating the GTPase activity of the photoreceptor-specific G protein, transducin. RGS9-Gbeta5L is tightly associated with the membranes of photoreceptor outer segments; however, the nature of this association remains unknown. Here we demonstrate that rod outer segment membranes contain a limited number of sites for high affinity RGS9-Gbeta5L binding, which are highly sensitive to proteolysis. In membranes isolated from bovine rod outer segments, all of these sites are occupied by the endogenous RGS9-Gbeta5L, which prevents the binding of exogenous recombinant RGS9-Gbeta5L to these sites. However, treating membranes with urea or high pH buffers causes either removal or denaturation of the endogenous RGS9-Gbeta5L, allowing for high affinity binding of recombinant RGS9-Gbeta5L to these sites. This binding results in a striking approximately 70-fold increase in the RGS9-Gbeta5L ability to activate transducin GTPase. The DEP (disheveled/EGL-10/pleckstrin) domain of RGS9 plays a crucial role in the RGS9-Gbeta5L membrane attachment, as evident from the analysis of membrane-binding properties of deletion mutants lacking either N- or C-terminal parts of the RGS9 molecule. Our data indicate that specific association of RGS9-Gbeta5L with photoreceptor disc membranes serves not only as a means of targeting it to an appropriate subcellular compartment but also serves as an important determinant of its catalytic activity.     

Absence of the RGS9.Gbeta5 GTPase-activating complex in photoreceptors of the R9AP knockout mouse

Timely termination of the light response in retinal photoreceptors requires rapid inactivation of the G protein transducin. This is achieved through the stimulation of transducin GTPase activity by the complex of the ninth member of the regulator of G protein signaling protein family (RGS9) with type 5 G protein beta subunit (Gbeta5). RGS9.Gbeta5 is anchored to photoreceptor disc membranes by the transmembrane protein, R9AP. In this study, we analyzed visual signaling in the rods of R9AP knockout mice. We found that light responses from R9AP knockout rods were very slow to recover and were indistinguishable from those of RGS9 or Gbeta5 knockout rods. This effect was a consequence of the complete absence of any detectable RGS9 from the retinas of R9AP knockout mice. On the other hand, the level of RGS9 mRNA was not affected by the knockout. These data indicate that in photoreceptors R9AP determines the stability of the RGS9.Gbeta5 complex, and therefore all three proteins, RGS9, Gbeta5 , and R9AP, are obligate members of the regulatory complex that speeds the rate at which transducin hydrolyzes GTP.     

Noncatalytic domains of RGS9-1.Gbeta 5L play a decisive role in establishing its substrate specificity

The complex between the photoreceptor-specific regulator of G protein signaling (RGS) protein, RGS9-1, and type 5 G protein beta-subunit, Gbeta5L, regulates the duration of the cellular response to light by stimulating the GTPase activity of G protein, transducin. An important property of RGS9-1.Gbeta5L is that it interacts specifically with transducin bound to its effector, cGMP phosphodiesterase, rather than with transducin alone. The minimal structure within the RGS9-1.Gbeta5L complex capable of activating transducin GTPase is the catalytic domain of RGS9. This domain itself is also able to discriminate between free and effector-bound transducin but to a lesser degree than RGS9-1.Gbeta5L. The goal of this study was to determine whether other, noncatalytic domains of RGS9-1.Gbeta5L enhance the intrinsic specificity of the catalytic domain or whether they set the specificity of RGS9-1.Gbeta5L regardless of the specificity of its catalytic domain. We found that a double L353E/R360P amino acid substitution reversed the specificity of the recombinant catalytic domain but did not reverse the specificity of RGS9-1.Gbeta5L. However, the degree of discrimination between free and effector-bound transducin was reduced. Therefore, noncatalytic domains of RGS9-1.Gbeta5L play a decisive role in establishing its substrate specificity, yet the high degree of this specificity observed under physiological conditions requires an additional contribution from the catalytic domain.     

Modulation of transducin GTPase activity by chimeric RGS16 and RGS9 regulators of G protein signaling and the effector molecule

RGS9, a member of the family of regulators of G protein signaling (RGS), serves as a GTPase-activating protein (GAP) for the transducin alpha-subunit (Gtalpha) in the vertebrate visual transduction cascade. The GAP activity of RGS9 is uniquely potentiated by the gamma-subunit of the effector enzyme, cGMP-phosphodiesterase (Pgamma). In contrast, Pgamma attenuates the GAP effects of several other RGS proteins, including RGS16. We demonstrate here that the Pgamma subunit exerts its effects on the GTPase activity of the Gtalpha-RGS complex via the C-terminal domain, Pgamma-63-87. The structural determinants that control the direction of Pgamma effects on the RGS-Gtalpha system are localized within the RGS domains. The addition of Pgamma caused an increase in the maximal stimulation of Gtalpha GTPase activity by RGS9d without affecting the EC50 value. Modulation of Gtalpha GTPase activity by chimeric RGS16 and RGS9 proteins and Pgamma has been investigated. This analysis suggests that in addition to the differences in primary structures, the overall conformations of the RGS fold in RGS9 and RGS16 are likely to be responsible for the opposite effects of Pgamma on the RGS9 and RGS16 GAP activity. The RGS9 alpha3-alpha5 region constituted the minimal insertion of the RGS9 domain into RGS16 that reversed the inhibitory effect of Pgamma. A model of the RGS9 complex with Gtalpha shows the alpha3-alpha5 helices in RGS9 facing the proximate Pgamma binding site on Gtalpha. Our results and this model demonstrate that the mechanism of potentiation of RGS9 GAP activity by Pgamma involves a more rigid stabilization of the Gtalpha switch regions when Gtalpha is bound to both RGS9 and Pgamma.     

Signal-dependent translocation of transducin, RGS9-1-Gbeta5L complex, and arrestin to detergent-resistant membrane rafts in photoreceptors

Many lines of evidence show that membranes contain microdomains, "lipid rafts", that are different from the rest of the membrane in specific lipid and protein composition. In several biological systems, they were shown to be necessary for trafficking and signal transduction. Here, we investigate if lipid rafts have a role in the regulation of the G protein-mediated pathway underlying vertebrate phototransduction. Photoreceptor membranes contain detergent-resistant membrane (DRM) rafts. Rhodopsin and cGMP phosphodiesterase are found in raft and nonraft portions of the membrane; guanylate cyclase is found exclusively in the raft. Distribution of these proteins does not change in the light or dark. In contrast, the G protein transducin, the RGS9-1-Gbeta5L complex, and the p44 isoform of arrestin undergo dramatic translocation to the raft upon illumination. Phosphorylation of RGS9-1 occurs exclusively in the raft. GTPgammaS or pertussis toxin prevent the light-mediated translocation of transducin and RGS9-1, whereas AlF(minus sign)(4) causes both proteins to move to the raft in the dark. This shows that the Galphat-RGS9-1-Gbeta5L complex has the highest affinity to rafts in the transition state of the GTPase. GTPgammaS binds to transducin at a significantly slower rate in the raft, indicating that this translocation results in a reduced rhodopsin-transducin coupling. Thus, an external signal can rearrange components of a G protein pathway in specific domains of the cell membrane, changing its signaling properties. These findings could reveal a novel mechanism utilized by the cells for regulation of G protein-mediated signal transduction.     

Phosphorylation of the regulator of G protein signaling RGS9-1 by protein kinase A is a potential mechanism of light- and Ca2+-mediated regulation of G protein function in photoreceptors

In vertebrate photoreceptors, photoexcited rhodopsin interacts with the G protein transducin, causing it to bind GTP and stimulate the enzyme cGMP phosphodiesterase. The rapid termination of the active state of this pathway is dependent upon a photoreceptor-specific regulator of G protein signaling RGS9-1 that serves as a GTPase activating protein (GAP) for transducin. Here, we show that, in preparations of photoreceptor outer segments (OS), RGS9-1 is readily phosphorylated by an endogenous Ser/Thr protein kinase. Protein kinase C and MAP kinase inhibitors reduced labeling by about 30%, while CDK5 and CaMK II inhibitors had no effect. cAMP-dependent protein kinase (PKA) inhibitor H89 reduced RGS9-1 labeling by more than 90%, while dibutyryl-cAMP stimulated it 3-fold, implicating PKA as the major kinase responsible for RGS9-1 phosphorylation in OS. RGS9-1 belongs to an RGS subfamily also including RGS6, RGS7, and RGS11, which exist as heterodimers with the G protein beta subunit Gbeta5. Phosphorylated RGS9-1 remains associated with Gbeta5L, a photoreceptor-specific splice form, which itself was not phosphorylated. RGS9-1 immunoprecipitated from OS was in vitro phosphorylated by exogenous PKA. The PKA catalytic subunit could also phosphorylate recombinant RGS9-1, and mutational analysis localized phosphorylation sites to Ser(427) and Ser(428). Substitution of these residues for Glu, to mimic phosphorylation, resulted in a reduction of the GAP activity of RGS9-1. In OS, RGS9-1 phosphorylation required the presence of free Ca(2+) ions and was inhibited by light, suggesting that RGS9-1 phosphorylation could be one of the mechanisms mediating a stronger photoresponse in dark-adapted cells.     

Dependence of RGS9-1 membrane attachment on its C-terminal tail.

RGS9-1 is a GTPase-accelerating protein (GAP) required for rapid recovery of the light response in vertebrate rod and cone photoreceptors. Similar to its phototransduction partners transducin (G(t)) and cGMP phosphodiesterase, it is a peripheral protein of the disc membranes, but it binds membranes much more tightly. It lacks the lipid modifications found on G(t) and cGMP phosphodiesterase, and the mechanism for membrane attachment is unknown. We have used limited proteolysis to generate a fragment of RGS9-1 that is readily removed from membranes under moderate salt conditions. Immunoblots reveal that this soluble fragment lacks a 3-kDa fragment from the C-terminal domain, the only domain within RGS9-1 that differs in sequence from the brain-specific isoform RGS9-2. Recombinant fragments of RGS9-1 with or without the partner subunit G beta(5L) were constructed with or without the C-terminal domain. Those lacking the C-terminal domain bound to photoreceptor membranes much less tightly than those containing it. Removal by urea of G beta(5L) from endogenous or recombinant RGS9-1 bound to rod outer segment membranes left RGS9-1 tightly membrane-bound, and recombinant RGS9-1 was urea-soluble in the absence of membranes. Thus the C-terminal domain of RGS9-1 is critical for membrane binding, whereas G beta(5L) does not play an important role in membrane attachment.     

The alpha-helical domain of Galphat determines specific interaction with regulator of G protein signaling 9

RGS proteins (regulators of G protein signaling) are potent accelerators of the intrinsic GTPase activity of G protein alpha subunits (GAPs), thus controlling the response kinetics of a variety of cell signaling processes. Most RGS domains that have been studied have relatively little GTPase activating specificity especially for G proteins within the Gi subfamily. Retinal RGS9 is unique in its ability to act synergistically with a downstream effector cGMP phosphodiesterase to stimulate the GTPase activity of the alpha subunit of transducin, Galphat. Here we report another unique property of RGS9: high specificity for Galphat. The core (RGS) domain of RGS9 (RGS9) stimulates Galphat GTPase activity by 10-fold and Galphai1 GTPase activity by only 2-fold at a concentration of 10 microM. Using chimeric Galphat/Galphai1 subunits we demonstrated that the alpha-helical domain of Galphat imparts this specificity. The functional effects of RGS9 were well correlated with its affinity for activated Galpha subunits as measured by a change in fluorescence of a mutant Galphat (Chi6b) selectively labeled at Cys-210. Kd values for RGS9 complexes with Galphat and Galphai1 calculated from the direct binding and competition experiments were 185 nM and 2 microM, respectively. The gamma subunit of phosphodiesterase increases the GAP activity of RGS9. We demonstrate that this is because of the ability of Pgamma to increase the affinity of RGS9 for Galphat. A distinct, nonoverlapping pattern of RGS and Pgamma interaction with Galphat suggests a unique mechanism of effector-mediated GAP function of the RGS9.     

Activation of RGS9-1GTPase acceleration by its membrane anchor,R9AP

The GTPase-accelerating protein (GAP) complex RGS9-1.G beta(5) plays an important role in the kinetics of light responses by accelerating the GTP hydrolysis of G alpha(t) in vertebrate photoreceptors. Much, but not all, of this complex is tethered to disk membranes by the transmembrane protein R9AP. To determine the effect of the R9AP membrane complex on GAP activity, we purified recombinant R9AP and reconstituted it into lipid vesicles along with the photon receptor rhodopsin. Full-length RGS9-1.G beta(5) bound to R9AP-containing vesicles with high affinity (K(d) < 10 nm), but constructs lacking the DEP (dishevelled/EGL-10/pleckstrin) domain bound with much lower affinity, and binding of those lacking the entire N-terminal domain (i.e. the dishevelled/EGL-10/pleckstrin domain plus intervening domain) was not detectable. Formation of the membrane-bound complex with R9AP increased RGS9-1 GAP activity by a factor of 4. Vesicle titrations revealed that on the time scale of phototransduction, the entire reaction sequence from GTP uptake to GAP-catalyzed hydrolysis is a membrane-delimited process, and exchange of G alpha(t) between membrane surfaces is much slower than hydrolysis. Because in rod cells different pools exist of RGS9-1.G beta(5) that are either associated with R9AP or not, regulation of the association between R9AP and RGS9-1.G beta(5) represents a potential mechanism for the regulation of recovery kinetics.     

RGS9 knockout causes a short delay in light responses of ON-bipolar cells

RGS9 and R9AP are components of the photoreceptor-specific GTPase activating complex responsible for rapid inactivation of the G protein, transducin, in the course of photoresponse recovery from excitation. The amount of this complex in photoreceptors is strictly dependent on the expression level of R9AP; consequently, the knockouts of either RGS9 or R9AP cause comparable delays in photoresponse recovery. While RGS9 is believed to be present only in rods and cones, R9AP is also expressed in dendritic tips of ON-bipolar cells, which receive synaptic inputs from photoreceptors. Recent studies demonstrated that knockouts of R9AP and its binding partner in ON-bipolar cells, RGS11, cause a small delay in ON-bipolar cell light responses manifested as a delayed onset of electroretinography b-waves. This led the authors to suggest that R9AP and RGS11 participate in regulating the kinetics of light responses in these cells. Here we report the surprising finding that a nearly identical b-wave delay is observed in RGS9 knockout mice. Given the exclusive localization of RGS9 in photoreceptors, this result argues for a presynaptic origin of the b-wave delay in this case and perhaps in the case of the R9AP knockout as well, since R9AP is expressed in both photoreceptors and ON-bipolar cells. We also conducted a detailed analysis of the b-wave rising phase kinetics in both knockout animal types and found that, despite a delayed b-wave onset, the slope of the light response is unaffected or increased, dependent on the light stimulus intensity. This result is inconsistent with a slowdown of response propagation in ON-bipolar cells caused by the R9AP knockout, further arguing against the postsynaptic nature of the delayed b-wave phenotype in RGS9 and R9AP knockout mice.     

Translational control by RGS2

The regulator of G protein signaling (RGS) proteins are a family of guanosine triphosphatase (GTPase)–accelerating proteins. We have discovered a novel function for RGS2 in the control of protein synthesis. RGS2 was found to bind to eIF2Bϵ (eukaryotic initiation factor 2B ϵ subunit) and inhibit the translation of messenger RNA (mRNA) into new protein. This effect was not observed for other RGS proteins tested. This novel function of RGS2 is distinct from its ability to regulate G protein–mediated signals and maps to a stretch of 37 amino acid residues within its conserved RGS domain. Moreover, RGS2 was capable of interfering with the eIF2–eIF2B GTPase cycle, which is a requisite step for the initiation of mRNA translation. Collectively, this study has identified a novel role for RGS2 in the control of protein synthesis that is independent of its established RGS domain function.     

Activation of the regulator of G protein signaling 14-Gαi1-GDP signaling complex is regulated by resistance to inhibitors of cholinesterase-8A

RGS14 is a brain scaffolding protein that integrates G protein and MAP kinase signaling pathways. Like other RGS proteins, RGS14 is a GTPase activating protein (GAP) that terminates Gαi/o signaling. Unlike other RGS proteins, RGS14 also contains a G protein regulatory (also known as GoLoco) domain that binds Gαi1/3-GDP in cells and in vitro. Here we report that Ric-8A, a nonreceptor guanine nucleotide exchange factor (GEF), functionally interacts with the RGS14-Gαi1-GDP signaling complex to regulate its activation state. RGS14 and Ric-8A are recruited from the cytosol to the plasma membrane in the presence of coexpressed Gαi1 in cells, suggesting formation of a functional protein complex with Gαi1. Consistent with this idea, Ric-8A stimulates dissociation of the RGS14-Gαi1-GDP complex in cells and in vitro using purified proteins. Purified Ric-8A stimulates dissociation of the RGS14-Gαi1-GDP complex to form a stable Ric-8A-Gαi complex in the absence of GTP. In the presence of an activating nucleotide, Ric-8A interacts with the RGS14-Gαi1-GDP complex to stimulate both the steady-state GTPase activity of Gαi1 and binding of GTP to Gαi1. However, sufficiently high concentrations of RGS14 competitively reverse these stimulatory effects of Ric-8A on Gαi1 nucleotide binding and GTPase activity. This observation correlates with findings that show RGS14 and Ric-8A share an overlapping binding region within the last 11 amino acids of Gαi1. As further evidence that these proteins are functionally linked, native RGS14 and Ric-8A coexist within the same hippocampal neurons. These findings demonstrate that RGS14 is a newly appreciated integrator of unconventional Ric-8A and Gαi1 signaling.     

Modules in the photoreceptor RGS9-1.Gbeta 5L GTPase-accelerating protein complex control effector coupling, GTPase acceleration, protein folding, and stability

RGS (regulators of G protein 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 G(gamma)-like domains that bind G(beta)(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 G(gamma)-like-G(beta)(5L) complex in regulation of GAP activity by an effector subunit, cGMP phosphodiesterase gamma 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, G(beta)(5S), and G(beta)(5L). The dependence of RGS9-1 on G(beta)(5) co-expression for folding, stability, and function has been confirmed in vivo using transgenic Xenopus laevis. These results reveal how multiple domains and regulatory polypeptides work together to fine tune G(talpha) inactivation.     

RGS6, RGS7, RGS9, and RGS11 stimulate GTPase activity of Gi family G-proteins with differential selectivity and maximal activity

Regulator of G-protein signaling (RGS) proteins are GTPase activating proteins (GAPs) of heterotrimeric G-proteins that alter the amplitude and kinetics of receptor-promoted signaling. In this study we defined the G-protein alpha-subunit selectivity of purified Sf9 cell-derived R7 proteins, a subfamily of RGS proteins (RGS6, -7, -9, and -11) containing a Ggamma-like (GGL) domain that mediates dimeric interaction with Gbeta(5). Gbeta(5)/R7 dimers stimulated steady state GTPase activity of Galpha-subunits of the G(i) family, but not of Galpha(q) or Galpha(11), when added to proteoliposomes containing M2 or M1 muscarinic receptor-coupled G-protein heterotrimers. Concentration effect curves of the Gbeta(5)/R7 proteins revealed differences in potencies and efficacies toward Galpha-subunits of the G(i) family. Although all four Gbeta(5)/R7 proteins exhibited similar potencies toward Galpha(o), Gbeta(5)/RGS9 and Gbeta(5)/RGS11 were more potent GAPs of Galpha(i1), Galpha(i2), and Galpha(i3) than were Gbeta(5)/RGS6 and Gbeta(5)/RGS7. The maximal GAP activity exhibited by Gbeta(5)/RGS11 was 2- to 4-fold higher than that of Gbeta(5)/RGS7 and Gbeta(5)/RGS9, with Gbeta(5)/RGS6 exhibiting an intermediate maximal GAP activity. Moreover, the less efficacious Gbeta(5)/RGS7 and Gbeta(5)/RGS9 inhibited Gbeta(5)/RGS11-stimulated GTPase activity of Galpha(o). Therefore, R7 family RGS proteins are G(i) family-selective GAPs with potentially important differences in activities.     

Palmitoylation of a conserved cysteine in the regulator of G protein signaling (RGS) domain modulates the GTPase-activating activity of RGS4 and RGS10

RGS4 and RGS10 expressed in Sf9 cells are palmitoylated at a conserved Cys residue (Cys(95) in RGS4, Cys(66) in RGS10) in the regulator of G protein signaling (RGS) domain that is also autopalmitoylated when the purified proteins are incubated with palmitoyl-CoA. RGS4 also autopalmitoylates at a previously identified cellular palmitoylation site, either Cys(2) or Cys(12). The C2A/C12A mutation essentially eliminates both autopalmitoylation and cellular [(3)H]palmitate labeling of Cys(95). Membrane-bound RGS4 is palmitoylated both at Cys(95) and Cys(2/12), but cytosolic RGS4 is not palmitoylated. RGS4 and RGS10 are GTPase-activating proteins (GAPs) for the G(i) and G(q) families of G proteins. Palmitoylation of Cys(95) on RGS4 or Cys(66) on RGS10 inhibits GAP activity 80-100% toward either Galpha(i) or Galpha(z) in a single-turnover, solution-based assay. In contrast, when GAP activity was assayed as acceleration of steady-state GTPase in receptor-G protein proteoliposomes, palmitoylation of RGS10 potentiated GAP activity >/=20-fold. Palmitoylation near the N terminus of C95V RGS4 did not alter GAP activity toward soluble Galpha(z) and increased G(z) GAP activity about 2-fold in the vesicle-based assay. Dual palmitoylation of wild-type RGS4 remained inhibitory. RGS protein palmitoylation is thus multi-site, complex in its control, and either inhibitory or stimulatory depending on the RGS protein and its sites of palmitoylation.     

G protein selectivity is a determinant of RGS2 function

RGS (regulator of G protein 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 Galpha 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 G(q) versus G(i) family members. RGS2 was 5-fold more potent than RGS4 as an inhibitor of G(q)-stimulated phosphoinositide hydrolysis in vivo. In contrast, RGS4 was 8-fold more potent than RGS2 as an inhibitor of G(i)-mediated signaling. RGS2 mutants were identified that display increased potency toward G(i) family members without affecting potency toward G(q). These mutations and the structure of RGS4-G(i)alpha(1) complexes suggest that RGS2-G(i)alpha interaction is unfavorable in part because of the geometry of the switch I binding pocket of RGS2 and a potential interaction between the alpha8-alpha9 loop of RGS2 and alphaA of G(i) class alpha 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 Galpha subunits.     

N-terminal half of the cGMP phosphodiesterase gamma-subunit contributes to stabilization of the GTPase-accelerating protein complex

In the visual signal terminating transition state, the cyclic GMP phosphodiesterase (PDE6) inhibitory γ-subunit (PDEγ) stimulates GTPase activity of the α-subunit of transducin (αt) by enhancing the interaction between αt and its regulator of G protein signaling (RGS9), which is constitutively bound to the type 5 G protein β-subunit (β5). Although it is known from a crystal structure of partial molecules that the PDEγ C terminus contacts with both αt and RGS9, contributions from the intrinsically disordered PDEγ N-terminal half remain unclear. In this study, we were able to investigate this issue using a photolabel transfer strategy that allows for mapping the interface of full-length proteins. We observed label transfer from PDEγ N-terminal positions 50, 30, and 16 to RGS9·β5 in the GTPase-accelerating protein (GAP) complex composed of PDEγ·αt·RGS9·β5. In support of a direct PDEγ N-terminal interaction with RGS9·β5, the PDEγ N-terminal peptide PDEγ(1-61) abolished label transfer to RGS9·β5, and another N-terminal peptide, PDEγ(10-30), disassembled the GAP complex in label transfer and pulldown experiments. Furthermore, we determined that the PDEγ C-terminal interaction with αt was enhanced whereas the N-terminal interaction was weakened upon changing the αt conformation from the signaling state to the transition state. This "rearrangement" of PDEγ domain interactions with αt appears to facilitate the interaction of the PDEγ N-terminal half with RGS9·β5 and hence its contribution to optimal stabilization of the GAP complex.     

Identification of protein kinase C isozymes responsible for the phosphorylation of photoreceptor-specific RGS9-1 at Ser475

Inactivation of the visual G-protein transducin by GTP hydrolysis is regulated by the GTPase-accelerating protein (GAP) RGS9-1. Regulation of RGS9-1 itself is poorly understood, but we found previously that it is subject to a light- and Ca(2+)-sensitive phosphorylation on Ser(475). Because there are much higher RGS9-1 levels in cones than in rods, we investigated whether Ser(475) is phosphorylated in rods using Coneless mice and found that both the phosphorylation and its regulation by light occur in rods. Therefore, we used rod outer segments as the starting material for the purification of RGS9-1 kinase activity. Two major peaks of activity corresponded to protein kinase C (PKC) isozymes, PKCalpha and PKCtheta. A synthetic peptide corresponding to the Ser(475) RGS9-1 sequence and RGS9-1 were substrates for recombinant PKCalpha and PKCtheta. This phosphorylation was removed efficiently by protein phosphatase 2A, an endogenous phosphatase in rod outer segments, but not by PP1 or PP2B. Phosphorylation of RGS9-1 by PKC had little effect on its activity in solution but significantly decreased its affinity for its membrane anchor protein and GAP enhancer, RGS9-1 anchor protein (R9AP). PKCtheta immunostaining was at higher levels in cone outer segments than in rod outer segments, as was found for the components of the RGS9-1 GAP complex. Thus, PKC-mediated phosphorylation of RGS9-1 represents a potential mechanism for feedback control of the kinetics of photoresponse recovery in both rods and cones, with this mechanism probably especially important in cones.