Mechanisms Governing Subcellular Localization and Function of Human RGS2*

Scott P. HeximerDagger, Han Lim, Jennifer L. Bernard, and Kendall J. Blumer§

From the Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, November 1, 2000, and in revised form, January 11, 2001




    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

RGS proteins negatively regulate heterotrimeric G proteins at the plasma membrane. RGS2-GFP localizes to the nucleus, plasma membrane, and cytoplasm of HEK293 cells. Expression of activated Gq increased RGS2 association with the plasma membrane and decreased accumulation in the nucleus, suggesting that signal-induced redistribution may regulate RGS2 function. Thus, we identified and characterized a conserved N-terminal domain in RGS2 that is necessary and sufficient for plasma membrane localization. Mutational and biophysical analyses indicated that this domain is an amphipathic alpha -helix that binds vesicles containing acidic phospholipids. However, the plasma membrane targeting function of the amphipathic helical domain did not appear to be essential for RGS2 to attenuate signaling by activated Gq. Nevertheless, truncation mutants indicated that the N terminus is essential, potentially serving as a scaffold that binds receptors, signaling proteins, or nuclear components. Indeed, the RGS2 N terminus directs nuclear accumulation of GFP. Although RGS2 possesses a nuclear targeting motif, it lacks a nuclear import signal and enters the nucleus by passive diffusion. Nuclear accumulation of RGS2 does not limit its ability to attenuate Gq signaling, because excluding RGS2 from the nucleus was without effect. RGS2 may nonetheless regulate signaling or other processes in the nucleus.




    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Many hormones, neurotransmitters, and sensory stimuli elicit specific physiological responses in target tissues by activating receptors that are coupled to heterotrimeric G proteins1 (1, 2). Activated receptors promote exchange of GTP for GDP on Galpha subunits leading to dissociation of GTP-bound Galpha subunits from Gbeta gamma heterodimers and activation of downstream effector pathways. Signals are terminated following Galpha -catalyzed hydrolysis of GTP and reformation of G protein heterotrimers. Thus, G proteins act as molecular switches to coordinate the wide range of responses elicited by extracellular stimuli.

The regulators of G protein signaling (RGS) proteins are a large family that regulate G protein signaling in part by acting as GTPase-activating proteins (GAPs) for several classes of G protein alpha  subunits (3-6). The GAP activity of RGS proteins shortens the half-life of active, GTP-bound Galpha subunits and leads to attenuation of a response during prolonged stimulation or accelerated termination of the signal following removal of agonist (7-11). Binding of RGS proteins to active Galpha subunits can also interfere with effector binding, thereby blocking activation and downstream signaling (12). These activities are mediated by the ~120-amino acid RGS domain, a conserved feature of this protein family.

Genetic studies in budding yeast (13) and Caenorhabditis elegans (14) indicate that eukaryotes express several types of RGS proteins to provide selective regulation of distinct G protein signaling pathways, and a means of differentially regulating a single pathway in response to various physiologic conditions. Similarly, certain mammalian RGS proteins have been shown to regulate selectively different G protein signaling pathways. For example, deletion of RGS2 in mice results in defects in T-cell activation, antiviral immunity, and hippocampal neuron function (15), whereas an RGS9L knockout shows a slowed recovery of rod photoresponse (7). Such differences in function are reflective of both differences in cell-specific RGS expression patterns as well as selectivity of RGS proteins for different signaling pathways. Many RGS proteins are co-expressed in a wide number of cell types and tissues. In such cases, specificity may be mediated in part through selectivity of RGS proteins for particular G protein substrates (16, 17). More generally, however, RGS proteins are relatively nonselective toward G protein substrates in vitro. This suggests that the regulatory selectivity observed in cells (18) is achieved by interaction with other signaling molecules. For example, the N terminus of RGS4 confers receptor-selective regulation of Gq-coupled responses (19, 20); the PDZ domain of RGS12 binds peptides from the C termini of the interleukin-8-RB receptor (21); the GGL domains of RGS6, RGS7, RGS9, and RGS11 selectively bind Gbeta 5 (22-29); and a Dbl-like domain in p115RhoGEF activates Rho GTPases (30, 31). Thus, modular domains of RGS proteins may act as scaffolds to recruit specific classes of signaling proteins or alternatively as motifs that target these molecules to specific subcellular compartments.

Accordingly, an emerging theme is that regulatory domains other than the RGS core domain mediate patterns of subcellular localization and that localization is a determinant of RGS protein function. Thus it is of interest that many of the small RGS proteins accumulate in compartments distinct from plasma membrane where their substrate G proteins reside (32). This has led to the hypothesis that RGS protein association with other factors in the cytoplasm, nucleus, and Golgi helps to maintain an inactive pool of RGS proteins that can be recruited to the plasma membrane in response to specific signals (32, 33). In support of this notion, G protein-mediated signals have been shown to induce plasma membrane translocation of RGS3 and RGS4 (34, 35). However, the mechanisms and functional importance of signal-induced recruitment to the plasma membrane of mammalian cells remain to be determined.

A common approach to address these questions has involved heterologous expression of mammalian RGS proteins in yeast (36-38). Such studies have shown that several RGS isoforms can associate avidly and constitutively with the plasma membrane and that plasma membrane localization is required for inhibition of G protein-dependent pheromone signaling. More recently, this system has been used to identify putative plasma membrane targeting domains of several RGS proteins, revealing that a conserved N-terminal amphipathic alpha -helical domain is required for plasma membrane targeting and inhibition of G protein signaling (37, 38). However, it has not been determined in mammalian cells whether these targeting motifs mediate stable or signal-induced association of RGS proteins with plasma membrane, and whether they are required for RGS proteins to attenuate G protein signaling.

RGS2, like RGS1, RGS4, RGS5, and RGS16, is a member of the "small" mammalian RGS protein subfamily and contains a short N-terminal extension to the RGS box. Our previous data in yeast suggest that the N-terminal sequences of human RGS2 may contain domains important for its subcellular localization and function (16). Accordingly, the goals of the present study were first to define the determinants that mediate plasma membrane localization of RGS2 in yeast and next to determine whether the N-terminal domain of RGS2 governs subcellular localization and attenuation of Gq signaling in mammalian cells. Because RGS2 has been shown to localize to the nucleus of mammalian cells (32, 33), we have also examined whether the nuclear localization is an important determinant of RGS2 function.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- The cytomegalovirus promoter on plasmid pEGFP-C1 (CLONTECH; Palo Alto, CA) was used to express various RGS2-GFP constructs and a constitutively active Gq mutant (Q209L). Constitutively active Gq(R183C) construct in pCIS was a kind gift from Dr. J. Hepler (Emory University, Atlanta, GA). The thymidine kinase promoter-driven Renilla luciferase reporter (pRL-TK) used as a transfection control in transient assays was a kind gift from K. Murphy (Washington University, St. Louis). Polyclonal GFP antibody was the kind gift of P. Silver (Dana Farber Cancer Institute, Boston). Gq antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Unless otherwise stated, all other reagents and chemicals were from Sigma.

cDNA Constructs-- Mammalian and yeast expression constructs were made by high fidelity polymerase chain reaction (Pfu, Stratagene, La Jolla, CA) and subsequent cloning into pEGFP-C1 or pVT102U, respectively (39). A strong translational initiation signal (Kozak consensus; GCCACCATGGCG) was incorporated at the initiator methionine of all clones to ensure robust expression. RGS2 point mutants were made using the QuikChange mutagenesis kit (Stratagene). All constructs were purified using an Endo-Free Maxi large scale DNA purification kit (Qiagen) and verified by DNA sequencing of the entire protein coding region.

Expression in Yeast and Halo Assays-- Expression of GFP-tagged cDNAs in the yeast Saccharomyces cerevisiae and determination of RGS function in halo assays were as described previously (16).

Preparation of Liposomes-- 1,2-Dipalmitoyl-sn-glycero-3-phosphatidylocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3[phosphatidyl-rac-(1-glycerol)] (DPPG) were from Avanti Polar Lipids, Inc. (Alabaster, AL), and were stored as chloroform stocks under argon at -80 °C. Small unilamellar liposomes were prepared as described previously (38). Lipid ratios of the resulting liposomes were monitored by high performance thin layer chromatography. Vesicle preparations were used on the same day that they were prepared.

Peptide Synthesis and Circular Dichroism Measurements-- Peptides corresponding to amino acid residues 34-57 in WT and mutant RGS2 were synthesized and purified using reverse phase high pressure liquid chromatography (PNACL, Washington University, St. Louis). In the case of WT and each mutant peptide (K42A/R44A, W41A/L45A/F48A/L49A, L45D, and T43P) purity was confirmed using electrospray mass spectrometry. Stock solutions of the peptides were made by dissolving 15 mg of purified peptide in 1 ml of double-distilled water. The concentration of each peptide stock was determined by amino acid analysis. Stock solutions were aliquoted, stored at -20 °C, and thawed only once prior to CD spectrometric analysis. Far UV CD spectra of peptide samples were collected on a Jasco J600 Spectropolarimeter using a 1-mm path length quartz cell at ambient temperature. Spectra were recorded from 250 to 180 nm in 0.4 nm steps at 50 nm/min. Data represent the average of five independent spectra. Peptide and liposomes were diluted in 20 mM sodium phosphate, pH 7, to final concentrations of 30 µM and 4 mM, respectively. Background signals for the buffers and lipids were not significant and were subtracted from peptide spectra prior to analysis. Data are expressed as mean molar ellipticity (degrees cm2/dmol). The percentage alpha -helical contents of RGS peptides was estimated from the molar ellipticity at 222 nm as described previously (38).

Tissue Culture-- HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum, 2 mM glutamine, 10 µg/ml streptomycin, and 100 units/ml penicillin at 37 °C in a humidified atmosphere with 5% CO2.

Assays of Gq-dependent Phosphoinositide Hydrolysis-- Cells (7 × 106 in 10-cm plates) were washed in serum-free media and incubated with 6.4 ml of Opti-MEM (Life Technologies, Inc.) containing a mixture of the following DNA constructs: 1 µg of pRL-TK; 1.5 µg of Gq(R183C); and 3 µg of RGS2-GFP or empty vector control (5.5 µg of total plasmid DNA/well) and 10 µl of LipofectAMINE. Following a 5-h incubation, the media were removed and replaced with maintenance media (described above) for recovery overnight. After 17 h of recovery, cells from each transfection were trypsinized and plated in 6-well (7 × 106 cells/well) and 10-cm dishes. For inositol hydrolysis assays, at 22 h post-transfection, culture media were replaced with labeling media containing 4 µCi/ml [3H]myoinositol in the presence of 10 mM LiCl and cells were incubated overnight. Phosphoinositide hydrolysis was determined as described previously (16), and values were normalized to Renilla luciferase levels to account for differences in transient transfection efficiency. For determination of Renilla luciferase levels cells at 22-25 h, post-transfections were washed twice in ice-cold phosphate-buffered saline, harvested in 250 µl of hypotonic lysis buffer (10 mM KH2PO4, pH 7.5, 1 mM EDTA), and spun at 12,000 × g to remove cellular debris. Duplicate 50-µl samples of the supernatant were measured for Renilla luciferase activity in a Optocomp II luminometer (MGM Instruments, Hamden, CT). Renilla luciferase substrate was diluted 1/25,000 in 10 mM KH2PO4, pH 7.5, 0.5 M NaCl, and 1 mM EDTA for the assays. Levels of RGS and G protein expression were monitored on Western blots using ECL detection (Amersham Pharmacia Biotech).

Confocal Fluorescence Microscopy-- Cells (2 × 105 per well on polylysine-coated coverslips) were transfected as described above using 1 µg of each construct to be studied. Following overnight recovery, maintenance media were replaced with microscopy media (maintenance media without phenol red and bicarbonate, containing 25 mM Hepes, pH 7.5). Confocal microscopy was performed on live cells using a Zeiss Axioplan microscope coupled to an MRC-1000 laser scanning confocal microscope (Bio-Rad). Images represent single equatorial planes obtained with a × 63 objective. Confocal images were processed with Adobe Photoshop 4.0.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mapping the Domain of Human RGS2 Required for Plasma Membrane Association and Function in Yeast-- The mating pheromone response pathway in the yeast S. cerevisiae is a G protein-coupled signaling pathway. Expression of mammalian RGS proteins in yeast cells lacking Sst2, the RGS protein homolog that normally attenuates pheromone signaling, permits rapid and quantitative analysis of function in pheromone-induced growth arrest (halo) assays (40, 41). RGS protein function in halo assays is dependent on domains that direct plasma membrane targeting (36-38). Indeed, our previous studies indicated that the N-terminal domain of RGS2 is necessary for plasma membrane localization and function in yeast (16). To identify the minimal region within the N-terminal domain required for targeting and function, we made a series of deletions within the N terminus of RGS2 and correlated their effects on membrane localization and attenuation of mating pheromone-induced G protein signaling in yeast (Fig. 1A). Deletion of the N-terminal 4, 15, or 32 amino acids result did not affect membrane localization or function. Deletion of the first 66 amino acids, however, resulted in loss of both membrane localization and inhibitory activity, as indicated by an increase in pheromone sensitivity (increased halo size). This result suggested that the domain in RGS2 necessary for membrane association maps between residues 32 and 66 in the N terminus (Fig. 2). When residues 33-67 were fused to the N terminus of GFP, the resulting protein localized to the plasma membrane, indicating that this sequence contained determinant(s) sufficient for plasma localization in yeast (Fig. 1B).



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Fig. 1.   Localization and attenuation of yeast mating pheromone response by GFP-tagged N-terminal deletion mutants of RGS2. A, yeast cells (SWY518) that carry plasmids expressing GFP-tagged RGS2 or the indicated N-terminal truncations were analyzed by confocal microscopy (right-hand panels). All images are shown at the same magnification. The ability of the indicated GFP fusions to inhibit the pheromone response (left-hand panels) was measured using growth arrest (halo) assays. For halo assays, a mutant yeast strain (BC180) that lacks Sst2, the RGS protein that normally regulates the mating response, was transformed with the indicated RGS2 constructs. Shown are the responses elicited by 0.5 nmol of pheromone; other doses were used to quantify the extent of inhibition by the various constructs. The data shown are representative of three transformants assayed in two independent experiments. B, the yeast strain BC180 carrying the indicated RGS2-(aa 33-67)-GFP fusion was analyzed as in A.



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Fig. 2.   Sequence and helical net comparisons of RGS2 with membrane association domains of RGS4 and RGS16. Shown is the schematic representation of the RGS2 membrane association domain (red), proximal to the N-terminal end of the RGS box (green). The corresponding sequence for this region was aligned to RGS4 and RGS16 by the CLUSTAL method, and the consensus is shown below. Shading indicates a hydrophobic core of aliphatic residues found along one face of the putative amphipathic alpha -helix identified in RGS4 and RGS16. Red sequences are conserved in all three sequences, and blue indicates conservation in two out of three sequences. The same region was represented as a helical net with residues flanking the shaded regions (hydrophobic core) shown in color. Aliphatic and nonpolar aromatic residues are shown as black, and basic residues are yellow.

Sequence alignments indicated that the apparent N-terminal membrane targeting domain of RGS2 is conserved in a subset of the lower molecular weight RGS family members (Fig. 2), which includes RGS1, RGS2, RGS3, RGS4, RGS5, and RGS16 (only alignments for RGS2, RGS4, and RGS16 are shown). Because this region of RGS2 is similar in sequence to the amphipathic alpha -helical domains of RGS4 and RGS16 implicated in plasma membrane localization and function in yeast cells, we compared helical net views of these domains (Fig. 2). This analysis indicated that the apparent membrane-targeting domain of RGS2 also has the potential to form an amphipathic alpha -helix. Indeed, RGS2, RGS4, and RGS16 share a hydrophobic (aliphatic) core along one face of the putative helix that is flanked at the N-terminal end of the helix by two or more basic residues. These observations suggested that the N-terminal domain of RGS2 may function as an amphipathic alpha -helical membrane-targeting domain similar to those of RGS4 and RGS16. Furthermore, putative N-terminal amphipathic helices of RGS2, RGS4, and RGS16 are positioned immediately proximal to the RGS core domain, suggesting a similar organization of functional domains among this subset of RGS proteins.

Interaction of the N-terminal Membrane Targeting Domain of RGS2 with Acidic Phospholipids-- To determine whether the apparent membrane-targeting domain of RGS2 can adopt a alpha -helical conformation and interact with membranes, we analyzed the helical content of synthetic peptides corresponding to this region by CD spectroscopy in the presence or absence of phospholipid vesicles. Bernstein et al. (38) used a similar approach to show that the N-terminal membrane-targeting domain of RGS4 adopts a alpha -helix upon binding to negatively charged phospholipid vesicles.

Several results indicated that, similar to RGS4, peptides derived from the N-terminal membrane-targeting domain of RGS2 adopt an alpha -helical conformation upon binding to vesicles containing anionic phospholipids (Fig. 3). A peptide corresponding to residues 34-57 of RGS2 adopted a random coil conformation in aqueous solution, indicated by the single minimum at 200 nm (Fig. 3B). The helix-forming potential of this peptide was characterized by minima at 208 and 222 nm in the presence of the helix-stabilizing solvent trifluoroethanol (data not shown). Addition of vesicles made from neutral lipids (DPPC) did not induce a helical conformation of the peptide (Fig. 3A). However, addition of phospholipid vesicles made with anionic (DPPG) and neutral (DPPC) phospholipids at a mole ratio of 10:90 caused the peptide to adopt a more alpha -helical conformation. Apparent alpha -helical content increased upon increasing the mole fraction of DPPG, reaching a maximum helical content at a 40:60 ratio of DPPG:DPPC (Fig. 3, A and B).



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Fig. 3.   Residues 34-57 in RGS2 adopt an amphipathic alpha -helix in the presence of anionic liposomes. A, values indicating the % alpha -helix for a peptide corresponding to RGS2 residues 34-57 were calculated from CD data collected at 222 nm as described previously (Bernstein et al. (38)) and under "Experimental Procedures." Values were determined in the presence of liposomes containing the indicated ratios of DPPC (nonpolar) and DPPG (anionic) lipids. B, CD spectra for the wild type RGS2 peptide was recorded as described above. Control spectrum for WT peptide in buffer alone is shown in black. Spectra for the WT peptide in buffer with liposomes containing 20 and 40% DPPG are shown as red and green, respectively. C, CD spectra for RGS2 mutant peptides were recorded as above. Spectra for WT (square), L45D (circle), W41A/L45A/F48A/L49A (triangle) and K42A/R44A (inverted triangle) were compared for peptides in buffer (black curves) and anionic liposomes (colored curves WT, red; L45D, cyan; W41A/L45A/F48A/L49A, green; K42A/R44A, purple). Each curve is the average of at least two independent spectra.

To determine whether an amphipathic character of the RGS2 peptide is required for helix formation and/or binding to phospholipid vesicles, we analyzed a series of peptides with amino acid substitutions predicted to affect the amphipathic nature of the helix (Fig. 3C). The mutations used were designed to reduce the aliphatic (W41A/L45A/F48A/L49A) or basic (K42A/ R44A/L45D) nature of the amphipathic helix core. These mutations did not alter the ability of the resulting peptides to form a alpha -helix since each mutant peptide showed similar helix-forming potential as the wild type in the presence of trifluoroacetic acid (data not shown). However, the mutant peptides did display an impaired ability to adopt an alpha -helical conformation in the presence of vesicles containing anionic phospholipids. Substitutions predicted to decrease or disrupt the aliphatic nature of the hydrophobic face decreased lipid-induced helix formation. Similarly, introducing a negatively charged residue along the putative membrane-binding interface resulted in reduced helix formation, whereas changing positive residues in the putative amphipathic helix to alanine had little effect in this assay. These data indicated that in the case of the RGS2 helix, the aliphatic interface is a primary determinant of its ability to interact with lipid vesicles.

The RGS2 N Terminus Mediates Association with the Nucleus and Plasma Membrane in Mammalian Cells-- Although previous studies have characterized N-terminal lipid association domains in other small RGS proteins, the functional importance of these domains in mammalian cells has not been rigorously addressed. Therefore, we set out to define the role of the N terminus of RGS2 in mediating localization and function in mammalian cells. We used a GFP tagging approach to determine the subcellular localization of RGS2 because it has not been possible to localize the protein expressed endogenously in primary cell cultures or cell lines. As indicated by confocal microscopy, a functional RGS2-GFP fusion expressed transiently in HEK293 cells localized primarily to the nucleus and was also detected in the cytoplasm and on the plasma membrane (Fig. 4A). Plasma membrane association of RGS2-GFP in these cells is indicated by membrane structures at the periphery of the cells, pronounced GFP fluorescence at cell-cell borders, and at the basal surface of the cells as determined by z-sectioning during confocal microscopy (data not shown). The subcellular localization of RGS2-GFP appeared to be specific because RGS4-GFP or GFP did not accumulate on the plasma membrane, and they were distributed uniformly throughout the cytoplasm and nucleus in HEK293 cells (Fig. 4B). The localization of RGS2-GFP in unstimulated HEK293 cells is similar to the localization of GFP-RGS2 localization in a pre-B cell line (33) but distinct from the exclusively nuclear localization of GFP-RGS2 in COS-7 cells (32). These differences suggest that the pattern of RGS2 localization is somewhat cell type-specific, although other explanations are possible. Nevertheless, we found that nuclear and plasma membrane accumulations of RGS2-GFP were reduced in the N-terminal truncation mutant, RGS2-(aa 78-211)-GFP, suggesting a role for the N terminus in mediating the subcellular localization of RGS2 in HEK293 cells.



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Fig. 4.   Definition of the subcellular localization of full-length and N-terminally truncated RGS2-GFP with and without co-expressed Gq(Q209L). A, localization of the indicated GFP fusion constructs was examined by transient transfection with and without Gq(Q209L) in HEK293 cells followed by confocal microscopy of live cells. Pictures are of cells with low to intermediate fluorescence intensity and are representative of at least 50 cells transfected with the same construct. Shown are GFP fluorescence images of the basal side (relative to nuclear equator) of the cell, as determined by a z axis series. B, localization of GFP and similarly tagged control proteins RGS4 and RGS2-(aa 78-211) were examined as in A.

The N Terminus of RGS2 Mediates Gq-induced Plasma Membrane Localization-- Studies (34, 35) on other small RGS protein family members have suggested an important role for the N terminus in signal-mediated translocation to the plasma membrane. The mechanism of this translocation is currently not well understood. To test whether RGS2 also undergoes a signal-dependent translocation, we co-expressed RGS2-GFP with Gq(Q209L), a constitutively active Gqalpha mutant (Fig. 4A). Indeed, RGS2-GFP accumulated more markedly at the plasma membrane when co-expressed with Gq(Q209L). This apparent subcellular redistribution in the presence of activated Gq was also indicated by a decrease in RGS2-GFP accumulation in the nucleus. To examine the role of the N terminus in RGS2-GFP translocation, we also studied an N-terminally truncated construct. Since translocation was not observed when the RGS2-(aa 78-211)-GFP fusion was co-expressed with Gq(Q209L), it appeared that the N terminus of RGS2 rather than RGS2-G protein binding is required for Gq-induced plasma membrane localization.

The Amphipathic alpha -Helical Domain of RGS2 Mediates Plasma Membrane Targeting in Mammalian Cells-- The preceding data show the N-terminal domain of RGS2 is necessary to mediate tonic and signal-induced plasma membrane association in HEK293 cells. To identify the domains that confer localization in mammalian cells amino acid residues 1-67 of RGS2 were fused to the N terminus of GFP (Fig. 5). As shown by confocal microscopy, this construct localized to the plasma membrane in HEK293 cells indicating that these residues directed plasma membrane association. Notably, this is the first demonstration of an RGS protein domain that can mediate tonic association with the plasma membrane in mammalian cells.



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Fig. 5.   Characterization of the N-terminal plasma membrane targeting motif of RGS2 in HEK293 cells. A, localization of N-terminal-GFP fusions of RGS2-(aa 1-67) and RGS4-(aa 1-42) and the indicated RGS2 truncation mutants were examined by confocal microscopy as described above. B, wild type and mutant (K42A/R44A; W41A/ L45A/F48A/L49A) RGS2 domains corresponding to residues 33-66 were cloned as fusions at the C-terminal end of GFP and analyzed as described above.

To characterize the features of the plasma membrane targeting signal of RGS2, we generated a series of truncation and point mutants in RGS2-(aa 1-67)-GFP. Deletion of the first 4 or 15 amino acids did not alter the subcellular localization of the fusion proteins in HEK293 cells. Surprisingly, deletion of the first 32 amino acids directed localization of the fusion to mitochondria. Since mitochondrial targeting signals frequently are N-terminal amphipathic alpha -helices (42), these data were consistent with the model that residues 33-67 in RGS2 form such a structure. To preclude mitochondrial localization and assess plasma membrane targeting by this fragment of RGS2, we fused residues 33-67 of RGS2 to the C terminus of GFP (Fig. 5B). As expected, the GFP-RGS2-(aa 33-67) fusion did not associate with mitochondria, but it did associate with the plasma membrane indicating that this region contains a functional targeting signal. We also noted that GFP-RGS2-(aa 33-67) accumulated in the nucleus, an observation that will be explored at the end of the "Results."

RGS2 Amphipathic Helix Is Required for Tonic Plasma Membrane Localization-- To define the boundaries of the helical domain, we generated a series of point mutations within the context of the RGS2-(aa 1-67)-GFP fusion. First, a series of proline substitution mutations were constructed to determine if helix-forming ability affects the function of the targeting signal. Introduction of proline residues between Asp-40 and Ser-46 on the putative hydrophilic face of the helix dramatically reduced association of the RGS2-(aa 1-67)-GFP fusion with the plasma membrane (Table I). This appeared to reflect the disruption of a alpha -helix because introduction of an alanine residue at any of these positions did not affect plasma membrane association. Second, a series of negatively charged amino acid substitutions were generated to determine if association of the targeting signal with phospholipids is important, as predicted by the amphipathic helix model. Introduction of acidic residues along the hydrophobic face of the presumptive helix between Leu-37 and Leu-49 resulted in severely decreased association with the plasma membrane (Table I). In contrast, introduction of acidic residues on the hydrophilic side of the helix had little effect on membrane association, further substantiating the model. Together these results suggest that an amphipathic membrane targeting helix extends from Leu-37 to Leu-49.


                              
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Table I
Subcellular localization of point mutations affecting the putative amphipathic alpha -helical domain in RGS2(aa 1-67)-GFP
RGS2-(aa 1-67)-GFP and corresponding mutant constructs were studied in live HEK293 cells following transient transfection. Subcellular localization phenotypes are compared to wild type RGS2-(aa 1-67)-GFP (WT) and are representative of 50 transfected cells. + indicates fluorescence >=  that of WT. ± indicates detectable fluorescence less than that for WT. - indicates no detectable fluorescence.

To determine whether the amphipathic helix is required for function of the RGS2 plasma membrane targeting signal in mammalian cells, we introduced point mutations into GFP-RGS2-(aa 33-67) equivalent to those shown previously that affect association with phospholipid vesicles (Fig. 5B). Confocal microscopy indicated that the mutant constructs displayed dramatically decreased association with the plasma membrane. These results suggested that positively charged and aliphatic residues are functional determinants of the plasma membrane targeting signal, consistent with the model that this signal functions as an amphipathic helix.

Do the Amphipathic Helices of RGS2 and RGS4 Behave Differently in Mammalian Cells?-- Although the N termini of RGS2 and RGS4 contain amphipathic helical domains that can associate with phospholipid vesicles in vitro and the yeast plasma membrane, they appear to function differently in HEK293 cells. In contrast to what we observed with the N-terminal domain of RGS2, the N-terminal domain of RGS4 (residues 1-42) did not target GFP to the plasma membrane or lead to nuclear accumulation (Fig. 4). These results are mirrored by the observed differences in localization between full-length RGS2-GFP and RGS4-GFP (Fig. 1).

Comparison of the apparent targeting signals of RGS2 and RGS4 was used to suggest why the RGS4 targeting signal might not function as efficiently as that of RGS2. We noted that the hydrophobic face of the RGS4 targeting domain has a serine and alanine at positions equivalent to Leu-37 and Leu-38 of RGS2 (Fig. 2). Thus, we hypothesized that the RGS4 signal may bind membranes less avidly because the membrane-binding surface of the helix is less hydrophobic. To test this idea, we replaced Leu-37 and Leu-38 of RGS2(1-67)-GFP with their RGS4 equivalents (Ser-15 and Ala-16, respectively). Indeed, this mutant displayed a defect in membrane association.2 However, this hypothesis appeared insufficient to explain the difference in the apparent strength of the plasma membrane targeting signals of RGS2 and RGS4. We suggest this because RGS4-(1-42)-GFP does not target the plasma membrane when leucine residues were introduced at positions 14 and 15 (data not shown). This hypothesis, however, assumes the RGS4 helix extends as far toward the N terminus as the RGS2 domain. Further studies are needed to determine whether the length of the helices are a key determinant of the function of these two lipid association domains.

The Amphipathic Helix Is Required for Signal-induced Plasma Membrane Association of RGS2 in Mammalian Cells-- The preceding data indicated that the N terminus, and more specifically the amphipathic helical domain, is sufficient to mediate tonic plasma membrane association of an RGS2-GFP fusion in HEK293 cells. We next determined whether the same helical domain was required for the observed Gq-induced translocation of full-length RGS2-GFP. Indeed, the same mutations that inhibited association with phospholipid vesicles in vitro and association of the N terminus with the plasma membrane in HEK293 cells also prevent Gq-induced translocation of RGS2-GFP (Fig. 6). These data suggest the alpha -helical domain is required for signal-induced translocation of RGS2. The same principle may hold for RGS proteins with related domains such as RGS4, RGS5, and RGS16.



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Fig. 6.   Determination of Gq(Q209L)-mediated plasma membrane translocation potential for RGS2 proteins with mutations in the amphipathic alpha -helix domain. Localization of GFP-tagged wild type or the indicated RGS2 mutants was studied in cells with and without a co-expressed constitutively active Gq mutant (Gq(Q209L). Confocal microscopy was performed as described in the legend of Fig. 4.

The Plasma Membrane Targeting Function of the RGS2 N-terminal Domain Is Not Required for Attenuation of Gq Signaling-- To determine whether stable association of RGS2 with plasma membrane is required for inhibition of Gq signaling in mammalian cells, we analyzed point mutations affecting the amphipathic helical domain. We used a constitutively active Gq mutant (R183C) whose signaling activity can be attenuated by the ability of RGS proteins to function as a GAP or an effector antagonist. Expression of Gq(R183C) in HEK293 cells resulted in an ~100-fold increase in phosphoinositide hydrolysis compared with vector alone (Fig. 7). When full-length RGS2-GFP was co-expressed with Gq(R183C), the extent of phosphoinositide hydrolysis was reduced by ~75%. Similarly, mutant forms of RGS2-GFP that were defective in membrane association assays (L45D; K42A/R44A; W41A/L45A/F48A/L49A) displayed nearly wild type activity in this assay when expressed at similar levels. We therefore suggest that plasma membrane association mediated by the N-terminal domain of RGS2 is not the major determinant required for attenuation of Gq signaling in mammalian cells.



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Fig. 7.   Ability of RGS2 amphipathic helix domain mutants to attenuate Gq-mediated phosphoinositide hydrolysis. HEK293 cells were co-transfected with control or constitutively active Gq(R183C), the indicated GFP control or GFP-tagged RGS2 wild type and amphipathic alpha -helical domain mutant constructs, and a Renilla luciferase control plasmid. Triplicate wells containing transfected cells (1 × 106) were labeled with [3H]inositol and 10 mM LiCl. Inositol 1,4,5-trisphosphate (IP3) levels were measured as described under "Experimental Procedures." Values represent the average of triplicate samples normalized for luciferase activity and were expressed as the percentages of soluble inositol 1,4,5-trisphosphate relative to total soluble inositol-containing material; S.E. are indicated by error bars. These data are representative of three independent experiments. The levels of RGS2-GFP constructs were determined by immunoblotting (inset).

To conclude that the plasma membrane targeting function of the N-terminal domain is not essential, we had to determine whether assays using overexpressed RGS2 mutants were capable of detecting a loss of function phenotype. One means of addressing this issue was suggested by the following observations: 1) complete deletion of the RGS2 N terminus (RGS- (aa 78-211)-GFP) resulted in mislocalization to the cytoplasm and loss of function as an inhibitor of yeast mating pheromone signaling (Ref. 16 and data not shown); and 2) appending a C-terminal Ras2 CAAX box to the RGS2 core domain resulted in plasma membrane targeting and complete restoration of its function in yeast. We therefore analyzed the function of these RGS2 constructs in HEK293 cells. Similar to results obtained in yeast, deletion of the N terminus from RGS2-GFP markedly reduce its ability to inhibit Gq(R183C)-mediated signaling in HEK293 cells (Fig. 8). However in contrast to what was observed in yeast, appending the polybasic region and prenylation signal of k-Ras to the C terminus of the RGS2 core domain (RGS2-(aa 78-211)-GFP-CAAX) did not completely restore inhibitory function despite very efficient plasma membrane association (Fig. 1B). These results therefore suggested that RGS2 function in mammalian cells may be governed by interaction of the N terminus with other factors or by post-translational modifications that are absent or inoperative in yeast.



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Fig. 8.   Ability of RGS2 core and plasma membrane-targeted core domains in to inhibit Gq-mediated phosphoinositide hydrolysis. HEK293 cells were co-transfected with control or constitutively active Gq, the indicated GFP control or RGS2 constructs, and a Renilla luciferase control. Phosphoinositide hydrolysis was measured as described in the legend to Fig. 7. IP3, inositol 1,4,5-trisphosphate.

Nuclear Accumulation of RGS2 Is Mediated by Its N-terminal Domain-- The observation that several RGS proteins including RGS2 accumulate in the nucleus suggests that nuclear import and/or export could determine whether an RGS protein is available to attenuate the activity of plasma membrane-bound G proteins. These findings also suggest that RGS proteins may possess signals that mediate nuclear import, export, or retention. Thus, the N terminus might direct RGS2 to the nucleus where it is inactive (sequestered) yet poised to respond to appropriate signals. This idea is supported by the observation that co-expression with activated Gq resulted in a decreased nuclear accumulation of RGS2-GFP (Figs. 1 and 6). To address these hypotheses, we first determined whether we could identify a domain responsible for nuclear accumulation of RGS2, and we then asked whether exclusion of RGS2 from the nucleus affected its function as an attenuator of Gq signaling.

The localization patterns of various RGS2-GFP fusions suggested that a nuclear accumulation signal maps within or near the plasma membrane targeting signal. First, deletion of the N-terminal domain of RGS2-GFP resulted in uniform distribution of the protein throughout the cytoplasm and nucleus, in contrast to the nuclear enrichment of the full-length protein (Fig. 4A). Second, the N-terminal domain of RGS2 (residues 1-67) was sufficient to direct the accumulation of GFP in the nucleus in structures resembling nucleoli (Fig. 5). Third, subfragments of the N-terminal domain that were active as plasma membrane targeting signals were also active as nuclear accumulation signals (residues 4-67, 15-67, or 33-67 appended to the C terminus of GFP).

Analysis of additional fusions indicated that the N-terminal domain of RGS2 does not mediate nuclear accumulation by functioning as an import signal, arguing that the N-terminal domain functions as a nuclear retention signal. Specifically, we found that nuclear accumulation was blocked by appending GST to GFP-RGS2 (Fig. 9A), making an ~80-kDa protein that is larger than the exclusion limit of nuclear pore complexes (~60 kDa). Similarly, we found that that RGS2-(aa 78-211)-GFP-GST (data not shown) and GFP-GST were also excluded. Rather than being actively transported, RGS2, therefore, appears to enter the nucleus by passive diffusion where it is retained.



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Fig. 9.   Localization and function of RGS2-GFP-GST constructs. A, confocal microscopy of GST-tagged control and RGS2 constructs. The indicated GST-tagged GFP control or RGS2 constructs were transfected into HEK293 cells and analyzed by confocal microscopy as described above. B, function of GST-tagged RGS2 in phosphoinositide signaling assays. The GST-tagged full-length RGS2-GFP construct was compared with RGS2-GFP in Gq(R183C)-mediated phosphoinositide hydrolysis assays as described in the legend to Fig. 7. IP3, inositol 1,4,5-trisphosphate.

Because full-length RGS2-GFP-GST was excluded from the nucleus, we used it to determine whether nuclear accumulation affects the function of RGS2. To address this question we determined whether RGS2-GFP and RGS2-GFP-GST differed in their ability to attenuate signaling by constitutively active Gq(R183C). The results indicated that these two fusion proteins were expressed at similar levels and attenuated Gq-mediated phosphoinositide hydrolysis to a similar extent (Fig. 9B). These results suggested that nuclear accumulation is not required for RGS2-mediated attenuation of Gq-directed signaling and that nuclear localization does not appear to impair significantly RGS2 function.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Subcellular Localization of RGS2-- Determining the subcellular localization patterns of RGS proteins in mammalian cells is required to understand the functions of these molecules and the mechanisms that regulate their activities. Recent studies indicate that RGS1, RGS3, RGS4, and RGS16 are cytoplasmic; RGS10 is nuclear, and GAIP and RGSZ associate with the Golgi apparatus (32). We have found that RGS2-GFP localizes to the nucleus, cytoplasm, and plasma membrane of HEK293 cells, similar to the localization of GFP-RGS2 in 3T3 fibroblasts and L1/2, a transformed pre-B-lymphocyte cell line (33). In COS-7 cells, GFP-RGS2 localizes exclusively to the nucleus (32), suggesting that RGS2 localization may be regulated in a cell type-specific manner.

Functional Domains Governing Subcellular Localization of RGS2-- By identifying features of RGS2 involved in plasma membrane and nuclear localization, we have addressed whether subcellular distribution governs the function of this molecule as an inhibitor of Gq signaling. This approach has indicated that RGS2 possesses an N-terminal domain that targets the protein to the plasma membrane. Similar to RGS4 and RGS16, the plasma membrane targeting signal of RGS2 appears to be an amphipathic alpha -helical domain located at or near the N terminus of the molecule. Indeed, the targeting signals of RGS2, RGS4, and RGS16 function similarly when analyzed by heterologous expression in yeast (36-38) and/or by their association with lipid vesicles in vitro. However, the targeting signals of these RGS proteins may not function identically. Whereas full-length RGS2 or its N-terminal domain alone can associate with the plasma membrane of unstimulated HEK293 cells, RGS4 and its N-terminal domain apparently do not. These differences suggest that RGS2 and RGS4 may have somewhat different functions. For example, in cells that constitutively express RGS2 and RGS4, the threshold of agonist needed to elicit a response in naive cells may be set in part by RGS2 because it can pre-associate with the plasma membrane, whereas RGS4 apparently does not.

RGS2 and several other RGS proteins have been suggested to possess a basic bipartite motif in their RGS domain that functions as a nuclear import signal (32). However, our studies indicate that this apparently is not the case as follows: 1) an RGS2 fusion protein larger than the exclusion limit of the nuclear pore complex does not enter the nucleus of HEK293 cells, suggesting that the RGS core domain or other regions of the molecule lack a functional nuclear import sequence; 2) RGS2 lacking its N-terminal domain but possessing its RGS domain does not accumulate preferentially in the nucleus; and 3) a GFP fusion bearing the N-terminal domain of RGS2 and lacking an RGS domain preferentially accumulates in the nucleus. Therefore, it appears that RGS2 enters the nucleus by passive diffusion. Whether other RGS proteins enter the nucleus by active transport or passive diffusion remains to be determined.

Does RGS2 Require Stable Association with the Plasma Membrane?-- Although RGS2 possesses an N-terminal domain capable of targeting the molecule to the plasma membrane, stable association of with plasma membrane may not be necessary for the ability of RGS2 to attenuate G protein signaling. Plasma membrane association does not appear to be essential because point mutations in the N-terminal domain that impair basal or Gq-induced plasma membrane localization nonetheless preserve the ability of RGS2 to attenuate Gq signaling in this system.

Whereas our results indicate that plasma membrane localization is not essential for attenuation of activated Gq by overexpressed RGS2 in mammalian cells, it remains possible that plasma membrane targeting activity is important for function of RGS2 expressed at endogenous levels. Addressing this issue would require analysis of RGS2 mutants expressed at wild type levels. However, the inability to detect RGS2 protein expressed endogenously in mammalian cells thus far has precluded such investigations.

Other lines of evidence indicate that the N-terminal domain of RGS2 has an essential function. Deletion of this domain inactivates the ability of RGS2 to attenuate Gq signaling, and forced targeting of this truncated protein to the plasma membrane fails to restore function in mammalian cells. Therefore, we speculate that the N-terminal domain of RGS2 may associate with receptors or other components of G protein signaling pathways at the plasma membrane, as has been proposed for RGS4 (19).

Signal-induced Recruitment of RGS Proteins to the Plasma Membrane-- Although RGS2 associates inefficiently with the plasma membrane in unstimulated cells, it is targeted more efficiently when activated Gq is expressed, similar to what has been reported for RGS3 and RGS4. Presumably, signal-induced recruitment of these RGS proteins to the plasma membrane provides a negative feedback loop that enables cells to desensitize. Once the signal decays, RGS proteins could dissociate from the plasma membrane, facilitating resensitization.

How Do Activated G Protein alpha  Subunits or Other Signals Induce Plasma Membrane Targeting of RGS Proteins?-- Although the mechanisms are not understood, it appears that the N-terminal non-catalytic domains of RGS2, RGS3, and RGS4 mediate signal-induced plasma membrane accumulation. Because the N-terminal domains of RGS proteins are not believed to interact with G protein alpha  subunits, signal-induced plasma membrane recruitment could be caused by regulating the availability or affinity of these domains for the lipid bilayer. We suggest that for RGS2 and other amphipathic helix-containing RGS proteins, the translocation observed in the presence of activated G protein alpha  subunits is a downstream consequence of G protein signaling. This conclusion stands in contrast to that suggested by analyzing RGS2 function in yeast, possibly because in mammalian cells the N-terminal domain of RGS2 interacts with other cellular factors that are absent or whose homologs are not recognized in yeast. For example, signal-induced recruitment of RGS proteins to the plasma could be caused by binding or release of accessory proteins, such as 14-3-3 (43) or calmodulin (44) which have been shown to bind RGS3, RGS4, or RGS7, and allow the plasma membrane targeting signals of these RGS proteins to function efficiently.

Roles of RGS2 in the Nucleus-- Our studies have begun to address potential functions of RGS proteins localized to the nucleus. First, it is unlikely that RGS2 transports cargo into the nucleus because it enters the nucleus by passive diffusion and is subject to the exclusion limit (~60 kDa) of nuclear pore complexes. Second, sequestration of RGS2 in the nucleus does not appear to limit the ability to attenuate Gq signaling, because exclusion of RGS2 from the nucleus does not increase its inhibitory potency. Although there is no evidence that RGS2 has a novel function in the nucleus, this is an intriguing hypothesis. Indeed, effectors of Gq such as phospholipase C beta -isoforms are present in the nucleus (45), suggesting that RGS2 or other RGS proteins in the nucleus have the potential to regulate novel intracellular signaling pathways or other processes that control gene expression, nucleocytoplasmic trafficking, chromosome replication, or nuclear architecture.

The redistribution of RGS2 out of the nucleus in response to activation of a G protein signaling pathway recalls a similar trafficking pattern for Ste5 (46), the MAP kinase pathway scaffold of the mating pheromone response pathway of yeast. However, unlike what has been proposed for Ste5, our results suggest that overexpressed RGS2 does not require nuclear entry and exit for activity as an inhibitor of Gq signaling. Nevertheless, nuclear entry and exit conceivably could modulate the function of RGS2 expressed at normal levels or impact novel functions of RGS2 yet to be discovered.


    ACKNOWLEDGEMENTS

We thank Andrew Grillo-Hill for consultation on CD spectroscopy experiments, Sharon Chinault for RGS2 constructs, and an anonymous reviewer for comments on the manuscript.


    FOOTNOTES

* This work was supported by grants from the National Institutes of Health, National Science Foundation, American Heart Association, and Monsanto.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Fellow of the American Heart Association. To whom correspondence should be addressed: Dept. of Cell Biology and Physiology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-1662; Fax: 314-362-7463; E-mail: sheximer@cellbio.wustl.edu.

§ Established Investigator of the American Heart Association.

Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M009942200

2 S. P. Heximer and K. J. Blumer, unpublished observations.


    ABBREVIATIONS

The abbreviations used are: G protein, guanine nucleotide binding regulatory protein; GAP, GTPase-activating protein; aa , amino acid; GFP, green fluorescent protein; RGS, regulator of G protein signaling; DPPC, 1,2-Dipalmitoyl-sn-glycero-3-phosphatidylocholine; DPPG, 1,2-dipalmitoyl-sn-glycero-3[phosphatidyl-rac-(1-glycerol)]; WT, wild type.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Bourne, H. R., Sanders, D. A., and McCormick, F. (1990) Nature 348, 125-132[CrossRef][Medline] [Order article via Infotrieve]
2. Hepler, J. R., and Gilman, A. G. (1992) Trends Biochem. Sci. 17, 383-387[CrossRef][Medline] [Order article via Infotrieve]
3. Berman, D. M., Wilkie, T. M., and Gilman, A. G. (1996) Cell 86, 445-452[Medline] [Order article via Infotrieve]
4. Watson, N., Linder, M. E., Druey, K. M., Kehrl, J. H., and Blumer, K. J. (1996) Nature 383, 172-175[CrossRef][Medline] [Order article via Infotrieve]
5. Ingi, T., Krumins, A. M., Chidiac, P., Brothers, G. M., Chung, S., Snow, B. E., Barnes, C. A., Lanahan, A. A., Siderovski, D. P., Ross, E. M., Gilman, A. G., and Worley, P. F. (1998) J. Neurosci. 18, 7178-7188[Abstract]
6. Kozasa, T., Jiang, X., Hart, M. J., Sternweis, P. M., Singer, W. D., Gilman, A. G., Bollag, G., and Sternweis, P. C. (1998) Science 280, 2109-2111[Abstract/Free Full Text]
7. Chen, C. K., Burns, M. E., He, W., Wensel, T. G., Baylor, D. A., and Simon, M. I. (2000) Nature 403, 557-560[CrossRef][Medline] [Order article via Infotrieve]
8. Doupnik, C. A., Davidson, N., Lester, H. A., and Kofuji, P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10461-10466[Abstract/Free Full Text]
9. Saitoh, O., Kubo, Y., Miyatani, Y., Asano, T., and Nakata, H. (1997) Nature 390, 525-529[CrossRef][Medline] [Order article via Infotrieve]
10. Chuang, H. H., Yu, M., Jan, Y. N., and Jan, L. Y. (1998) Proc. Natl. Acad. Sci. U. S. A 95, 11727-11732[Abstract/Free Full Text]
11. Zerangue, N., and Jan, L. Y. (1998) Curr. Biol. 8, R313-R316[Medline] [Order article via Infotrieve]
12. Hepler, J. R., Berman, D. M., Gilman, A. G., and Kozasa, T. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 428-432[Abstract/Free Full Text]
13. Versele, M., de Winde, J. H., and Thevelein, J. M. (1999) EMBO J. 18, 5577-5591[Abstract/Free Full Text]
14. Dong, M. Q., Chase, D., Patikoglou, G. A., and Koelle, M. R. (2000) Genes Dev. 14, 2003-2014[Abstract/Free Full Text]
15. Oliveira-Dos-Santos, A. J., Matsumoto, G., Snow, B. E., Bai, D., Houston, F. P., Whishaw, I. Q., Mariathasan, S., Sasaki, T., Wakeham, A., Ohashi, P. S., Roder, J. C., Barnes, C. A., Siderovski, D. P., and Penninger, J. M. (2001) Proc. Natl. Acad. Sci. U. S. A. 97, 12272-12277[Abstract/Free Full Text]
16. Heximer, S. P., Srinivasa, S. P., Bernstein, L. S., Bernard, J. L., Linder, M. E., Hepler, J. R., and Blumer, K. J. (1999) J. Biol. Chem. 274, 34253-34259[Abstract/Free Full Text]
17. Beadling, C., Druey, K. M., Richter, G., Kehrl, J. H., and Smith, K. A. (1999) J. Immunol. 162, 2677-2682[Abstract/Free Full Text]
18. Zhang, Y., Neo, S. Y., Han, J., Yaw, L. P., and Lin, S. C. (1999) J. Biol. Chem. 274, 2851-2857[Abstract/Free Full Text]
19. Xu, X., Zeng, W., Popov, S., Berman, D. M., Davignon, I., Yu, K., Yowe, D., Offermanns, S., Muallem, S., and Wilkie, T. M. (1999) J. Biol. Chem. 274, 3549-3556[Abstract/Free Full Text]
20. Zeng, W., Xu, X., Popov, S., Mukhopadhyay, S., Chidiac, P., Swistok, J., Danho, W., Yagaloff, K. A., Fisher, S. L., Ross, E. M., Muallem, S., and Wilkie, T. M. (1998) J. Biol. Chem. 273, 34687-34690[Abstract/Free Full Text]
21. Snow, B. E., Hall, R. A., Krumins, A. M., Brothers, G. M., Bouchard, D., Brothers, C. A., Chung, S., Mangion, J., Gilman, A. G., Lefkowitz, R. J., and Siderovski, D. P. (1998) J. Biol. Chem. 273, 17749-17755[Abstract/Free Full Text]
22. Witherow, D. S., Wang, Q., Levay, K., Cabrera, J. L., Chen, J., Willars, G. B., and Slepak, V. Z. (2000) J. Biol. Chem. 275, 24872-24880[Abstract/Free Full Text]
23. Snow, B. E., Krumins, A. M., Brothers, G. M., Lee, S. F., Wall, M. A., Chung, S., Mangion, J., Arya, S., Gilman, A. G., and Siderovski, D. P. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13307-13312[Abstract/Free Full Text]
24. Levay, K., Cabrera, J. L., Satpaev, D. K., and Slepak, V. Z. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 2503-2507[Abstract/Free Full Text]
25. Snow, B. E., Betts, L., Mangion, J., Sondek, J., and Siderovski, D. P. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6489-6494[Abstract/Free Full Text]
26. Posner, B. A., Gilman, A. G., and Harris, B. A. (1999) J. Biol. Chem. 274, 31087-31093[Abstract/Free Full Text]
27. Makino, E. R., Handy, J. W., Li, T., and Arshavsky, V. Y. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1947-1952[Abstract/Free Full Text]
28. Cabrera, J. L., de Freitas, F., Satpaev, D. K., and Slepak, V. Z. (1998) Biochem. Biophys. Res. Commun. 249, 898-902[CrossRef][Medline] [Order article via Infotrieve]
29. Zhang, J. H., and Simonds, W. F. (2000) J. Neurosci. 20, RC59[Medline] [Order article via Infotrieve] (1-5)
30. Rumenapp, U., Blomquist, A., Schworer, G., Schablowski, H., Psoma, A., and Jakobs, K. H. (1999) FEBS Lett. 459, 313-318[CrossRef][Medline] [Order article via Infotrieve]
31. Mao, J., Yuan, H., Xie, W., and Wu, D. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12973-12976[Abstract/Free Full Text]
32. Chatterjee, T. K., and Fisher, R. A. (2000) J. Biol. Chem. 275, 24013-24021[Abstract/Free Full Text]
33. Bowman, E. P., Campbell, J. J., Druey, K. M., Scheschonka, A., Kehrl, J. H., and Butcher, E. C. (1998) J. Biol. Chem. 273, 28040-28048[Abstract/Free Full Text]
34. Dulin, N. O., Sorokin, A., Reed, E., Elliott, S., Kehrl, J. H., and Dunn, M. J. (1999) Mol. Cell. Biol. 19, 714-723[Abstract/Free Full Text]
35. Druey, K. M., Sullivan, B. M., Brown, D., Fischer, E. R., Watson, N., Blumer, K. J., Gerfen, C. R., Scheschonka, A., and Kehrl, J. H. (1998) J. Biol. Chem. 273, 18405-18410[Abstract/Free Full Text]
36. Srinivasa, S. P., Bernstein, L. S., Blumer, K. J., and Linder, M. E. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5584-5589[Abstract/Free Full Text]
37. Chen, C., Seow, K. T., Guo, K., Yaw, L. P., and Lin, S. C. (1999) J. Biol. Chem. 274, 19799-19806[Abstract/Free Full Text]
38. Bernstein, L. S., Grillo, A. A., Loranger, S. S., and Linder, M. E. (2000) J. Biol. Chem. 275, 18520-18526[Abstract/Free Full Text]
39. Vernet, T., Dignard, D., and Thomas, D. Y. (1987) Gene (Amst.) 52, 225-233[CrossRef][Medline] [Order article via Infotrieve]
40. Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211-212[Medline] [Order article via Infotrieve]
41. Druey, K. M., Blumer, K. J., Kang, V. H., and Kehrl, J. H. (1996) Nature 379, 742-746[CrossRef][Medline] [Order article via Infotrieve]
42. Robinson, A., and Austen, B. (1987) Biochem. J. 246, 249-261[Medline] [Order article via Infotrieve]
43. Benzing, T., Yaffe, M. B., Arnould, T., Sellin, L., Schermer, B., Schilling, B., Schreiber, R., Kunzelmann, K., Leparc, G. G., Kim, E., and Walz, G. (2000) J. Biol. Chem. 275, 28167-28172[Abstract/Free Full Text]
44. Popov, S. G., Krishna, U. M., Falck, J. R., and Wilkie, T. M. (2000) J. Biol. Chem. 275, 18962-18968[Abstract/Free Full Text]
45. Cocco, L., Martelli, A. M., Mazzotti, G., Barnabei, O., and Manzoli, F. A. (2000) Adv. Enzyme Regul. 40, 83-95[CrossRef][Medline] [Order article via Infotrieve]
46. Mahanty, S. K., Wang, Y., Farley, F. W., and Elion, E. A. (1999) Cell 98, 501-512[Medline] [Order article via Infotrieve]


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