Regulator of G Protein Signaling 8 (RGS8) Requires Its NH2 Terminus for Subcellular Localization and Acute Desensitization of G Protein-gated K+ Channels*

Functional roles of the NH2-terminal region of RGS (regulators of G protein signaling) 8 in G protein signaling were studied. The deletion of the NH2-terminal region of RGS8 (ΔNRGS8) resulted in a partial loss of the inhibitory function in pheromone response of yeasts, although Gα binding was not affected. To examine roles in subcellular distribution, we coexpressed two fusion proteins of RGS8-RFP and ΔNRGS8-GFP in DDT1MF2 cells. RGS8-RFP was highly concentrated in nuclei of unstimulated cells. Coexpression of constitutively active Gαo resulted in translocation of RGS8 protein to the plasma membrane. In contrast, ΔNRGS8-GFP was distributed diffusely through the cytoplasm in the presence or absence of active Gαo. When coexpressed with G protein-gated inwardly rectifying K+ channels, ΔNRGS8 accelerated both turning on and off similar to RGS8. Acute desensitization of G protein-gated inwardly rectifying K+current observed in the presence of RGS8, however, was not induced by ΔNRGS8. Thus, we, for the first time, showed that the NH2terminus of RGS8 contributes to the subcellular localization and to the desensitization of the G protein-coupled response.


Functional roles of the NH 2 -terminal region of RGS (regulators of G protein signaling) 8 in G protein signal-
ing were studied. The deletion of the NH 2 -terminal region of RGS8 (⌬NRGS8) resulted in a partial loss of the inhibitory function in pheromone response of yeasts, although G␣ binding was not affected. To examine roles in subcellular distribution, we coexpressed two fusion proteins of RGS8-RFP and ⌬NRGS8-GFP in DDT1MF2 cells. RGS8-RFP was highly concentrated in nuclei of unstimulated cells. Coexpression of constitutively active G␣ o resulted in translocation of RGS8 protein to the plasma membrane. In contrast, ⌬NRGS8-GFP was distributed diffusely through the cytoplasm in the presence or absence of active G␣ o . When coexpressed with G protein-gated inwardly rectifying K ؉ channels, ⌬NRGS8 accelerated both turning on and off similar to RGS8. Acute desensitization of G protein-gated inwardly rectifying K ؉ current observed in the presence of RGS8, however, was not induced by ⌬NRGS8. Thus, we, for the first time, showed that the NH 2 terminus of RGS8 contributes to the subcellular localization and to the desensitization of the G protein-coupled response.
RGS 1 (regulators of G protein signaling) proteins comprise a large family of more than 20 members that modulate heterotrimeric G protein signaling (1,2). This protein family was originally identified as a pheromone desensitization factor in yeast (3). Many members of RGS protein family were subsequently identified by virtue of a common stretch of 120 amino acids termed the RGS domain in organisms ranging from yeast to human (1,2,4,5). It was shown that several RGS proteins (RGS1, RGS3, RGS4, and GAIP) attenuate G protein signaling in cultures (4,6,7). Biochemical studies demonstrated that RGS members function as a GTPase-activating protein for ␣ subunits of heterotrimeric G proteins (8,9,10). Therefore, RGS proteins are proposed to down-regulate G protein signaling in vivo by enhancing the rate of G␣ GTP hydrolysis.
We previously searched for RGS proteins specifically expressed in neural cells using a culture system of neuronally differentiating P19 cells. We isolated cDNA of RGS8 and identified it as a RGS protein induced in differentiated P19 cells (11). In addition, since RGS7 had been reported to be expressed predominantly in the brain (5), we also isolated a full-length cDNA of RGS7 (12). Biochemical studies indicated that RGS8 binds to G␣ o and G␣ i3 , and that RGS7 binds to G␣ o , G␣ i3 , and G␣ z . To examine effects of each RGS protein on G protein signaling, we coexpressed a G protein-coupled receptor and a G protein-coupled inwardly rectifying K ϩ channel (GIRK1/2) (13)(14)(15) in Xenopus oocytes and analyzed the activation and deactivation kinetics. We observed that RGS8 significantly speeds up both activation and deactivation of GIRK current (11). Doupnik et al. (16) reported the similar accelerated kinetics of GIRK current by RGS1, RGS3, or RGS4. We further observed that RGS8 induces acute desensitization of receptor-activated GIRK current in the presence of ligands (11). In the case of RGS7, activation of GIRK current was clearly accelerated as with RGS8, but the acceleration effect on deactivation was significantly weaker than that of RGS8. The acute desensitization of receptor-activated GIRK observed with RGS8 was not apparent with RGS7. Thus, RGS7 and RGS8 were shown to accelerate G protein-mediated modulation of GIRK current differentially (12). What is the structural basis for the weaker off acceleration and reduced desensitization in the case of RGS7? One possibility is that a difference in the NH 2 -terminal domain contributes to the differential modulation of GIRK current. The conserved NH 2 -terminal domains of RGS4 and RGS16 were recently shown to be important for membrane association and biological function by the analysis of the ability to inhibit pheromone signaling in the budding yeast (17)(18)(19). A homologous domain was also found in the NH 2 terminus of RGS8. Therefore, we investigated functional roles of the NH 2terminal domain of RGS8 in G protein signalings. We examined effects of deletion of this NH 2 -terminal domain on the pheromone signaling of yeasts, the G␣ binding, the subcellular distribution, and the modulation of the receptor-activated GIRK current.

EXPERIMENTAL PROCEDURES
Yeast Pheromone Response Assay-A bioassay was used to measure the sensitivity of the pheromone response in yeast that expresses RGS proteins as described (20). A DNA fragment containing the Myc tag (MEQKLISEEDLSRGS) was introduced into pTS210 yeast expression vector under the control of a galactose-inducible promoter. By polymerase chain reaction amplification, cDNA fragments containing the coding sequence of RGS8 or ⌬NRGS8 (mutant RGS8 which lacks NH 2terminal 35 amino acids) were isolated. After confirmation by sequencing analysis, they were fused in-frame immediately downstream of the Myc tag in pTS210. The sst2 deletion mutant yeast SNY86 (21) was transformed with each cDNA in pTS210 and selected on ura Ϫ dropout plates. Independent colonies of each yeast transformant were grown and a halo bioassay was performed.
Western Blotting of Epitope-tagged RGS Proteins-Single colonies of yeasts transformed with Myc-tagged RGS constructs were inoculated into ura Ϫ dropout medium supplemented with 2% galactose or glucose and were grown to an identical density (A 600 ϭ 1). Yeast cells were precipitated and lysed in SDS sample buffer. Proteins were extracted by sonication, separated on SDS-polyacrylamide gels, and transferred to nitrocellulose membranes. Expression levels of Myc-tagged RGS proteins were then examined by Western blotting using anti-Myc tag monoclonal antibody (9E10, BabCO). Signals were detected with an ECL system (Amersham Pharmasia Biotech).
Immunoprecipitation-By polymerase chain reaction amplification, cDNA fragments for RGS8 and ⌬NRGS8 were isolated. For ⌬NRGS8, a forward primer that included the ATG start codon was used. After confirmation of their nucleotide sequences, they were cloned into pCXN2 expression vector provided by Professor Miyazaki (22). Biotinylated proteins were generated by in vitro transcription/translation of the resultant plasmid DNAs using the TNT-coupled reticulocyte system (Promega) and biotinylated lysine-tRNA complex (Transcend TM tRNA, Promega). Each biotinylated protein was mixed with purified bovine G␣ o in binding buffer containing 20 mM HEPES, pH 8.0, 0.1 M NaCl, 1 mM dithiothreitol, 6 mM MgCl 2 , 10 M GDP, 30 M AlF 4 Ϫ (30 M AlCl 3 , 10 mM NaF), and 0.1% polyoxyethylene 10-lauryl ether (C 12 E 10 ). After incubation for 4 h at 4°C, the reaction mixture was precleared with 50% (v/v) protein G-Sepharose for 1 h at 4°C, incubated with anti-bovine G␣ o antibody for 2 h at 4°C, and then cleared with 50% (v/v) protein G-Sepharose for an additional 2 h. Protein G beads were washed four times in the binding buffer, suspended in SDS sample buffer, and boiled for 5 min. Proteins were separated by SDS-polyacrylamide gel and transferred to nitrocellulose paper. Co-immunoprecipitation of biotinylated protein was examined with streptavidine-horseradish peroxidase (Promega). Immunoprecipitation of G␣ o was confirmed by Western blotting using anti-G␣ o antibody (Santa Cruz Biotechnology). Signals were detected with an ECL system (Amersham Pharmasia Biotech).
Cell Fractionation-A Syrian hamster leiomyosarcoma cell line, DDT1MF2, was grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. RGS8 and ⌬NRGS8 expression constructs were described in the previous section on immunoprecipitation. The plasmid DNAs of these expression constructs were transfected into DDT1MF2 cells by the CaPO 4 methods (23). After selection in the presence of G418 (0.8 mg/ml, Life Technologies, Inc.), stable lines were established. The cultured cells were sonicated in 50 mM Tris acetate buffer, pH 7.5, containing 10 mM MgCl 2 , 1 mM dithiothreitol, 1 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride and the homogenate was centrifuged (44,000 ϫ g, 20 min, 4°C). The resultant supernatant and precipitate were mixed with SDS sample buffer and used as cytosolic and particulate fractions.
Chimeric cDNAs of RGS8 and Fluorescent Protein-We expressed RGS8 and ⌬NRGS8 as a chimeric protein with fluorescent protein at the carboxyl terminus. By polymerase chain reaction amplification, cDNA fragments for RGS8 and ⌬NRGS8 were isolated. For ⌬NRGS8, a forward primer that included the ATG start codon was used. After confirmation by sequencing analysis, RGS8 cDNA was fused in-frame to red fluorescent protein (RFP) in pDsRed1-N1 (CLONTECH) or in one particular experiment to green fluorescent protein (GFP) in pEGFP-N1 (CLONTECH). ⌬NRGS8 was ligated in-frame to the beginning of GFP in pEGFP-N1. Resultant plasmid DNAs were transiently transfected into DDT1MF2 cells using FuGENE 6 (Roche Molecular Biochemicals). At 48 h after the transfection, transfected cells were fixed by treatment with 4% paraformaldehyde for 30 min. Confocal microscopy was performed with a Zeiss microscope connected with an LSM410 Laser Scan-ning Confocal Microscope (Zeiss). The confocal images were collected using a ϫ63 objective. For a three-dimensional reconstructed image, transfected cells were examined with a LSM510 Laser Scanning Confocal Microscope (Zeiss) and stacks of 35 images spaced by 0.35 m were recorded.
Two-electrode Voltage Clamp-Two-electrode voltage clamp analysis was done as described previously (11). The curve fittings of turning on and off phases were done by a fitting function of pClamp software (Axon) based on the Simplex method. The turning on phases were fitted with a single-exponential function. As the turning off phases of RGS8 and ⌬NRGS8 data were fitted better with a two-exponential function, the fittings of them were done with a two-exponential function. When the turning off time course of the data of RGS(Ϫ) was fitted with a double-exponential function, the contribution of the slower component was small, and the time constant could not be determined reliably. Thus, the turning off phases of the data of RGS(Ϫ) were fitted with a single-exponential function.

The NH 2 -terminal Domain of RGS8 Is Required for Its Full
Ability to Function in Yeasts-In the NH 2 -terminal region of RGS4 and RGS16 outside of the RGS domains, the sequence conservation was found, and it was shown that the deletion of this region reduced the effect to attenuate pheromone signaling in yeasts (17,18). Sequence comparison revealed that the NH 2 terminus of RGS8 is similar to the conserved NH 2 -terminal sequences found in RGS4, RGS16, and also RGS5 (Fig. 1). However, the conserved cysteine residues (Cys-2, Cys-12) which were reported to be palmitoylated (18) are absent in the NH 2 terminus of RGS8. To define the role of the NH 2 -terminal domain of RGS8, we analyzed the ability of RGS8 mutant lacking the NH 2 -terminal 35 amino acids (⌬NRGS8) to inhibit the pheromone response pathway in Saccharomyces cerevisiae, as shown in Fig. 2A. Wild type RGS8 inhibited the growth arrest response to mating pheromone. Deletion of the NH 2 terminus 35 amino acids resulted in a partial loss of function; i.e. ⌬NRGS8 could not efficiently attenuate the effect of pheromone signaling. This effect of removal of the NH 2 terminus on RGS8 function was confirmed by comparing the size of the halos of growth inhibition of cell lawns grown on agar plates (Fig. 2B). Thus, it is clear that the NH 2 -terminal region of RGS8 is required for its full ability to function in yeasts. Expression levels of wild and mutant RGS8 proteins in yeast cells grown in galactose were determined by Western blotting using anti-Myc antibody (Fig. 2C). Similar expression levels were detected, indicating that the functional difference was not due to impairment of expression. The specificity of the antibody was tested by using yeasts grown in glucose (data not shown).
⌬NRGS8 Retains G protein Binding Activity-We examined whether NH 2 -terminal deletion affects the G protein binding activity of RGS8. Biotin-labeled proteins of the wild type and ⌬NRGS8 were generated by in vitro transcription and translation. Each labeled RGS8 protein was incubated with purified bovine G␣ o in the presence of GDP and AlF 4 Ϫ . The complex containing G␣ o was immunoprecipitated using anti-G␣ o antibody. Similar to wild type RGS8, ⌬NRGS8 was recovered with G␣ o (Fig. 3). From these results, it was clearly demonstrated that the NH 2 -terminal deletion of RGS8 does not affect G protein binding activity.  (28), mouse RGS5 (28), and rat RGS8 (11) are aligned. Conserved amino acid residues are boxed in black. NH 2 -terminal Deletion Shifts the Distribution of RGS8 from the Particulate to Cytosolic Fraction-How can the NH 2 -terminal domain modulate the function of RGS8 without changing the properties of interaction with G␣? It has been demonstrated that the NH 2 -terminal domains of RGS4 and RGS16 serve as a membrane targeting sequence in yeasts (18,19). To determine whether the NH 2 -terminal domain of RGS8 influences its intracellular distribution or not, wild type RGS8 or ⌬NRGS8 cDNA was transfected to DDT1MF2 cells, which do not express endogenous RGS8, and their cell homogenates were fractionated. Cytoplasmic and particulate fractions were subjected to Western blotting analysis using the RGS8-specific antibody (Fig. 4). The specificity of the antibody was determined by Western blotting of whole cell extracts from control cells and cells transfected with RGS8 cDNA. Wild type RGS8 was present in the particulate fraction. In contrast, ⌬NRGS8 was abundantly detected in the cytoplasm. In both transfected cells, G␣ q/11 was exclusively present in the particulate fraction. These results clearly demonstrated that the NH 2 -terminal domain of RGS8 is required for its subcellular distribution. NH 2 -terminal Domain Is Required for the Nuclear Localization and the G Protein-activated Translocation of RGS8 -To further investigate the cellular distribution of RGS8 in cultured cells and also to examine roles of its NH 2 terminus, two different fluorescent proteins were fused to RGS8 and ⌬NRGS8, respectively, and they were coexpressed in DDT1MF2 cells. RGS8 was expressed as a chimeric protein with RFP at the carboxyl terminus and ⌬NRGS8 was coexpressed as a chimeric protein with GFP. Surprisingly, RGS8-RFP was highly concentrated in nuclei of most transfected cells (Fig. 5, upper). On the other hand, ⌬NRGS8-GFP was diffusely distributed through the cytoplasm within identical cells (Fig. 5,  middle). These observations were consistent with the analysis using the cell fractionation method (Fig. 4), since the particulate fraction contains nuclei and cell membranes. These results clearly demonstrated that RGS8 is localized in nuclei in DDT1MF2 cells, and that the NH 2 -terminal domain of RGS8 is required for this unique nuclear localization. Sequence comparison revealed that the NH 2 terminus of RGS8 is similar to the conserved NH 2 -terminal sequences of RGS4 and RGS16, but that the first 7 amino acids of RGS8 are unique (Fig. 1). A search for the subcellular localization sites of protein identified a putative nuclear localization signal sequence, PRRNK at amino acids 7-11. This nuclear localization signal sequence was thought to function in the distribution of RGS8 in nonactivated DDT1MF2 cells. The nuclear localization of RGS8 was further confirmed by an immunofluorescence method. DDT1MF2, NIH3T3, and HEK293 cells were transfected with cDNA of wild type RGS8 without fluorescent protein and were immunostained with anti-RGS8 antibody. In all cases, similar nuclear localization of RGS8 was observed (data not shown).
RGS proteins interact directly with G␣ in its active state (9, 10) and the heterotrimeric G protein complex is mainly associated with cell membranes. Since RGS8 was found to be localized to nuclei in DDT1MF2 cells, it is considered to be unable to efficiently interact with G␣ in the cells activated by stimulation of G proteins. To investigate the subcellular localization of RGS8 after G protein activation, a constitutively active G␣ was further coexpressed in DDT1MF2 cells. We previously showed that RGS8 binds preferentially to G␣ o and G␣ i3 (11). When G␣ o Q205L was further coexpressed for 48 h, a marked translocation of RGS8 to the plasma membrane was observed (Fig. 6,  upper). In many cells, RGS8-RFP was concentrated to unique membrane structures that may correspond to membrane ruffles or microprojections and to the cell periphery. The nuclear localization of RGS8-RFP, which was intense in unstimulated cells, tended to decrease or disappear in G␣ o Q205L-expressing cells. The distribution of ⌬NRGS8-GFP, however, was not significantly influenced by expression of active G␣ o and ⌬NRGS8-GFP showed a relatively uniform pattern of distribution (Fig. 6, middle). In control cells transfected with RFP cDNA, a diffuse and sometimes punctuate pattern was observed and the distribution was clearly different from that of RGS8-RFP. Coexpression of G␣ o Q205L had almost no effect on the distribution of RFP. Thus, RGS8 was shown to be translocated to plasma membrane by G protein-activated signalings. It is clear that the NH 2 -terminal domain of RGS8 is required for this translocation. ⌬NRGS8 could bind to G␣ o , but it could not translocate to the membrane on coexpression of G␣ o Q205L. Therefore, it was thought that the membrane recruitment of RGS8 was not a direct result of the physical association with G␣. We further investigated the morphological structure of the region to which RGS8 was translocated on coexpression of G␣ o Q205L by confocal microscopy. Three-dimensional reconstitution of confocal slice images revealed that the structures where RGS8 accumulated as indicated in Fig. 6, left, were fine projections or microspikes present on the surface of DDT1MF2 cells (Fig. 7).

RGS8-induced Desensitization of the Receptor-activated GIRK Current Was Abolished by Deletion of Its NH 2 -terminal
Domain-GIRK channels are known to be activated directly by G␤␥ subunits released by the G i family. They are activated by G protein-coupled receptors such as m2 muscarinic and D2 dopamine receptors. We previously coexpressed GIRK1/GIRK2 heteromultimer and m2 muscarinic receptor with or without RGS8 in Xenopus oocytes and analyzed the effects of RGS8 on G protein-mediated modulation of K ϩ currents. We reported FIG. 4. NH 2 -terminal deletion shifts the distribution of RGS8 from the particulate to cytosolic fraction. A, DDT1MF2 cells expressing RGS8 or ⌬NRGS8 were cultured and fractionated. The resultant cytosolic (C) and particulate (P) fractions were separated on SDSpolyacrylamide gel and stained with Coomassie Brilliant Blue. B, after electrophoresis, both fractions were also transferred to nitrocellulose paper. Western blotting using anti-RGS8 antibody (top) and anti-G␣ q/11 antibody (bottom) was performed.
FIG. 5. Nuclear localization of RGS8. RGS8 and ⌬NRGS8 were transiently coexpressed in DDT1MF2 cells. RGS8 was expressed as a chimeric protein with RFP at the carboxyl terminus (RGS8) and ⌬NRGS8 was coexpressed as a chimeric protein with GFP (⌬NRGS8). At 48 h after transfection, confocal images were collected using a ϫ 63 objective lens. Bar, 10 m. that RGS8 speeds up the activation and deactivation kinetics of GIRK upon receptor stimulation. We also reported the RGS8induced acute desensitization of the receptor-activated GIRK current (11,12). Here, we examined the functional role of the NH 2 -terminal domain, which determines subcellular localization, in the RGS8-mediated modulation of on-off kinetics of GIRK current. We coexpressed RGS8 or ⌬NRGS8 with GIRK1/ GIRK2 heteromultimer and m2 muscarinic receptor in Xenopus oocytes. ⌬NRGS8 coexpression accelerated both the turning on and off to a similar extent as the wild type RGS8. The acute desensitization of receptor-activated GIRK current, however, was much less clear in oocytes expressing ⌬NRGS8 (Fig.  8A). These observations were confirmed quantitatively by comparing the time constants of the exponential functions used for the fitting of the activation ( on ) and deactivation ( off ) phases (detail in the legends of Fig. 8B) and the extent of desensitization (Fig. 8B). Thus, we identified a novel function of the NH 2 -terminal domain of RGS8 to cause acute desensitization of GIRK current activated by receptor stimulation.

DISCUSSION
In this study, we demonstrated the functional significance of the NH 2 -terminal domain of RGS8. First, we compared this region of RGS8 with those of RGS4, RGS5, and RGS16 and identified a conserved sequence in the NH 2 terminus. By using a yeast halo assay, we showed that the deletion of the NH 2terminal domain of RGS8 results in a partial loss of function to inhibit the pheromone response pathway. We used pTS210, a single-copy expression vector under the control of a galactoseinducible promoter. When a higher concentration of galactose or multicopy vector was used, we could not detect a clear difference in the sensitivity to the mating pheromone of yeasts carrying RGS8 and ⌬NRGS8. Under these conditions of overexpression, a proper subcellular distribution might not be nec-essary for sufficient function of RGS8 because of high amount within the cells.
We unexpectedly found that RGS8 is predominantly localized in the nuclei of DDT1MF2 cells. This nuclear localization of RGS8 was shown by its fusion protein with a fluorescent protein (GFP or RFP) and further confirmed immunocytochemically with anti-RGS8 antibody. Similar nuclear localization of RGS8 was observed in 3T3 and HEK293 cells transfected with RGS8 cDNA. We, moreover, showed that the NH 2 -terminal domain of RGS8 is required for its nuclear localization within cells. In the case of RGS4, despite having a similar conserved sequence in the NH 2 terminus (Fig. 1), a predominant localization in the cytoplasm has been reported using HEK293 cells transfected with RGS4 cDNA (24). Considering the subcellular distributions of RGS8 and RGS4, it is possible that the first 7 amino acids in the NH 2 terminus of RGS8 are responsible for the unique nuclear localization. Indeed, we found a potential NLS sequence at amino acids 7-11. Quite recently, a truncated isoform of RGS3, termed RGS3T, has been reported to be localized to the nucleus and induce apoptosis in RGS3T-transfected Chinese hamster ovary cells (25). Two potential nuclear localization signal sequences were found in the NH 2 terminus of RGS3T and truncation of the NH 2 terminus resulted in a reduction of nuclear localization. RGS8 in nucleus might also function in cellular processes such as apoptosis.
We showed that RGS8 is translocated from the nucleus to the plasma membrane structures by coexpression of G␣ o Q205L in DDT1MF2 cells. The NH 2 -terminal domain of RGS8 was shown to play critical roles in this membrane recruitment, since mutant RGS8 lacking the NH 2 -terminal sequence of 35 amino acids could not translocate to the membrane within identical cells. This ⌬NRGS8 contains an intact RGS domain, which is sufficient for the interaction with G␣. Therefore, the mechanism of the membrane translocation was considered to be independent of RGS8-G protein interaction. RGS4 was also reported to be recruited from the cytoplasm to the plasma membrane on the expression of activated, GTPase-deficient G␣ i2 (24). It was shown that a non-G␣ binding mutant of RGS4 could translocate like wild type RGS4, indicating that this translocation was not a direct result of physical association with an activated G␣. It is possible that G protein activation triggers binding of unidentified cellular factors to the subcellular localization signals of the NH 2 terminus of RGS proteins for translocation to the membrane.
When coexpressed in Xenopus oocytes with a G proteincoupled receptor and GIRK1/2, ⌬NRGS8 accelerated the turning on and off of GIRK current upon receptor stimulation similar to wild type RGS8. Wild type RGS8 induced significant levels of acute desensitization of the response during receptor activation, but the desensitization in oocytes expressing ⌬NRGS8 was similar to that in control oocytes. These results demonstrated that the NH 2 -terminal domain of RGS8 is required to cause acute desensitization of GIRK. Indeed, we previously reported that RGS7, which contains the characteristic long NH 2 terminus different from that of RGS8, showed slower desensitization. Kovoor et al. (26) also made similar recordings of receptor-activated GIRK current in Xenopus oocytes expressing RGS proteins. Their data indicated that RGS4 induces acute desensitization, but that RGS7 and RGS9 do not cause significant desensitization. RGS4 has a similar NH 2terminal sequence to RGS8, and both RGS7 and RGS9 possess the long NH 2 terminus containing DEP (Dishevelled/EGL10/ pleckstrin homology) and GGL (G protein ␥ subunit-like) domains instead of the short conserved NH 2 terminus of RGS8.
What is the mechanism behind the desensitization in the presence of RGS8? If we assume that there are only two forms of G protein (nonactive and active) and two rate constants (␣, on-rate; ␤, off-rate), the current amplitude is expected to increase single exponentially with a time constant of 1/(␣ϩ␤). In this case, no desensitization is expected to occur. To explain the presence of desensitization in the two-state model, it is neces-sary to assume a gradual decrease in the on-rate or a gradual increase in the off-rate in the course of the receptor stimulation. As the turn-on speed, determined dominantly by ␣, was not significantly decreased by the second ligand application immediately after the initial trial (data not shown), it appears FIG. 7. Three-dimensional reconstructed image of RGS8 translocated by G protein activation. RGS8 was expressed as a chimeric protein with GFP in DDT1MF2 cells. A constitutively active G␣ o cDNA (RGS8 ϩ G␣ o Q205L) or control vector (RGS8ϩvector) was co-transfected. At 48 h after transfection, confocal images were collected and a three-dimensional reconstructed image was obtained. The obtained image was rotated.
FIG. 8. Effects of RGS8 and ⌬NRGS8 on turning on and turning off kinetics, and on the desensitization of GIRK current upon stimulation of m2 muscarinic receptor. A, GIRK1/2 and m2 muscarinic receptor without (upper trace) or with RGS8 (middle trace) or with ⌬NRGS8 (lower trace) were coexpressed in Xenopus oocytes. Current traces at a holding potential of Ϫ80 mV are shown. 10 M ACh was applied at the time indicated by the bars. B, Comparison of the time constants on (top) and off (middle), and the desensitization level (bottom) of the GIRK 1/2 current upon stimulation of m2 muscarinic receptor in the absence (left) or presence of RGS8 (middle) or ⌬NRGS8 (right). The turning on phases of three groups were fitted with a single-exponential function, and the time constants of them were compared ( on ). The turning off phases of RGS(Ϫ) data were fitted with a single exponential function. In contrast, a two-exponential function was used to fit those of the data with RGS8 and ⌬NRGS8 satisfactorily. The time constants of the fast (and major) component of them were compared with that of the single time constant of RGS(Ϫ) data ( off ). on and off values of RGS8 and ⌬NRGS8 were significantly different from those without RGS protein (average and S.D. are shown, n ϭ 9 or 10, p value Ͻ 0.05) by Student's unpaired t test. The level of desensitization was measured as a percentage of the reduction of the induced current after 1 min of ligand application. Values of RGS8 were significantly different from those with ⌬NRGS8 or from those without RGS (average and S.D. are shown, n ϭ 9 or 10, p value Ͻ 0.05) by Student's unpaired t test. that the on-rate was not decreased during the ligand stimulation. If the off-rate is increased during the response, the turning off after ligand washout should be faster with RGS8, which showed a more intense desensitization. As the turning off speed after ligand washout was similar between RGS8 and ⌬NRGS8, the off-rate was not increased during the response. Thus, the desensitization could not be explained by a gradual change of the on-or off-rate. What then is the mechanism of desensitization in the presence of RGS8? With the assumption that there are three states, a nonactive nonready state, a nonactive ready state, and an active state, the desensitization could be explained by the depletion of the ready pool as discussed by Chuang et al. (27). The presence of a large ready pool enables the acceleration of the turning on, but at the same time the depletion of the ready pool during the response could result in the acute desensitization even if the on-rate and off-rate are not changed. If this is the case, the NH 2 -terminal region of RGS8 is thought to be necessary for the formation of the ready pool and for its depletion during the response. It is speculated that the NH 2 terminus of RGS8 controls the G protein pool by controlling its subcellular distribution, although further study is required to understand how this enlarged G protein pool is regulated by RGS proteins.