INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The G-protein-linked signaling strategy has evolved to sense chemoattractants in cells ranging from Dictyostelium discoideum to mammalian leukocytes (Devreotes and Zigmond, 1988). In these cells, chemoattractants elicit sharp rises in the proportion of polymerized actin as well as the phosphorylation of myosins, the production of cyclic nucleotides, and the activation of mitogen-activated protein kinases and STATs (Berlot et al., 1985; Caterina and Devreotes, 1991; Thompson et al., 1994; Lai et al., 1996; Maeda et al., 1996; Parent and Devreotes, 1996; Krump et al., 1997; Araki et al., 1998). Evidence suggests that it is the -complexes, released by receptor-catalyzed exchange of GTP for GDP on -subunits, which directly regulate effectors (Wu et al., 1995; Neptune et al., 1997; Jin et al., 1998). The specific G-proteins involved in chemotaxis consist of _i in mammals and 2 or 4 in D. discoideum (Kumagai et al., 1989; Hadwiger et al., 1994). In mammals, it is not certain which of the -complexes is key, whereas in D. discoideum, there is a single -subunit. Genetic evidence shows that 2 and  are essential for many responses to chemoattractants; it is expected that the G is also essential (Kumagai et al., 1989; Wu et al., 1995). However, the anticipated G has not been identified in spite of considerable effort by degenerate PCR analysis and database searching.

G-subunits play multiple roles in G-protein signal transduction. Not only are they required for proper G folding and function, the sequence diversity of Gs also contributes to the specificity of G-protein signaling (Iniguez-Lluhi et al., 1992). Specific G subtypes appear to interact with different effectors (Yan et al., 1996). Perhaps most significantly, -subunits determine the subcellular distribution of the G complex because isoprenylation of the COOH-terminal of Gs targets G to the membrane (Muntz et al. 1992). Interestingly, a G-like-domain (GGL) has been found in a subgroup of mammalian RGSs, indicating that -subunits can also have additional partners in the cytosol (Cabrera et al., 1998).

The structures and functions of heterotrimeric G-proteins are highly conserved from yeast to human, but the sequences of -subunits are quite divergent among different species (Gautam et al., 1998). Gs have seven WD repeats at their COOH-terminal that fold into -propeller structures, containing the binding sites for Gs and effectors (Ford et al., 1998; Jin et al., 1998; Li et al., 1998). The NH₂-terminal regions of Gs are variable and form tight coiled-coil structures with the diverse Gs. Because Gs share little homology among diverse species, our strategy was to purify G, isolate a DNA probe based on its amino acid sequence, and then obtain its cDNA by library screening. A g cell line was rescued with a His-tagged G, and the resulting cell line was used to purify the functional His-G heterodimer. The isolated G allowed us to characterize its function and explore its role in chemoattractant and G-protein-mediated responses.

G-protein-mediated responses can be induced by stimuli presented either as increments or as spatial gradients. Increments of chemoattractant elicit uniform responses around the cell periphery, whereas gradients elicit responses on the side of the cell that faces the higher chemoattractant concentration (Parent et al., 1998). When there is a polarized distribution of Gs, uniform increments of stimuli result in a similar polarized recruitment of downstream signaling molecules (Jin et al., 2000). Endogenous G proteins have been found both on the membrane and in the cytosol. Membrane localization may be critical for localized activation of cells in gradients. Previous studies have shown that nonisoprenylated G₁ mutant can interact with G₁ and is confined in cytosol (Simonds et al., 1991; Iniguez-Lluhi et al., 1992). To test the functional role of membrane localization, a CSVL-deleted G (G) construct was designed to compete with endogenous G and to shift the membrane G subunits to the cytosol. The consequences of G on responses to increments and gradients were examined.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials

Restriction enzymes were purchased from New England Biolabs (Beverly, MA), Boehringer Mannheim (Indianapolis, IN), or Promega Inc. (Madison, WI); Taq polymerase from Perkin Elmer-Cetus (Norwalk, CT); TA cloning kit from Invitrogen (San Diego, CA); Chelating-Sepharose and DEAE-Sepharose from Pharmacia Biotech (Piscataway, NJ); BCA protein assay kit from Pierce (Rockford, IL); cyclic-AMP ³H assay system and cyclic-GMP ³H assay system from Amersham (Arlington Heights, IL). Polyclonal anti-G and anti-G2 antibodies were made in our laboratory. All other reagents were reagent grade and were obtained from standard suppliers.

Cell Culture and Development

Cells were grown in shaken suspension (Watts and Ashworth, 1970) or on SM agar plates with Klebsilla aerogenes lawn (Kay, 1987). Cells were developed in shaken suspension at 2 × 10⁷/ml in development buffer (DB; 10 mM Na/K phosphate, 0.2 mM CaCl₂, 2 mM MgSO₄, pH 6.5) for 1 h and then pulsed with 50 nM cAMP at a 6-min intervals for 5 h.

Protein Assays, SDS-PAGE, and Western Blotting

Protein assays were carried out according to the protocol provided by Pierce. High percentage SDS-PAGE that separated low-molecular-weight proteins was carried out as described by Hoefer Scientific Instruments' protocol (San Francisco, CA). Western blotting was carried out as described in Current Protocol in Molecular Biology with minor modification. PVDF membrane was substituted for nitrocellulose; the transfer was performed at 300 mA at 4°C for 3 h; the secondary antibody was a horseradish peroxidase (HRP), anti-rabbit IgG conjugate (Sigma Chemical Co., St. Louis, MO). Band detection was achieved with the use of enhanced chemiluminescence (ECL).

Purification of G-subunit

G-subunits were copurified with HisG-subunits. The G cDNA, with six histidines fused to its NH₂-terminal, was cloned into the expression vector p88d1 (Hughes et al., 1994). The construct was transformed into g cells to create a HisG/g cell line. HisG/g cells were used to purify G-subunit. During the purification process, the total amount of protein was assayed by BCA protein assay (Pierce). The amount of G-subunits was estimated by Western analysis against G-subunit. Protease inhibitor cocktails, PIC I (1 mg/ml antipain, 2 mg/ml leupeptin, and 2 mg/ml aprotinin) and PIC II (1 mg/ml chymostatin and 1 mg/ml pepstatin in DMSO), were added to buffers to protect the G-protein subunits from degradation.

HisG/g cells were cultured in HL5 medium with 20 µg/ml G418, shaking at 200 rpm room temperature. When the cell density reached 5 × 10⁶ per ml, 24 liters of HisG/g were harvested. The harvested cells were collected by flow-through centrifugation, resuspended in DB to 2 × 10⁷ per ml, and shaken at room temperature for 3 h to reduce protease activities. The following purification steps were carried out at 4°C. Cells were washed with 16 liters of DB twice and resuspended into 1 liter of lysis buffer (LB; 2 mM EDTA, 200 mM sucrose, and 10 mM Tris, pH 8), and cells were lyzed by Parr Bomb. Lysates were centrifuged at 17,000 × g for 40 min. After discarding the supernatant, the pellet was washed with 1 liter LB. The cell pellet was then extracted in 3 liters of running buffer I (RBI; 250 mM NaCl, 1% NP-40, 0.1% of PIC I, 0.2% of PIC II, and 20 mM Tris, pH 7.5), centrifuged at 17,000 × g for 40 min, and the supernatant was saved for the following purification. The supernatant was mixed with 50 ml Ni²⁺-chelating resin. The mixture was stirred for 0.5 h to achieve the optimal binding and then loaded on a Bio-Rad column. The column was then washed with 250 ml RBI followed by 250 ml RBI plus 20 mM imidazole. The G-protein was eluted with 150 ml elution buffer I (EBI: RBI plus 250 mM imidazole). The eluate was diluted 10-fold with running buffer II (RBII; 10 mM NaCl, 1 mM EDTA, 1% NP-40, and 20 mM Tris, pH 7.5) and loaded on to a 30 ml DEAE-Sepharose column. The column was washed with 300 ml running buffer III (RBIII; 50 mM NaCl, 1 mM EDTA, 0.1% NP-40, and 20 mM Tris, pH 7.5). The G-protein-containing fraction was then eluted with RBII with 250 mM NaCl. The DEAE column eluates were dialyzed against running buffer IV (RBIV; RBI with 0.1% NP-40) and loaded on 1.5 ml freshly made Ni²⁺-chelating column. After washing the column with 15 ml of RBIV, the G-protein was eluted with 5 ml elution buffer III (EBIII; RBIV in which 0.1% NP-40 was replaced with 0.1% SDS). One hundred fifty microliters of eluates was loaded on a high percentage SDS-PAGE. There were three closely spaced Commassie Blue-stained bands in the 7- to 10-kDa region of the gel. The two upper bands were cut and sent for sequencing (Figure 1A).

View larger version (26K): [in this window] [in a new window]   Figure 1.   Isolation of G cDNA (A) A NH2-terminal 21 amino acid sequence of G was obtained and used to design PCR primers. A DNA probe between primers was isolated for the screening of a cDNA library. (B) The G cDNA was isolated and sequenced.

PCR and Screen cDNA Library

The PCR was performed in a 50 µl reaction mixture as described in PCR protocols with minor modifications. The sequences of primers were as follows:



The mixture contained 5 µl 10× PCR buffer (Life Technologies), 200 µM each dNTPs, 1.0 mM MgCl₂, 0.5 µM primers, template, and 3 U of Taq polymerase. The samples were placed in a DNA thermal cycler from Perkin Elmer-Cetus. The program comprised 5 cycles of low-temperature (40°C) annealing and 30 cycles of high-temperature (46°C) annealing. Screening cDNA was carried out as described in Current Protocols in Molecular Biology (Ausubel et al., 1987).

Isolation of G cDNA

Based on the NH₂-terminal amino acid sequence of G, four sets of degenerate PCR primers were designed. With the use of D. discoideum genomic DNA as templates, the PCR was carried out with four possible combinations of primers: 1A with 2A, 1A with 2B, 1B with 2A, and 1B with 2B. A 20-nucleotide sequence was isolated and used as a probe to screen cDNA library. The sequences of the probe were as follows:
Approximately 1.5 × 10⁵ phages from gt11 cDNA library, prepared from mRNA isolated at 2-4 h of development, were screened (Lilly et al., 1993). Fifty-two positive clones were detected and 4 independent clones were obtained after secondary and tertiary screens. They were picked for subcloning and DNA sequencing (Figure 1B).

Developmental Northern Blotting

Nine 35-mm tissue culture plates were filled with 2 ml DB agar each. Each plate was then covered with 1 × 10⁷ vegetative growing AX3 cells. Cell samples were collected at time 0, 2, 4, 6, 9, 12, 16, 20, and 30 h. Total RNA from each sample was isolated with the use of the Trizol reagents (BRL-Life Technologies, Rockville, MD). Twenty micrograms of total RNA from each time point was loaded onto a 1% formaldehyde gel and blotted onto nylon membrane. The full-length cDNA was random prime labeled, and the blot was probed according to the method described in Current Protocols in Molecular Biology. The quantitative measurement of bands was carried out by a FUJIFILM BAS1500 PhosphorImager (Fuji, Stamford, CT).

Plasmid Construction and Transformation

The hexyl-histidine was fused to the NH₂-terminal of G by PCR, and the resulting DNA fragment was cloned into D. discoideum extrachromosomal expression plasmid, p88d1. Four COOH-terminal amino acid residues of G were removed by PCR, and the resulting DNA fragment was cloned into p88d1 to make G plasmid. G cDNA was cloned into p88d1 to make G plasmid. PHcrac-GFP was the same as that in Parent et al. 1998. The transformation was carried out as described previously (Insall et al., 1994). The plasmid was electroporated into cells with the use of a Bio-Rad gene pulser, and the stable transformants were selected in 20 µg/ml G418.

Functional Assays

Amoebae aggregation was assayed on 35-mm nonnutrient agar as described previously (Devreotes et al., 1987). Chemotaxis assays and cAMP-binding assays were carried out according to the protocols by Xiao et al. (1997). In vitro adenylyl cyclase assays were performed as described by Theibert et al. (1986). In vivo adenylyl cyclase assays were performed as described by Parent et al. (1995). The production of cGMP was assayed by Amersham kits. Fluorescent microscopy analysis on live PHcrac-GFP:G/AX2 cells was performed as described (Parent et al., 1998).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Purification of the G-subunit and Isolation of Its cDNA

We reasoned that a hexylhistidine-tagged G-subunit, HisG, would form a tight association with endogenous G and could be used to isolate the complex. A plasmid expressing HisG was transformed into g cells, and its functionality and level of expression was assessed. Although g cells were completely defective in aggregation and most signaling events (Wu et al., 1995), HisG/g cells developed appropriately into spores and stalks (our unpublished results). The HisG protein was not largely overexpressed, consistent with previous data suggesting tight control of G expression (Lilly et al., 1993). Through purification of HisG, G was isolated from the membrane fractions of 10¹¹ cells (Table 1). The purification used sequential Ni²⁺-chelating, DEAE-Sepharose, and Ni²⁺-chelating columns, yielding an overall purification of 2000-fold. A high percentage SDS-PAGE provided an additional 10-fold of purification. There were three Commassie blue-stained bands in the 7- to 10-kDa region of the gel. These bands were absent in parallel samples from wide-type cells that lack HisG, indicating that they were released from the HisG by SDS (our unpublished results). The purified G-subunits were sequenced and found to contain identical NH₂-terminal sequences (Figure 1A).

View this table: [in this window] [in a new window]   Table 1.  G isolated from membrane fractions

The amino acid sequence data were used to isolate the G cDNA. Based on the NH₂-terminal amino acid sequence, four sets of degenerate PCR primers were designed. A 20-bp region of novel sequence isolated from PCR was used as a probe to screen the cDNA library. Four positive clones with identical sequences were isolated. The completed cDNA sequence is shown in Figure 1B. The cDNA sequence encodes a protein of 68 amino acids. Its COOH-terminal contains the sequence CSVL, which is the characteristic signal for isoprenylation. When compared with G-subunits from other species, the D. discoideum G shared 10-18% identity with Gs from Bos taurus, Rattus norvegicus, Caenorhabditis elegans, and Drosophila melanogaster (Figure 2A). The G-subunits from Saccharomyces cerevisiae showed less homology; only three residues could be aligned. Interestingly, a number of identical amino acid residues were conserved with the GGL domain of mammalian RGSs. A phylogenetic tree is shown in Figure 2B. The secondary structure program, PHD, predicted 2.5 alpha-helixes, which presumably would form coiled-coil domains with the NH₂-terminal of G.

View larger version (33K): [in this window] [in a new window]   Figure 2.   Homology of the Dictyostelium G-subunit to that of other species (A) D. discoideum, B. taurus, R. norvegicus, C. elegans, D. melanogaster G-subunits, and GGL domain of RGSs are aligned. (B) A phylogenetic analysis was carried out on G-subunits.

To investigate the expression of G throughout development, Northern analyses were carried out (Figure 3). Based on quantitative phosphorimage analyses, G and G showed identical patterns of expression. G and G mRNAs were expressed in vegetative cells, increased twofold within 6 h after the initiation of development, and gradually decreased after passing the mound stage at 10 h, reaching 20% of the growth level at the final stage. When exogenous cAMP was added at 6-min intervals to assist development, the increase of G expression was accelerated while the maximum expression levels remained unchanged.

View larger version (83K): [in this window] [in a new window]   Figure 3.   Northern blotting of G and G in development. Total RNA was isolated from cells at 0, 2, 4, 6, 9, 12, 16, 20, and 30 h of development. Twenty micrograms of RNA was loaded on each lane, and the blot was probed with the mixture of 32P-labeled G and G cDNA.

Overexpression of CSVL-deleted G Impairs Cell Aggregation

The terminal CSVL of G was deleted, and G was used as a tool to study G function. The G construct was transformed into wild-type AX2 cells. Sixteen clones were screen by Western blotting analysis, and the one with highest level of G was designated G/AX2 cell line (our unpublished results). We initially chose AX2 rather than AX3 cells because the partial duplication of chromosome 2 in AX3 might have complicated the analyses of G dominant negative effects. Later, we found that overexpression of the G construct caused similar defects in AX3 cells. Wild-type G was transformed into AX2 to create G/AX2. In all of the following experiments, both AX2 and G/AX2 cells were used as controls. Expression of G caused the endogenous membrane-associated G to shift significantly to the cytosol. In wild-type cells, 70% of G was on the membrane, whereas in G/AX2 cells, only 20% of G remained on membrane (Figure 4A). When G/AX2 cells were plated on the surface of nonnutrient agar, they failed to aggregate and remained as a smooth monolayer (Figure 4C). Repeated cAMP stimuli at 6-min intervals for 6 h did not alter this phenotype. These observations suggested that G/AX2 cells were defective in certain G-protein-mediated responses required for aggregation.

View larger version (38K): [in this window] [in a new window]   Figure 4.   Expression of G impairs membrane G-protein function. (A) Expression of G shifts G-subunit to cytosol. The distribution of G protein between membrane (M) and cytosol (C) in AX2 and G/AX2 cells was examined with Western blotting, with the use of G antibody as a probe. (B) The coupling of cAR1 and membrane G-protein in AX2, G/AX2, G/AX2, g, and cAR1/g cells was examined by the GTPS-induced inhibition of 3H-cAMP binding assays. The average results of three representative experiments were presented. (C) The aggregation assays were carried out on nonnutrient plates. The photo was taken at 0 and 24 h of development. Each assay was repeated at least three times, and their average is presented.

The shift of a portion of G to the cytosol significantly reduced receptor/G-protein coupling. We examined the ability of membrane associated G-proteins to regulate the binding of cAMP to cAR1. Typically, membranes contain a mixture of high- and low-affinity cAMP binding sites (Van Haastert et al., 1992). In the presence of GTPS, the high-affinity binding sites are lost because of the dissociation of cAR1 from the G-proteins. In membranes from g or cAR1/g cells, cAMP binding sites are insensitive to GTPS because in the absence of functional G-proteins, all the cAMP binding sites are low affinity (Figure 4B; Wu et al., 1995). As shown in Figure 4B, with wild-type and G/AX2 cells, ~70% of the apparent cAMP binding sites detected with 2 nM ³H-cAMP were sensitive to GTPS. With G/AX2 cells, only 30% of cAMP binding sites were sensitive to GTPS, suggesting that the functional membrane-associated G-protein level was significantly reduced.

G/AX2 Cells Fail to Carry Out Chemotaxis but Can Respond to Stimulus Increments

At the 5-h stage of development, wild-type cells acquire a series of characteristics associated with the differentiated state. A set of genes required for chemotaxis and aggregation are fully expressed (Firtel, 1995). The cell shape changes from rounded to elongated, and the cells show increased sensitivity toward external gradients. These cells carry out vigorous chemotaxis toward cAMP at concentrations from 10¹⁰ to 10⁶ M. As shown in Figure 5, when exposed to a cAMP gradient from a micropipette containing 1 µM cAMP, AX2 and G/AX2 cells moved toward the source and began to form an aggregate at the micropipette tip within 15 min. The g cells did not acquire an elongated shape and failed to carry out chemotaxis. In contrast, G/AX2 cells did acquire the characteristic elongated shape and motility of wild-type cells. In addition, cAR1 was expressed when they were stimulated repeatedly with cAMP (AX2: 1.0 ± 0.2 × 10⁵ cAMP-binding sites/cell; G/AX2: 1.3 ± 0.2 × 10⁵ cAMP-binding sites/cell). However, the G/AX2 cells were severely impaired in chemotaxis. They maintained random movements in the presence of a cAMP gradient and did not accumulate at the pipette tip (Figure 5). The same chemotaxis defects were observed in G/AX2 cells by repeating the assays with 0.1 or 10 µM cAMP in the micropipette (our unpublished results).

View larger version (75K): [in this window] [in a new window]   Figure 5.   G/AX2 cells fail to carry out chemotaxis. Cells at the 6-h stage of development were plated on a cover slide and a micropipette containing 1 µM cAMP was touched to its surface. The cell-movement images were taken by a CCD camera with Zeiss microscope. The left panels were taken when the micropipette first contacted the cover slide. The right panels were taken in 10 min later. Each chemotaxis assay was repeated at least three times.

We also monitored several biochemical responses of the cells to stimulus increments. Receptor-induced actin polymerization and the production of cAMP and cGMP are mediated by G-proteins. These responses are absent in g cells (Wu et al., 1995, Jin et al., 1998). In the G/AX2 cells, the cAMP-induced F-actin formation response was ~30% as large as that of AX2 (our unpublished results). Surprisingly, chemoattractant-induced adenylyl cyclase activation in G/AX2 cells reached nearly the same levels as that in wild-type cells (Figure 6A). The similar results were obtained by assaying adenylyl cyclase activation by GTPS in vitro (Figure 6B). Furthermore, in response to 100 nM cAMP stimuli, G/AX2 cells produced the same amounts of cGMP as AX2 cells (Figure 6C). Apparently, in G/AX2 cells, the adenylyl and guanylyl cyclase activation pathways are functional even when the majority of the G-protein is in cytosol.

View larger version (21K): [in this window] [in a new window]   Figure 6.   Adenylyl cyclase A and guanylyl cyclase assays. AX2, G/AX2, and G/AX2 cells were allowed to differentiate for 6 h with cAMP. (A) The adenylyl cyclase activity was recorded after stimulation with 100 nM cAMP stimuli at time 0 s. (B) The activation of adenylyl cyclase by GTPS was measured. (C) The accumulation of cGMP was recorded after stimulation with 100 nM cAMP. Each assay was repeated at least three times, and their average is presented.

YFP-G:G/AX2 Cells Fail to Carry Out Chemotaxis

To visualize the redistribution of G from membranes to cytosol, we expressed YFP-G in the presence and absence of G in g cells. When expressed in wild-type cells, the distribution of YFP-G alone is similar in all cells and reflects the 70% membrane/30% cytosol observed for endogenous G distribution (Jin et al., 2000; also see Figure 4A). Inclusion of G caused a variation among clones in the partitioning of YFP-G between the membrane and cytosol. Higher levels of G caused more G to shift to the cytosol. Among 16 independent YFP-G:G/AX2 clones examined, clone 12 had the lowest expression of G and clone 5 had eightfold higher expression (our unpublished results). In clone 12 cells, a large portion of YFP-G remained on the membrane, whereas in clone 5, most of YFP-G was in the cytosol (Figure 7, A and B). As shown in Figure 7, C and D, clone 12 cells were able to carry out normal chemotaxis, whereas clone 5 cells were severely impaired in chemotaxis. Furthermore, we examined the activation of adenylyl cyclase by GTPS. Even although the majority of YFP-G was in cytosol, the adenylyl cyclase of clone 5 was activated to the same level as that of clone 12 (our unpublished results). The YFP-G:G/AX2 data further confirmed that the expression of G could selectively impair chemotaxis, as opposed to the activation of adenylyl cyclase.

View larger version (36K): [in this window] [in a new window]   Figure 7.   YFP-G: G/AX2 cells fail to carry out chemotaxis. Two representative clones of YFP-G: G/AX2 were allowed to differentiate for 7 h. (A) YFP-G was predominantly distributed in the cytosol of clone 5 cells. (B) Clone 12 showed wild-type-like distribution of YFP-G. (C) Chemotaxis assay of clone 5 cells was carried out under a Zeiss inverted fluorescent microscope. At time 0 min, a micropipette containing 1 µM cAMP was positioned in a field of cells as indicated by the white star. The photographs were taken at times 0 and 15 min. (D) Chemotaxis assay of clone 12 cells was recorded. At time 0 min, the micropipette was positioned at the white star. These assays were repeated at least three times, and a representative assay is presented.

Incremental versus Gradient Stimulation

Cells are able to respond to chemoattractants presented as either increments or as stable gradients. As shown by Parent et al., GFP-tagged PH domains are useful tools for directly comparing the response to these two different types of stimulation (Parent et al. 1998). When an increment of cAMP is added, PH domains such as that of CRAC and protein kinase B (PKB) transiently associate with plasma membrane at the cell perimeter (Meili et al., 1999). In contrast, in an external cAMP gradient, the PH domains are persistently localized at the side of the cell facing the higher concentration of chemoattractant. These redistributions require functional G-proteins (Parent et al., 1998).

To examine the response of GFP-tagged PH domains in the G/AX2 cells, we coexpressed PHcrac-GFP and G in AX2 cells. The clone with highest expression of G was selected. When a 100 nM cAMP increment was applied uniformly to these cells, the cytosolic PHcrac-GFP transiently associated with the membrane (Figure 8A). Such translocation to the membrane was consistent with the fact that adenylyl cyclase can be activated in the G/AX2 cells. The dose dependence of this response was examined with the use of cAMP increments ranging from 10¹² to 10⁵ M. The translocation of PHcrac-GFP in mutant cells could be induced by as low as 10⁸ M cAMP, about one magnitude higher than that in wild-type cells (Figure 8B). In contrast, when exposed to a cAMP gradient from a micropipette containing 1 µM cAMP, the cells failed to carry out chemotaxis and the PHcrac-GFP remained in cytosol and did not associate with the membrane (Figure 8C). Changing the chemoattractant gradient with the use of 0.1 or 10 µM cAMP did not alter these assay results (our unpublished results). These data suggest that, although less sensitive, G/AX2 cells can sense stimulus increments. However, they are severely impaired in detecting shallow gradients of chemoattractant.

View larger version (25K): [in this window] [in a new window]   Figure 8.   Incremental versus gradient activation. PHcrac: G/AX2 cells were allowed to differentiate for 7 h with cAMP stimuli as described above. (A) At time 0 s, cells in 200 µl DB were stimulated with 20 µl of 1 µM cAMP and photographs were taken at 0 and 5 s. (B) The dose dependence of PHcrac-GFP translocation were assayed in PHcrac-GFP: G/AX2 (dashed line) and PHcrac-GFP/AX2 cells (solid line). The results were rated subjectively, with "" as no response and "+++" as maximum response. (C) A cell was moving in a cAMP gradient created by a micropipette containing 1 µM cAMP located 20 µm to the left of the cell. The photographs were taken at 5-s intervals. These assays were repeated more than six times on different cells, and no exception was observed.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We have isolated a putative G cDNA from D. discoideum based on its association with G. Its cDNA predicts a protein of 68 amino acids in length, similar to most G-subunits. The purification approach was the most expedient method to identify the G gene. When compared with other Gs sequences, the D. discoideum G contains few identical amino acids, the longest stretch of identity being three residues. In retrospect, the unsuccessful database searches and PCR approaches are understandable. The predicted secondary structure of G contains 2.5 -helixes as does transducin G (Sondek et al., 1996). In addition, a CSVL motif is found in its COOH-terminal, which is conserved in all G-subunits. Expression of a G containing a deletion of this motif causes the endogenous G of wild-type cells to relocate to the cytosol, providing convincing evidence of a functional interaction of G and G (Simonds et al., 1991). Furthermore, fluorescent resonant energy transfer (FRET) studies demonstrate the direct interaction of G and G in a living cell (Zhang et al., manuscript in preparation).

Our data suggest that there is one G-dimer in D. discoideum. The genome sequencing project has provided >90% of the genomic sequence, including a single G sequence. Previous studies have shown that Gs from the same species usually share certain level of homology (Gautam et al., 1998). When we used our G cDNA to search the databases, we found multiple cDNA and genomic clones, all with the same sequence. Of cause, blast-search results cannot rule out the possibility of another G with a very different sequence. However, when we purified G complex, we found there was only one G associated with G. Moreover, our northern analyses showed that the mRNA levels of G and G at different development stages rose and fell in parallel.

Functional studies of G/AX2 cells indicate that responses to stimulus increments and gradients, although closely related, display different sensitivities. During development, both responses are manifested and utilize the same G-proteins. When G is disrupted, both responses are eliminated. The G/AX2 cells enabled us to separate the responses. Redistribution of G to the cytosol impairs the capacity to sense the gradients, as indicated by the inability to carry out chemotaxis or relocate PHcrac-GFP to the cell's leading edge. However, these cells were able to respond to incremental stimuli. A possible explanation is that both membrane-associated and cytosolic -subunits can participate in incremental sensing, but a full complement of membrane-tethered G-subunits is required for gradient sensing. Recent FRET studies showed cAMP-induced G-protein dissociation both on the membrane and in the cytosol, suggesting that cytosolic G protein might be activated by an incremental stimulus (our unpublished results).

The selective inhibition of gradient sensing in cells lacking a full complement of membrane-associated G can also be explained by hybrid spatial-temporal models for directional sensing (Fischer, 1990; Parent et al., 1999). In these schemes, receptor occupancy regulates two counteracting biochemical processes: local excitation and global inhibition. When an incremental stimulus is encountered, occupied receptors trigger a rapid excitation followed by a slower rise of inhibition (Figure 9A). Both processes eventually reach the same steady-state level of activity determined by receptor occupancy, and the response subsides. In a gradient, after an initial transient response, a steady state is reached where excitation slightly exceeds inhibition at the front of the cell, whereas inhibition exceeds excitation at the back (Figure 9B). In G/AX2 cells, the redistribution of G to cytosol selectively lowers the level of excitation but does not impair inhibition. On addition of an incremental stimulus, the decreased excitation briefly exceeds inhibition, and the decreased membrane G is sufficient to mediate transient responses such as activation of ACA and translocation of PHcrac-GFP (Figure 9C). However, in a gradient at steady state, the magnitude of excitation above inhibition depends on the gradient across one single cell length of ~10-20 µm, which is usually very shallow. In the G/AX2 cells, this difference in excitation levels is further decreased (Figure 9D). Thus, after one initial transient response, the G left on the membrane of G/AX2 cells is not enough to sense gradients and to mediate chemotaxis.

View larger version (15K): [in this window] [in a new window]   Figure 9.   Schematic representation of model for gradient sensing. (A) In wild-type cells, upon an incremental activation, a transient response (R) results from a balance between rapid excitation (E) and slower inhibition processes (I). (B) For a wild-type cell in a chemoattractant gradient, excitation is higher than the inhibition at the front and lower than the inhibition at the back at the steady state. (C) In G/AX2 cells, the receptor-specified levels of excitation (E') are lower because less G-protein is on the membrane. On an incremental activation, the response (R') is slightly impaired. (D) In a chemoattractant gradient, after the initial transient response, the inhibition exceeds the decreased excitation (E') at the front of the G/AX2 cells, and gradient is not sensed.

Our isolation of -subunit provided a long missing member of G-protein family in D. discoideum. Further analysis of this gene should help us to understand the roles of G-proteins in chemotaxis and to identify other components located downstreams of -subunits. Furthermore, we propose that dominant properties of the CXXL-deletions make these constructs useful tools for studying the functions of G-subunits in mammalian systems. In mammals, there are 20 -subunits, 11 -subunits, and 5 -subunits. This large number of subunits contributes to an even larger number of possible combinations and complicates functional analysis. Although it would be very challenging to generate clones containing disruptions of multiple G genes, it might be feasible to select clones expressing specific combinations of COOH-terminal truncated G genes. According to our data, these Gs are likely to compete with specific endogenous Gs, shift the corresponding G-subunits to cytosol, and selectively impair functions. Such cell lines or transgenic animals can be used to carry out the studies on the specific functions of each subtype of G.