Gap junction assembly: PTX-sensitive G proteins regulate the distribution of connexin43 within cells

Paul D. Lampe1, Qiu Qiu2, Rita A. Meyer2, Erica M. TenBroek2, Timothy F. Walseth3, Todd A. Starich2, Haiying L. Grunenwald2, and Ross G. Johnson2

1 Fred Hutchinson Cancer Research Center, Seattle, Washington 98109; and 2 Departments of Genetics, Cell Biology, and Development, and 3 Pharmacology, University of Minnesota, St. Paul, Minnesota 55108


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cells expressing connexin43 are able to upregulate gap junction (GJ) communication by enhancing the assembly of new GJs, apparently through increased connexin trafficking. Because G proteins are known to regulate different aspects of protein trafficking, we examined the effects of pertussis toxin (PTX; a specific inhibitor of certain G proteins) on GJ assembly. Dissociated Novikoff hepatoma cells were reaggregated for 60 min to form nascent junctions. PTX inhibited GJ assembly, as indicated by a reduction in dye transfer. Electron microscopy also revealed a 60% decrease in the number of GJ channels per cell interface. Importantly, PTX blocked the twofold enhancement in GJ assembly found in the presence of low-density lipoprotein. Two Gialpha proteins (Gialpha 2 and Gialpha 3), which have been implicated in the control of membrane trafficking, reacted with PTX in ADP-ribosylation studies. PTX and/or the trafficking inhibitors, brefeldin A and monensin, inhibited GJ assembly to comparable degrees. In addition, assays for GJ hemichannels demonstrated reduced plasma membrane levels of connexin43 following PTX treatment. These results suggest that PTX-sensitive G proteins regulate connexin43 trafficking, and, as a result of inhibition with PTX, the number of plasma membrane hemichannels available for GJ assembly is reduced.

gap junctions; pertussis toxin; protein trafficking; connexin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GAP JUNCTIONS (GJs) are thought to play an important role in the control of cell proliferation, embryonic development, and the regulation of differentiated function in postmitotic cells by providing for the direct, intercellular exchange of small molecules (19, 27). In vertebrates, the transfer of molecules between adjacent cells occurs via cell-to-cell channels formed by members of the "connexin" protein family. Connexins have been shown to form hexamers, termed "connexons" or GJ hemichannels, within cytoplasmic membranes (1, 25, 45). After oligomerization, these channels travel to the cell surface (22, 36), where a hemichannel in one plasma membrane pairs with another in an apposed membrane to form a complete cell-to-cell channel (20, 39).

The level of GJ communication between cells appears to be carefully regulated, via several different mechanisms that vary widely as a function of cell type. There are functional implications associated with the different levels of GJ communication, because quantitative changes can apparently influence biological processes such as cell growth (e.g., see Refs. 10, 14, 46), development (26, 35, 37, 64), and secretion (69).

A number of previous studies have emphasized the role of channel gating mechanisms, including phosphorylation effects, in the control of GJ communication (5). For example, agents that elevate cAMP levels within cells have been reported to positively or negatively alter junctional permeability, depending on the cell type (33, 59). Cell-cell communication can also be sharply depressed in the presence of an active tyrosine protein kinase (34). Thus the gating of GJ channels provides one important mechanism for regulating the extent of GJ communication.

Regulation of GJ assembly also represents a potential means of controlling junctional communication between cells. For example, the short half-lives (1-5 h) of connexins in tissues (4, 17) and in cell culture (12, 29, 30, 42) imply that GJ assembly is continuous and thus potentially sensitive to regulatory intervention. Moreover, the process of GJ assembly likely includes checkpoints at different steps, which would involve multiple regulatory mechanisms. The substantial amounts of connexins within cytoplasmic membranes, often observed in immunofluorescence experiments, would appear to be an important store of junctional protein (28, 47, 67). The utilization of such sources is likely regulated when GJ assembly is enhanced (40, 47). The assembly of connexins into GJ structures is also likely to be regulated at the cell surface. Activation of protein kinase C has been found to dramatically inhibit GJ assembly at or near the plasma membrane in cells expressing connexin43 (Cx43) (30). In addition, phosphorylation of Cx43 by an unspecified kinase(s) has been correlated with both the incorporation of Cx43 into junctional plaques and the acquisition of Triton insolubility by Cx43 (44).

In the present study, we have utilized pertussis toxin (PTX), a highly specific inhibitor of heterotrimeric G proteins of the Gi class (65), to examine the potential role of G proteins in the process of GJ assembly. Although nothing was previously known regarding the role of G proteins in connexin trafficking or assembly, G protein-coupled receptors had previously been implicated in the regulation of channel gating in GJs via Src tyrosine kinase activation (49). We reasoned that PTX could aid in the identification of previously unrecognized events in GJ assembly and/or controls exerted over the process. Thus the toxin could prove to be a valuable experimental agent in the molecular dissection of the assembly process, by virtue of inhibiting a specific step(s) in assembly. We took advantage of the process of enhanced GJ assembly, where different treatments can increase the assembly of junctions severalfold within 1 h (40, 41, 47). This upregulation does not involve an increase in cellular levels of Cx43, but it is sensitive to several different trafficking inhibitors, including brefeldin A (BFA) and nocodazole (47). Thus the present experiments were designed to determine whether PTX blocked enhanced assembly, implicating heterotrimeric G proteins in its regulation. In addition, the quantitative assays employed here for GJs were capable of detecting small changes in assembly, which might result from PTX treatments under basal conditions.

We report here that PTX treatment reduced the size, i.e., the growth, of junctions that form in an assembly assay, with a corresponding reduction in cell-cell communication. However, PTX did not alter the percentage of cells forming GJs or the number of GJs that were assembled. Of particular interest, PTX completely blocked the twofold enhancement of GJ assembly observed in the presence of low-density lipoprotein (LDL). Through the use of physiological assays for Cx43 hemichannels and quantitative electron microscopy (EM) assays for GJs, we found that PTX acts by affecting the distribution of Cx43 within the cell, along with its transport to the plasma membrane. In this regard, the results resembled the effects of the trafficking inhibitors BFA and monensin. Consistent with an effect on trafficking, we also found that two Gialpha proteins in Novikoff cells are ADP-ribosylated in the presence of PTX. One of these (Gialpha 3) has been reported to function in the control of membrane trafficking and regulated exocytosis (2, 63, 66). In another study, inhibition of this same G protein with either PTX or synthetic peptides reduced the transport of a channel protein to the plasma membrane (66). We show that PTX-sensitive G proteins in Novikoff hepatoma cells influence the number of channels incorporated into newly forming GJs, likely by controlling the number of Cx43 hemichannels delivered to the plasma membrane. We suggest that this regulatory system modulates the distribution of Cx43 within the cell, with an effect on overall levels of GJ communication.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture, dissociation, recovery, and reaggregation. Tissue culture reagents were purchased from GIBCO BRL (Grand Island, NY). The Novikoff hepatoma cell line (N1S1-67) was maintained and dissociated as previously described (39). Cells in logarithmic growth were dissociated by centrifugation from standard growth medium (S210 media supplemented with 10% newborn calf serum), resuspended in S210 with 10 mM EDTA at 6 × 105 cells/ml, and placed in a 37°C gyratory shaker-incubator (200 rpm) for 15 min. The EDTA treatment was repeated, resulting in >95% single cells. The cells were "recovered" by shaking in S210 medium with 5% BSA (A7030; Sigma Chemical, St. Louis, MO) for 90 min, which eliminated remnants of previously existing junctions (50). The recovered cells were "reaggregated" by means of two different methods. In the first case, cells were reaggregated for EM experiments by centrifugation at 30 g for 5 min to yield loose pellets in 17 × 100 mm round-bottom tubes, which were then incubated for 60 min at 37°C. For microinjection experiments, 8 × 105 cells were settled out in 35-mm petri dishes for 60 min at 37°C.

Treatment with PTX, forskolin, monensin, BFA, and LDL. The effects of PTX, forskolin, and LDL on GJ assembly were compared with the effects of two reagents that have been shown to affect protein trafficking: BFA and monensin. PTX, forskolin, and monensin were purchased from Calbiochem (San Diego, CA). LDL and BFA were from Sigma Chemical. Forskolin, monensin, and BFA were dissolved in absolute ethanol at 1,000 times the final concentration. Control samples also contained 0.1% ethanol. PTX was dissolved in sterile, deionized water at 100 µg/ml and added directly to cells to obtain the concentrations indicated. The various reagents were added to Novikoff cells at different times during the recovery and reaggregation periods as indicated below. In experiments on the concentration dependence of PTX, the cells were treated with a range of concentrations (2.5, 5, 10, and 30 ng/ml) during the standard recovery and reaggregation periods (2.5 h). In time course experiments, the cells were treated with 30 ng PTX/ml for varying times (5, 15, 30 min) at the beginning of the 60-min reaggregation period. In the LDL experiments, PTX was present during the recovery and reaggregation periods (2.5 h) and LDL was present (2.5 µg protein/ml) during only the reaggregation period. Forskolin (10 µM), when utilized, was also added at the beginning of the reaggregation period. For monensin treatment, cells were allowed a 90-min recovery period to allow internalization and degradation of preexisting connexin protein before addition of monensin (5 µM), which remained in the media for an additional 30 min of recovery, followed by the normal 60 min of reaggregation. BFA (36 µM), which should not inhibit internalization, was added at the beginning of the 2-h recovery. When treatments combined PTX with either BFA or monensin, the inhibitors were also added as just described.

Microinjection of dye. Dye injection studies were performed to evaluate intercellular dye permeability between reaggregated Novikoff cells that had been settled out in petri dishes. One member of a cell pair was microinjected with a 4-ms pulse of 1% aqueous Lucifer yellow CH (Sigma) under 25 psi using a General Valve picospritzer. We recorded dye injection and subsequent cell-to-cell transfer of dye on a Zeiss IM35 microscope equipped with a Dage silicon intensified target camera, Panasonic video recorder, and monitor. The degree of dye transfer was estimated by measuring the time elapsed from the beginning of dye injection into the impaled cell to detection of fluorescence in the adjacent cell of the pair. These "transfer time intervals" were determined by analysis of the videotapes. Values are reported as means ± SE. With both untreated and PTX-treated cells, dye failed to transfer to the neighboring cell by 5 min in ~20% of the injections; these injections were not included in the calculations of dye transfer times. At least 18 microinjections were obtained for each data point from three separate injection sessions. A Student's t-test was used to determine whether the differences were significant.

Freeze fracture and EM to quantify GJ structures. Cell pellets were fixed in 2.5% glutaraldehyde in S210 medium for freeze fracture and EM. Further processing was performed as previously reported (39). In each of four experiments addressing the effects of PTX, we examined at least two replicas per treatment.

Methods of quantitation have previously been described (51). Briefly, an "interface" was defined as a fractured membrane area comprising at least 57 µm2 (i.e., filling the screen at 10,000× on our microscope) and containing an indication of cell apposition. Interfaces were scored according to the presence or absence of one or more GJs. "Formation plaques" were defined as specialized membrane areas with clusters or arrays of 9- to 11-nm intramembranous particles and a paucity of small particles. Individual 9- to 11-nm particles present within GJ aggregates are equated here with individual GJ channels. Formation plaque area and particle number measurements were performed as reported in detail elsewhere (51). Measurements were made with the aid of a microcomputer equipped with a Houston Instruments Hipad digitizing tablet and morphometry software. Values for a number of structural parameters are presented as means ± SE. A Student's t-test was used to determine whether statistical differences were found in the means, with a value of P < 0.05 considered significant.

The EM benefited from a multilayered cell preparation that minimized searching for interfaces. In contrast, cell pairs were ideal for unambiguous dye permeability data on a single cell-cell interface. Nevertheless, the two sets of data were qualitatively consistent. However, the values cannot be strictly compared quantitatively, because the cells for EM and dye injection were not reaggregated in the same manner.

Preparation of membranes from control and PTX-treated Novikoff cells. Novikoff cells were cultured under normal conditions in the absence or presence of 10 ng/ml PTX for 16 h. Cells were pelleted by centrifugation at 800 g for 8 min. Cell lysis was accomplished by homogenization of the cell pellets in 2 ml of a buffer consisting of 20 mM HEPES, pH 7.5, 1 mM EDTA, 2 mM EGTA, 2.5 µg/ml leupeptin, 2.5 µg/ml pepstatin A, 5 µg/ml soybean trypsin inhibitor, and 10 mM benzamidine-HCl (HEE buffer) using a loose-fitting glass homogenizer. Cells and debris were removed by centrifugation at 1,000 g for 10 min. The 1,000 g supernatant was then centrifuged at 38,000 g for 20 min, and the resulting pellet was resuspended in HEE buffer and used in experiments to identify PTX substrates.

PTX-catalyzed [32P]ADP-ribosylation of Novikoff cell membranes. Membranes from control or PTX-treated cells were treated with PTX and [32P]NAD as described previously (70). Briefly, 160 µg of membrane protein were incubated in the presence of 20 mM HEPES, pH 7.4, 1 mM ATP, 0.1 mM GTP, 0.25% (wt/vol) lubrol PX, 10 mM thymidine, 20 mM arginine, 1 µCi of [32P]NAD (800 Ci/mmol; NEN DuPont, Boston, MA), and 7.5 µg/ml activated PTX for 60 min at 37°C in a final volume of 100 µl. PTX was activated by incubation of 75 µg/ml PTX in 30 mM HEPES, pH 7.4, and 20 mM dithiothreitol for 20 min at 37°C. The PTX-catalyzed reaction was terminated by the addition of 300 µl of ice-cold acetone. The acetone-treated samples were centrifuged for 15 min at 15,000 g and the supernatant discarded. The precipitated proteins were resuspended in 75 µl of 1× sample buffer, boiled for 5 min, and analyzed by SDS-PAGE using 9% acrylamide gels containing 6 M urea (11, 58). Labeled proteins were detected by exposure to DuPont Cronex-4 X-ray film after transfer of the separated proteins from the gel to Immobilon-P membranes using a semidry electroblotter.

G protein identification by Western immunoblotting. Membrane protein (160 µg) derived from control or PTX-treated cells, as described above, was subjected to electrophoresis and electroblotting. A published immunoblotting procedure was followed (70), except that primary antibody incubations were for 12-16 h and detection utilized an alkaline phosphatase-conjugated goat anti-rabbit IgG (1:3,000 dilution; Bio-Rad, Hercules, CA) incubated for 60 min.

The primary antibodies used were TG976 (1:8,000 dilution), a rabbit antiserum that was raised against the COOH-terminal sequence of Gialpha 3, which recognizes Gialpha 3 as well as Goalpha isoforms; TG977 (1:1,000 dilution), an antiserum also raised against the COOH-terminal sequence of Gialpha 3, which is specific for Gialpha 3; TG978 (1:1,000 dilution), an antiserum raised against an internal sequence of Gialpha 1 (amino acids 159-168); and TG982 (1:2,000 dilution), an antiserum raised against the COOH-terminal sequence of Gialpha 1 and Gialpha 2, which recognizes both Gialpha 1 and Gialpha 2. These antisera were generous gifts from Thomas Gettys (53). AS/7 (1:1,000 dilution, NEN DuPont), an antiserum raised against the COOH-terminal sequence of transducin, which recognizes Gialpha 1 and Gialpha 2 and also cross-reacts weakly with Gialpha 3 (61), was also used.

Cx43 labeling, immunoprecipitation, and Western transfer. Novikoff cells were labeled with 32PO4 by dissociating cells as described above, recovering the cells at 1 × 106 cells/ml in S210 medium containing 2.5% BSA and 0.25 mCi 32PO4/ml for 3 h (with shaking at 200 rpm to maintain a single cell suspension and prevent junction formation), and reaggregating 2 million cells/sample in the presence or absence of PTX for 60 min. Cx43 was immunoprecipitated and run on SDS-PAGE and was subjected to autoradiography as previously described (54), except that 2 µg of monoclonal antibody 3068 (Chemicon, Temecula, CA) specific for residues 252-270 of Cx43 was used for each treatment.

For Western immunoblotting of Novikoff cells, SDS-PAGE was performed on 10% polyacrylamide gels. Approximately 1.5 million cells were treated with PTX (30 ng/ml) for 60 min during the reaggregation period. Control and treated cells were washed once with PBS (Celox, Minneapolis, MN), centrifuged, and solubilized directly with 2% SDS sample buffer that contained protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin, 10 µg/ml aprotinin, 1 µg/ml leupeptin). The samples were sonicated before being loaded onto the gel. Protein transfer was performed as previously described, and Cx43 was detected using an antibody prepared to the NH2-terminal 20 residues (generous gift of Barbara Yancey; described in Ref. 73). Primary antibody was detected using 125I-labeled protein A (3 µCi /blot; ICN, Costa Mesa, CA). Autoradiography was performed using Kodak XAR 5 film overnight with an intensifying screen.

Assays for cAMP and GJ hemichannels in Novikoff cells. cAMP levels in Novikoff cells were measured by radioimmunoassay (56). Novikoff cells (2 × 106) were treated in the absence or presence of 30 ng/ml PTX for 2.5 h. Metabolism was quenched by adding perchloric acid to a final concentration of 0.5 M. Protein was removed by centrifugation for 10 min at 15,000 g. The supernatant was neutralized with 2 M KHCO3, and cAMP was assayed after acetylation of the samples (56).

Nonjunctional hemichannels were assayed in control or PTX- or BFA-treated cells with a dye uptake method in the presence of reduced extracellular calcium as previously reported (36). Cells were dissociated and recovered as above, and then single cells were incubated with PTX or BFA as described in RESULTS. Results are expressed here as the percentage of dye-positive cells out of the total number of cells (n) examined. At least three experiments were performed for each of the different PTX treatments. Chi-square analysis was used to determine the statistical significance of these results, with a value of P < 0.05 considered significant.

RNA isolation from Novikoff cells and Northern blots. Novikoff cells were grown, dissociated, and recovered as described above. Cells were allowed to reaggregate for 1 or 2 h with or without 30 ng/ml PTX. Cells were harvested by centrifugation at 1,000 g for 5 min. The cell pellet was lysed and homogenized in a Dounce homogenizer, with the addition of 7.5 ml cold 5 M guanidine-HCl, 0.2 M sodium acetate, 0.5% N-laurylsarcosine, 1 mM EDTA, and 0.5 M 2-mercaptoethanol. The suspension was repeatedly extracted with phenol-chloroform-isoamyl alcohol (25:24:1), followed by a single chloroform extraction, and RNA was selectively precipitated by adding one-half volume 95% ethanol overnight at -20°C. After centrifugation at 12,000 g, pelleted RNA was resuspended in water treated with diethyl pyrocarbonate. Northern blots were hybridized in 0.25 M NaCl, 7% SDS, 1 mM EDTA, 0.25 M sodium phosphate, pH 7.0, 150 mg/ml herring sperm DNA, and 50% formamide at 48°C. Filters were washed at 55°C with 0.2× sodium chloride-sodium phosphate-EDTA, 0.1% SDS.

PCR amplification and sequencing of Cx43 cDNA in Novikoff cells. To isolate expressed sequences in Novikoff cells hybridizing to the Cx43 probe, cDNA was synthesized from 1 µg of total RNA using avian myeloblastosis virus (AMV) reverse transcriptase (USB, Cleveland, OH). The reaction was carried out in a volume of 20 µl in 1× PCR buffer (50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 10 mM Tris, pH 9.0, at 25°C) supplemented with 10 mM MgCl2, 40 units of RNasin, and 2 mM of each dNTP. A gene-specific primer (0.5 µM) complementary to nucleotide positions 1358-1375 of Cx43 plus an EcoRI restriction site (5'-GCGAATTCCTCCATAATCGACAGC-3') was used. RNA was heated to 65°C for 5 min and quenched on ice before the addition of 13 units of AMV reverse transcriptase. The mixture was incubated at 42°C for 1 h and then shifted to 52°C for 30 min. After cDNA synthesis, the reaction mix was diluted with 400 µl of 0.1 mM EDTA, 10 mM Tris, pH 8.0.

PCR amplification of cDNA products utilized the above primer plus a specific primer corresponding to Cx43 nucleotide positions 117-134 plus an EcoRI site (5'-CCGAATTCAGCCTCCAAGGAGTT-3'). Both of these primers lie outside the predicted protein coding sequence of Cx43. Typically, 2 µl of the cDNA reaction mix were used in a 50-µl volume including 1× PCR buffer, 0.2 mM of each dNTP, 0.5 µM of each primer, and 2.5 units of Taq polymerase. Amplifications usually employed 35 cycles, with primer annealing at 57°C, extensions carried out at 72°C for 2 min, and double-stranded products melted at 93°C for 1 min after each extension. To verify that PCR products were dependent on an original RNA template provided during cDNA synthesis and not on contaminating genomic DNA, mock cDNA synthesis reactions were performed without an RNA template. Such samples failed to produce any PCR products after amplification.

PCR products were cloned into pUC118 (68) and sequenced by employing the dideoxy chain-termination method on double-stranded templates (9). To generate nested sets of deletions for sequencing templates, controlled exonuclease III digestion was used. Three clones from independent PCR amplifications (representing two separate cDNA synthesis reactions) were isolated, and their sequences were compared and checked for any errors possibly introduced by Taq polymerase during PCR amplification.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Inhibition of cell communication in a GJ assembly assay. We began by investigating the effect of PTX on basal levels of GJ assembly. Subsequent experiments addressed the role of G proteins in enhanced GJ assembly. Novikoff cells were dissociated with EDTA, allowed to recover as single cells, and then reaggregated to form nascent junctions. After treating with a range of PTX concentrations for the 90-min recovery and the 60-min reaggregation periods, we microinjected cells with 1% Lucifer yellow. We observed a decrease in intercellular permeability that was dependent on the concentration of PTX (Fig. 1, solid bars). Decreased permeability was expressed as increased dye transfer time, i.e., the time elapsed from the beginning of dye injection into one member of a cell pair to detection of fluorescence in the adjacent cell. At a concentration as low as 5 ng/ml, PTX significantly (P < 0.001) inhibited intercellular dye transfer, as reflected by the 1.6-fold increase in mean dye transfer time. The effect of PTX leveled off at 10-30 ng/ml, with an approximate twofold increase in dye transfer time. Concentrations of PTX as high as 300 ng/ml showed no additional increase in dye transfer times. Therefore, we routinely did not exceed 30 ng/ml.


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Fig. 1.   Concentration dependence of pertussis toxin (PTX) inhibition: cell-cell dye transfer times (means ± SE) observed between Novikoff cell pairs treated with a range of PTX concentrations. Experiments were performed with 2.5 µg/ml low-density lipoprotein (LDL; hatched bars) or without LDL (solid bars). PTX was present during the recovery and reaggregation periods (a total of 150 min), and LDL was added at the beginning of the reaggregation period. The level of dye transfer was measured as the time elapsed from the beginning of injection of 1% Lucifer yellow into 1 cell to detection of fluorescence in the adjacent cell of a cell pair (dye transfer time).

To examine the time course of PTX inhibition, Novikoff cells were treated with PTX at 30 ng/ml for varying times at the beginning of the reaggregation period, washed with media in the absence of PTX, allowed to finish a 60-min reaggregation period, and then injected with 1% Lucifer yellow. Because PTX inhibition of G proteins is irreversible, this provides a sensitive means of detecting any PTX effects with short-term treatments. Fifteen minutes of PTX treatment time significantly (P < 0.01) decreased dye permeability as shown by the increase in dye transfer time (Fig. 2). The increase in dye transfer times was approximately linear up to 30 min. After 30 min of PTX treatment, near-maximal inhibition of dye transfer was obtained. This time course and the irreversible inhibition of GJ communication are consistent with PTX binding, entry into the cell, and the well-characterized reaction with Gi proteins.


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Fig. 2.   Time course of PTX inhibition. Reaggregating cells were treated with PTX at 30 ng/ml for varying times (as indicated on the x-axis), washed, and incubated for a total of 60 min and then injected with 1% Lucifer yellow in the absence of PTX. A nearly maximal effect was achieved when cells were treated with PTX for a minimum of 30 min, reflected by the longer mean dye transfer time (means ± SE). The 150-min time point corresponds to PTX treatment throughout the period of recovery and reaggregation.

Inhibition of GJ assembly by PTX: freeze-fracture EM. Freeze-fracture EM was performed to determine whether the decreased intercellular dye permeability we observed was associated with a decrease in GJ assembly, an effect on channel gating, or some combination of the two. Clumps of Novikoff cells from suspension cultures were dissociated to single cells and recovered to eliminate preexisting GJs (51), as in the dye injection experiments. Cells were reaggregated in loose pellets for 60 min, and cell interfaces were analyzed as described in MATERIALS AND METHODS. GJ assembly was evaluated by determining the number of GJ formation plaques (the specialized membrane areas that contain arrays of newly forming GJs) per cell interface and the number of aggregated 9- to 11-nm intramembranous particles, both per formation plaque and per interface (Table 1). These aggregated membrane particles represent the cell-to-cell channels found in GJs. Figure 3 provides examples of freeze-fracture replicas of control (A) and PTX-treated (B) cells. A significant (P < 0.001) decrease in the number of GJ channels per interface to 40% of the control value was observed in freeze-fracture samples of cells treated with PTX (Table 1). Because no GJs or formation plaques were detected at the beginning of the 60-min reaggregation period, the GJs observed at the end of this period were newly formed structures. PTX inhibited GJ assembly, specifically by reducing the growth of the GJs observed. No significant change was observed in the percentage of cell-cell interfaces, which contained GJ formation plaques (Table 1). Consistent with a reduction in channel number, we also observed a PTX-dependent decrease in the area of formation plaques, both per plaque and per interface. The inhibition of GJ assembly mediated by PTX was sufficient to explain the reduced levels of dye transfer detected with PTX.

                              
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Table 1.   PTX effects on gap junction assembly: freeze-fracture EM analysis



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Fig. 3.   Freeze-fracture electron microscopy of Novikoff cells in the absence (A) and presence (B) of 30 ng/ml PTX (bar = 100 nm). Note the smaller size of the developing gap junctions (GJs) in the presence of PTX.

Inhibition by PTX of LDL-enhanced GJ formation. LDL treatment has been shown to increase the degree of dye transfer between reaggregated Novikoff cells (i.e., to shorten dye transfer times) and to enhance GJ assembly severalfold (40). We used the GJ assembly assay to examine the effect of PTX on dye permeability in the presence of LDL to determine whether PTX was capable of blocking enhanced assembly. In the absence of PTX, LDL caused a significant (P < 0.001) reduction in the mean dye transfer time (Fig. 1, hatched bar at 0 PTX) to 50% of the control value (Fig. 1, solid bar at 0 PTX). However, when PTX was present, as little as 2.5 ng/ml inhibited the LDL-mediated enhancement of GJ communication (Fig. 1); in Fig. 1, the hatched bar at 2.5 ng/ml is significantly different from the hatched bar in the absence of PTX (P < 0.001).

We also performed freeze-fracture analysis on Novikoff cells treated with LDL (Table 1) in the presence or absence of PTX. LDL enhanced GJ formation (compare the number of GJ channels for LDL with control), whereas in the combined presence of LDL and PTX the number of GJ channels per interface was reduced to a level indistinguishable from that with PTX alone. Thus, when analyzed by both dye transfer and EM methods, the LDL-dependent increase in GJ formation was found to be completely blocked by PTX.

PTX effects on Cx43 mRNA, Cx43 protein, and Cx43 phosphorylation levels. We first demonstrated that Cx43 is expressed in the Novikoff cell line. RT-PCR was employed following the isolation of total RNA. With a pair of gene-specific primers for Cx43, PCR products were obtained, cloned, and sequenced. The derived sequence matched exactly that obtained for rat uterine Cx43 expression (32). These results confirmed that Cx43 and not a closely related connexin is expressed in Novikoff cells. Northern blot analysis had earlier failed to detect seven other connexins (30). Based on these data, as well as previous reports with immunological and antisense methods (36, 39), we conclude that Cx43 is responsible for most, if not all, of the GJ communication observed in Novikoff cells. Northern blot analysis detected no effects of PTX treatments on levels of Cx43 transcripts (data not shown).

Considerable evidence indicates that the gating of GJ channels and possibly GJ assembly is regulated by phosphorylation of connexins. Thus we next evaluated potential variations in both Cx43 protein and phosphorylation levels with Western immunoblot methods, because changes in SDS-PAGE mobility have been correlated with the phosphorylation of Cx43 (6, 8, 23, 29, 30, 42). We evaluated connexin phosphorylation with and without PTX treatment by labeling Novikoff cells with 32PO4 during a 3-h recovery and a 1-h reaggregation period. Immunoprecipitation yielded several bands (Mr = 42-49 kDa) representing multiple phosphorylation states of Cx43 as has been previously shown (e.g., Ref. 42). No significant differences (Fig. 4A) in the levels of Cx43 phosphorylation were observed when the control cells were compared with cells treated with PTX for the 1-h reaggregation period (mean ratio of PTX/control = 1.02 ± 0.08, n = 5). Note that when Cx43 antibody was omitted from the immunoprecipitation protocol, no Cx43 was immunoprecipitated. We also attempted to detect changes in Cx43 levels and phosphorylation with Western immunoblot methods. The presence of PTX did not appear to have any effect on the number or intensity of the putative nonphosphorylated (Mr = 40 kDa) or phosphorylated bands (Mr = 42-46 kDa) in an immunoblot (Fig. 4B). Densitometry confirmed that no change in phosphorylation occurred and that the total intensity of all labeling with the Cx43-specific antibody (phosphorylated and nonphosphorylated bands) also was unchanged on PTX treatment (mean ratio of PTX/control = 0.96 ± 0.09, n = 7). Thus PTX did not appear to change the level of Cx43 protein nor its extent of phosphorylation. Novikoff cells do not apparently accumulate the P2 phosphoisoform of Cx43 in GJs that are Triton X-100 insoluble (47). The absence of changes in Cx43 protein levels emphasizes the importance of the redistribution of existing Cx43 in the process of enhanced GJ assembly.


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Fig. 4.   Phosphorylation of connexin43 (Cx43). A: Novikoff cells were labeled with 32PO4 and either untreated [control (CON) or no antibody (No Ab)] or treated with PTX. Cx43 was immunoprecipitated as described in MATERIALS AND METHODS, except Cx43 antibody was left out of the No Ab control immunoprecipitation reaction. B: Cx43 Western immunoblot. Control cells and cells that were treated with PTX were pelleted, treated with sample buffer that contained protease inhibitors, sonicated, and loaded on SDS-PAGE. After electrophoresis and Western transfer, the blot was probed with a rabbit antibody for Cx43, which was detected with 125I-labeled protein A. Note the lack of changes in Cx43 with PTX treatment.

Effects of forskolin, PTX, BFA, and monensin on GJ assembly. In some cell types, the inhibitory action of certain Gi proteins on adenylyl cyclase is relieved by PTX (65), and thus PTX might have been predicted to increase cAMP levels. However, previous reports on cell systems expressing Cx43, from our laboratory (3, 47) and others (38, 71), indicate that elevated cAMP levels actually lead to increased GJ assembly and communication. To directly answer this question, we examined cAMP levels in Novikoff cells, and they did not appear to be significantly affected by PTX (control: 30.6 ± 1.6 pmol; PTX: 30.3 ± 1.8 pmol/million cells; n = 3 and 4, respectively). Although in this study, forskolin provided for a less significant (P < 0.1) increase in the number of GJ channels (Fig. 5), it clearly failed to inhibit GJ assembly. Thus PTX effects on GJ assembly are not likely to be related to cAMP levels. In addition, our data are consistent with the cAMP regulatory element being proximal to the site of PTX action, since PTX could be added during the reaggregation period only (Figs. 2 and 5) and still have the maximal inhibitory effect on the level of GJ assembly, indicating that the PTX-sensitive event was close to or at the plasma membrane.


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Fig. 5.   Freeze-fracture electron microscopy analysis of GJ assembly in the presence of PTX, forskolin, brefeldin A (BFA), and/or monensin. The number of GJ channels per interface (means ± SE; n >=  46 interfaces/treatment) observed in Novikoff cells is shown. Compared with control, PTX (P < 0.01), BFA (P < 0.001), and BFA + PTX (P < 0.005) significantly reduced the number of assembled GJ channels.

One interpretation of the findings with PTX is that it reduces the size of forming GJs by inhibiting the process of Cx43 trafficking to the plasma membrane. To examine this possibility, reagents that block protein trafficking (i.e., BFA and monensin) were studied alone and in combination with PTX. Freeze-fracture and EM methods were utilized because they provide the most unambiguous assay for determining the number of channels within assembling GJs. Figure 5 provides data on the number of aggregated GJ particles present after treatment of Novikoff cells with forskolin, PTX, BFA, and monensin. When Novikoff cells were treated with PTX only during the 60-min reaggregation period, the mean number of aggregated particles per interface (reflecting largely GJ size) was significantly (P < 0.01) reduced to 40% of the control level. Studies with higher concentrations of PTX (see Fig. 1) and longer treatments (up to 3 h) yielded no additional PTX effects on assembly. In studies with BFA or monensin, the numbers of aggregated GJ particles per interface were reduced to comparable levels (Fig. 5). Combining either of these reagents with PTX led to no further reductions. Therefore, the effects of PTX on assembly resemble those of known trafficking inhibitors. Moreover, these studies provide no support for separate trafficking pathways to the cell surface, since the effects with different inhibitors were not additive. The limited GJ assembly that occurs with each of the three inhibitors (amounting to 30-40% of the control levels) could represent channels that were downstream of the sites of inhibition, when the inhibitors were added (see DISCUSSION).

In these studies with trafficking inhibitors, the EM methods monitored the end result of GJ assembly as the numbers and sizes of GJs. One would also anticipate a reduction in plasma membrane levels of Cx43 with BFA, as reported earlier (45), along with depressed GJ communication (28). Moreover, if the inhibition of assembly by PTX results from decreased Cx43 transport to the plasma membrane, one would also expect a reduction in plasma membrane levels of Cx43 with PTX. Therefore, we next applied a dye uptake assay for nonjunctional, GJ hemichannels in Novikoff cells, in the presence of low extracellular (nM) calcium (36). Cells were treated with either PTX or BFA for 2.5 h, as in the EM experiments on GJ assembly, and then assayed for dye uptake activity. Cells assayed at normal, external calcium levels displayed dye uptake in only 4 ± 1% (n = 298) of the cases. However, when calcium was reduced to nanomolar levels with EGTA, 45 ± 2% (n = 266) of the cells were dye positive in a manner that has been shown to be dependent on GJ hemichannel activity and Cx43 concentration (36). If PTX or BFA was present, this level was reduced to about one-half with only 22 ± 1% (n = 443) and 27 ± 4% (n = 153), respectively. These significant decreases with PTX (P < 0.0001) and BFA (P < 0.001) in dye-uptake activity are comparable to the levels of inhibition seen in basal GJ assembly in the presence of the two reagents. These findings support the idea that PTX inhibits Cx43 trafficking to the plasma membrane. The close relationship between trafficking and assembly further strengthens the interpretation that PTX inhibits assembly by means of its effect on Cx43 transport.

PTX-dependent ADP-ribosylation of G proteins. We sought to identify the PTX-sensitive proteins present in Novikoff cells to help interpret the action of the toxin in the inhibition of GJ assembly. PTX is known to inactivate various Gi proteins via ADP-ribosylation of their alpha -subunits. Figure 6 reveals the presence of two PTX-sensitive G proteins in Novikoff cells when analyzed by a modified SDS-PAGE method. This protocol has been shown previously to resolve ADP-ribosylated PTX substrates from their non-ADP-ribosylated forms (55) and various Galpha subtypes from each other (11, 58, 60). Figure 6, A and B, shows immunoblot analysis of membranes isolated from control or PTX-treated Novikoff cells. An antiserum, TG977, specific for Gialpha 3, was used in A, and TG982, specific for Gialpha 2 and Gialpha 1, was used in B. Because Gialpha 1 migrates more slowly than Gialpha 3 on this gel system (11), we conclude that the band detected by TG982 is Gialpha 2 (Fig. 6B, CON) and that by TG977 is Gialpha 3 (Fig. 6A, CON). This conclusion is supported by the failure of an antiserum specific for Gialpha 1 (TG978) to detect anything in these membranes (not shown). An antiserum that reacts with both Gialpha 3 and Goalpha behaved identically with TG977. The band detected by TG982 in Fig. 6B was also strongly reactive with AS/7, an antiserum that recognizes Gialpha 1 and Gialpha 2, and comigrated with Gialpha 2 identified in other cell systems (data not shown). We conclude that PTX treatment of Novikoff cells leads to the ADP-ribosylation of Gialpha 2 and Gialpha 3.


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Fig. 6.   Characterization of PTX-sensitive G proteins in Novikoff cells. Membranes from control or PTX-treated Novikoff cells were resolved on a modified SDS-PAGE system (see MATERIALS AND METHODS). A: immunoblot analysis using TG977, an antiserum specific for Gialpha 3. Treatment of isolated membranes with PTX resulted in a complete shift of these proteins to the characteristic position of ADP-ribosylated Gialpha 3. B: immunoblot analysis using TG982, an antiserum specific for Gialpha 2 and Gialpha 1. C: autoradiograph of PTX-catalyzed [32P]ADP-ribosylation of membranes derived from control or PTX-pretreated Novikoff cells. Because the pretreatment led to efficient ADP-ribosylation in these cells, no labeling occurred with in vitro PTX treatment, and both bands in the control lane displayed the reduced migration rates of the ADP-ribosylated forms. The 46-kDa marker represents the migration position of prestained ovalbumin (Rainbow standards; Amersham), and the 39-kDa marker represents the migration position of a fluorescent alcohol dehydrogenase standard (Integrated Separation Sciences).

Figure 6, A and B, shows that the G protein immunoreactivity detected in control membranes migrated faster than the immunoreactivity detected in membranes isolated from PTX-treated cells. The faster migrating bands in these two panels (CON) represents non-ADP-ribosylated Galpha , while the slower migrating band in each panel (PTX) represents ADP-ribosylated Galpha . PTX treatment of the cells was complete since each antibody detected a single band under each condition in A and B.

In Fig. 6C, membranes isolated from control or PTX-treated cells were subsequently treated in vitro with PTX and [32P]NAD. Two radiolabeled PTX substrates (Gialpha 2 and Gialpha 3) were observed in the autoradiograph when control membranes (Fig. 6C) were ADP-ribosylated. PTX pretreatment of the cells completely prevented additional ADP-ribosylation of the G proteins when the membrane preparation was subsequently exposed to PTX (Fig. 6C). These results provide further evidence that PTX pretreatment of the cells completely ADP-ribosylated all available substrates. Note that the two ADP-ribosylated bands in the control lane of Fig. 6C comigrated with the slower migrating (ADP-ribosylated) bands detected in the PTX lanes of the immunoblots (Fig. 6, A and B). Whether the G proteins were ADP-ribosylated in cells or in the isolated membrane preparations, ADP-ribosylation leads to slower migration.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The regulation of GJ assembly provides cells with an important mechanism for controlling the level of communication they have with neighboring cells. The observation of enhanced GJ assembly in cultured cells illustrates the potential associated with this mechanism, enabling cells to elevate levels of GJ communication severalfold within 1 h (40, 41, 47). Studies on the role of GJ communication in various cell processes emphasize the physiological relevance of such changes (10, 26, 35, 46, 64, 69). An important feature of enhanced assembly is that cells rely on preexisting GJ proteins in the early stages of the response (47). However, connexins must be redistributed to cell-cell interfaces during enhanced assembly. The related connexin trafficking is sensitive to well-known inhibitors, including BFA and nocodazole. In the present study, we have shown that Gialpha proteins play a role in the process of GJ assembly, particularly in the regulation of connexin trafficking during enhanced GJ assembly.

We relied on the well-established observation that PTX inactivates certain heterotrimeric G proteins in a highly specific manner by transferring an ADP-ribose unit to the Gialpha subunit (65). Using structural and functional assays, we have demonstrated that PTX inhibits GJ assembly. Both the concentration dependence and the time course for PTX inhibition of assembly were consistent with inactivation of a Gialpha protein (65). Two Novikoff Gialpha proteins, Gialpha 2 and Gialpha 3, were shown to be completely ADP-ribosylated in the presence of PTX and thus represent likely candidates for the sites of PTX action. Alternatively, we cannot rule out the possibility that the beta gamma -subunits play a role, since they have been shown to have independent activity in other systems (72).

This work on the inhibition of GJ assembly by PTX included the following observations: 1) there was an ~60% decrease in the number of aggregated GJ particles per cell-cell interface with PTX and a parallel decrease in intercellular permeability, following a 60-min reaggregation of unstimulated cells, 2) the effect of PTX was more striking in the case of enhanced GJ assembly, where it thoroughly blocked a stimulation of assembly resulting from LDL treatment, 3) there was no change, however, in the percentage of cells forming junctions or in the number of GJ plaques observed per cell, 4) there was also no apparent inhibition of membrane particle aggregation in GJ formation plaques, 5) the reduced numbers of aggregated GJ particles in the presence of PTX were indistinguishable from those obtained with either BFA or monensin or with PTX in combination with either of these agents, implicating an effect on Cx43 trafficking, and 6) assays for GJ hemichannels indicated that the plasma membrane levels of Cx43 were reduced in the presence of PTX, as with BFA. Thus exposure to PTX reduces nonjunctional levels of Cx43 in the plasma membrane, which, in turn, decreases the incorporation of Cx43 into GJs. Taken together, these results suggest that GJ assembly is influenced by PTX-sensitive G proteins that regulate the level of Cx43 trafficking to the plasma membrane.

A variety of studies indicate that cells contain substantial levels of cytoplasmic connexins. In addition to the characteristic punctate staining of GJs, large amounts of Cx43 are immunolocalized to perinuclear locations and other sites in Novikoff cells (40, 47), normal rat kidney (28), and many other cell systems (13, 52, 57). Transport of Cx43 from these cytoplasmic membrane pools to the plasma membrane was likely affected by BFA, monensin, and PTX in the experiments described in this report. BFA is known to specifically inhibit protein transport between the endoplasmic reticulum and Golgi (24), and it has been shown specifically to inhibit Cx43 trafficking to the plasma membrane (28, 45). The quantitative analysis of the effects of BFA, monensin, and PTX on junction assembly in the present EM experiments revealed that none of these reagents acted to reduce the percentage of cells forming junctions. However, when BFA and PTX were tested, they both led to a reduction in the level of plasma membrane Cx43, as revealed by hemichannel assays, and a decrease in the size of GJs, as demonstrated with EM methods.

Both structural and functional assays demonstrated consistently that some channels escaped inhibition and were incorporated into developing junctions. This could be explained in one of three possible ways. First, there may have been only a partial inhibition by PTX of the relevant G proteins. This seems unlikely, given that extended treatment times and 10-fold higher concentrations were no more effective than 30 ng/ml of PTX for 15-30 min. Moreover, the biochemical analysis of PTX effects in Novikoff cells indicated that Gialpha 2 and Gialpha 3 were fully ADP-ribosylated. Second, there may be multiple pathways for the trafficking of Cx43 to the plasma membrane, with PTX affecting only one or a subset. For example, alternative insertion pathways for connexins have been demonstrated in vitro (1). However, the findings with BFA, monensin, and PTX are consistent with these three agents acting on the same pathway. Third, if PTX inhibits the trafficking of Cx43 en route to the plasma membrane, some Cx43 will be downstream from the site of PTX inhibition (e.g., already in the plasma membrane but not in the GJ). This Cx43 could correspond to the small amount of connexin that assembles into GJs even at 4°C (21) or when cellular ATP levels have been sharply reduced (15). Although we favor the third interpretation, we recognize that multiple pathways for trafficking Cx43 to the plasma membrane and Cx43 recycling could also exist.

The hypothesis that PTX is acting on one or more Gi protein-dependent steps in the trafficking of Cx43 from the cytoplasm to the plasma membrane is consistent with a number of observations in the literature (7, 48, 62). G proteins could regulate the significant cytoplasmic pools of Cx43. The strongest support for this Cx43 model comes from a report that suggests an interesting parallel between the trafficking of aquaporin 2 (66) and the present findings on Cx43. Treatment with PTX inhibited both the redistribution of aquaporin from an intracellular compartment to the plasma membrane and the associated increase in water permeability, which was induced by vasopressin. Consistent with other reports (16, 63), both Gialpha 2 and Gialpha 3 were detected and found to reside largely within the cell (66). In addition, the redistribution of aquaporin was inhibited by Gialpha 3 peptides. These same peptides were used in an earlier study of mast cells to inhibit secretion (2). The highly regulated trafficking of Cx43 and aquaporin would appear to equip cells for important homeostatic functions.

We have suggested that GJ assembly involves a series of stages (21), including initiation (the appearance of GJ formation plaques), maturation (the aggregation of junctional particles in formation plaques), and growth (the addition of channels to existing GJ aggregates). Presumably, the number of cells participating in GJ assembly and the number of developing GJs per cell are regulated at the plasma membrane through some form of cell signaling. For example, we have shown that cell-cell contact influences Cx43 trafficking (47). It is also known that protein kinase C exerts a strong regulatory influence over Cx43 in the process of GJ assembly, inhibiting it in this case (30), possibly through a direct phosphorylation of Cx43 within the plasma membrane, since PKC apparently phosphorylates Cx43 at multiple sites within cells (31). The present study indicates that the signaling process driven by cell-cell contact is not sensitive to PTX, as illustrated by the fact that PTX had no effect on the percentage of cells involved in GJ assembly. From the results in this study, we conclude that PTX acts to inhibit GJ assembly by reducing the number of GJ channels available for the growth phase. We further postulate the existence of a cytoplasmic, PTX-sensitive regulatory site(s), which controls the trafficking of connexins through the cell.

It is possible that PTX is acting indirectly by inhibiting the transport or action of another protein that is critical for the growth of GJs. At first glance, cell adhesion molecules would be a logical possibility, since they are known to be important in GJ formation (39, 43). However, defective cell-cell adhesion (43) was shown to result in a major reduction in the percentage of cells engaged in GJ assembly (i.e., in the initiation of GJ formation). The same is true of experiments where antibodies for cadherins were used to inhibit GJ assembly (39). Therefore, if PTX acts via a loss of adhesive function, we would expect EM methods to detect a decrease in the percentage of cells forming GJs and the number of junctions per cell interface in the presence of PTX. Such effects were not seen. In addition, if Cx43 trafficking continued unabated, but PTX inhibited transport of a critical adhesion protein, then one would expect to see an increase in plasma membrane levels of Cx43, or at least steady levels. Yet, we actually observed a decrease in cell surface Cx43 as measured by hemichannel assays. The simplest explanation for all of the PTX findings on GJ assembly is that PTX causes a reduction in plasma membrane levels of Cx43 by inhibiting the trafficking of Cx43, with a subsequent decrease in the incorporation of hemichannels into GJ structures.

In conclusion, experiments indicate that GJ assembly is controlled at a number of sites within the cell, ranging from transcriptional regulation (18, 57) to modulation at the cell surface (30). The studies reported here suggest that the distribution of Cx43 is regulated at a critical, intracellular site (or sites) by Gialpha proteins. The transport of Cx43 beyond this site is an integral part of both basal and enhanced forms of GJ assembly. Future studies are required to identify the specific trafficking pathways utilized by cells for transporting Cx43 from the endoplasmic reticulum to the cell surface. Given the sizeable stores of connexins within the cytoplasm, the potential for upregulated communication is significant. Furthermore, this mechanism could complement transcriptional controls, in that the response time would be significantly shorter. In cases where even more immediate control is required, cells could utilize channel gating to control communication. Thus we can envision a number of complementary mechanisms, which likely exist to regulate GJ communication between cells.


    ACKNOWLEDGEMENTS

We thank Judson Sheridan and Michael Atkinson for comments on this manuscript and Barbara Yancey, David Paul, Eric Beyer, and Tom Gettys for their valuable reagents.


    FOOTNOTES

This work was supported by National Institute of General Medical Sciences Grants GM-46277 (to R. G. Johnson) and GM-55632 (to P. D. Lampe).

Current address of R. A. Meyer: Department of Biomedical Sciences, Creighton University, Omaha, NE 68178.

Address for reprint requests and other correspondence: P. Lampe, Fred Hutchinson Cancer Research Center, DE-320, 1100 Fairview Ave. North, Seattle, WA 98109 (E-mail: plampe{at}fhcrc.org).

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.

Received 30 January 2001; accepted in final form 29 May 2001.


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RESULTS
DISCUSSION
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