Dictyostelium cells bind a secreted autocrine factor that represses cell proliferation

Abstract

Background: Dictyostelium cells secrete the proteins AprA and CfaD. Cells lacking either AprA or CfaD proliferate faster than wild type, while AprA or CfaD overexpressor cells proliferate slowly, indicating that AprA and CfaD are autocrine factors that repress proliferation. CfaD interacts with AprA and requires the presence of AprA to slow proliferation. To determine if CfaD is necessary for the ability of AprA to slow proliferation, whether AprA binds to cells, and if so whether the binding requires the presence of CfaD, we examined the binding and effect on proliferation of recombinant AprA.

Results: We find that the extracellular accumulation of AprA increases with cell density and reaches a concentration of 0.3 microg/ml near a stationary cell density. When added to wild-type or aprA- cells, recombinant AprA (rAprA) significantly slows proliferation at 0.1 microg/ml and higher concentrations. From 4 to 64 microg/ml, the effect of rAprA is at a plateau, slowing but not stopping proliferation. The proliferation-inhibiting activity of rAprA is roughly the same as that of native AprA in conditioned growth medium. Proliferating aprA- cells show saturable binding of rAprA to 92,000 +/- 11,000 cell-surface receptors with a KD of 0.03 +/- 0.02 microg/ml. There appears to be one class of binding site, and no apparent cooperativity. Native AprA inhibits the binding of rAprA to aprA- cells with a Ki of 0.03 mug/ml, suggesting that the binding kinetics of rAprA are similar to those of native AprA. The proliferation of cells lacking CrlA, a cAMP receptor-like protein, or cells lacking CfaD are not affected by rAprA. Surprisingly, both cell types still bind rAprA.

Conclusion: Together, the data suggest that AprA functions as an autocrine proliferation-inhibiting factor by binding to cell surface receptors. Although AprA requires CfaD for activity, it does not require CfaD to bind to cells, suggesting the possibility that cells have an AprA receptor and a CfaD receptor, and activation of both receptors is required to slow proliferation. We previously found that crlA- cells are sensitive to CfaD. Combined with the results presented here, this suggests that CrlA is not the AprA or CfaD receptor, and may be the receptor for an unknown third factor that is required for AprA and CfaD activity.

Figure 1

The concentration of extracellular AprA increases with cell density. A) An SDS-polyacrylamide gel of recombinant AprA (left lane) and molecular mass standards (right lane) was stained with Coommassie.B) Using different concentrations or recombinant AprA to make a standard curve, Western blots were used to determine the extracellular concentration of AprA as a function of cell density. Values are mean ± SEM (n = 3). The absence of error bars indicates that the error was smaller than the plot symbol.

Figure 2

rAprA slows cell proliferation. A) Different concentrations of rAprA were added to the indicated cell types, and after 12 hours cells were collected and counted. WT is wild-type. Values are mean ± SEM (n = 3). The absence of error bars indicates that the error was smaller than the plot symbol. The lines are sigmoidal dose response curve fits to the data; the dashed line is the fit to aprA^-. The inhibition of wild type and aprA^- proliferation is significant with p < 0.01 at 0.1 μg/ml and higher rAprA concentrations (1-way ANOVA, Dunnett's test). B) A 2:1 mixture of rAprA and rCfaD was added to wild-type cells as in A above, so that at, for instance, 0.32 μg/ml rAprA there was an additional 0.16 μg/ml rCfaD. Values are mean ± SEM (n = 3). The line is a sigmoidal dose response curve fit to the data. The inhibition of wild type proliferation is significant with p < 0.05 at 0.02 μg/ml rAprA/0.01 μg/ml rCfaD and higher concentrations (t test).

Figure 3

Wild-type conditioned growth medium slows cell proliferation. Conditioned growth medium was collected and diluted with fresh growth medium to the indicated concentrations. Wild-type (WT) and aprA^- cells were then grown in the mixed media, and cells were counted after 12 hours. Values are mean ± SEM (n = 3). The absence of error bars indicates that the error was smaller than the plot symbol. Lines are curve fits of a sigmoidal dose response curve; the dashed line is the fit to aprA^-. The inhibition of WT and aprA^- proliferation is significant with p < 0.05 at 30% and higher conditioned growth medium concentrations (1-way ANOVA, Dunnett's test).

Figure 4

Binding of rAprA to cells. For a rAprA binding assay, wild-type cells were incubated for 10 minutes with the indicated concentrations ("Added to cells" in ng/ml) of myc-tagged rAprA. The cells were then washed to remove unbound rAprA, and were solubilized in SDS sample buffer. A western blot of the solubilized cells electrophoresed alongside different amounts of myc-tagged rAprA ("Standards") was stained with anti-myc antibodies (upper panel) while a duplicate gel of the cell samples was stained with Coomassie (lower panel). The heavy band in the Coomassie-stained samples is actin.

Figure 5

Time course of rAprA binding to cells. Cells of the indicated strains (WT is wild-type) were incubated with 150 ng/ml of rAprA for the indicated times at 4°C. Cells were collected and the bound myc-tagged rAprA was quantitated by western blots (staining for the myc tag), using known amounts of rAprA as standards. The plot symbols are the same as those in Figure 2. Values are mean ± SEM (n = 3). Lines are curve fits of an association binding curve; the dashed line is the fit to aprA^-.

Figure 6

Cells bind physiological concentrations of rAprA. Cells of the indicated strains (WT is wild-type) were incubated with different concentrations of rAprA. After 10 minutes, cells were collected and the bound rAprA was measured as in Figure 5. The plot symbols are the same as in Figure 2. Values are mean ± SEM (n = 3). For the 60 kDa rAprA, a bound rAprA value of 1.0 ng/5 × 10⁵ cells is equivalent to 2.0 × 10⁴ molecules/cell. The lines are curve fits to a one-site binding model with no cooperative binding.

Figure 7

Endogenous AprA competes with rAprA for binding to cells. The binding of rAprA to aprA^- cells was measured in the presence of the indicated concentrations of wild-type conditioned growth medium from cells at 10⁷ cells/ml, where the AprA concentration is 0.3 μg/ml. Values are mean ± SEM (n = 4). The line is a curve fit of a sigmoidal dose response curve.