Protein interacting with C-kinase 1/protein kinase Calpha-mediated endocytosis converts netrin-1-mediated repulsion to attraction

Abstract

In vertebrates, the receptor families deleted in colorectal cancer (DCC) and UNC5 mediate responses to the bifunctional guidance cue netrin-1. DCC mediates attraction, whereas a complex of DCC and UNC5 mediates repulsion. Thus, a primary determinant of the responsiveness of an axon to netrin-1 is the presence or absence of UNC5 family members on the cell surface. Currently, little is known about the role of receptor trafficking in regulating neuronal responses to netrin-1. We show that protein interacting with C-kinase 1 (PICK1) recruits activated protein kinase Calpha (PKCalpha) to MycUNC5A at the plasma membrane, stimulating its endocytosis. We identify two PKCalpha phosphorylation sites at serines 408 and 587, as well as dileucine internalization motifs, which are required for this endocytosis. We find that PKCalpha-stimulated internalization of UNC5A alters the functional response of developing hippocampal axons to netrin-1, preventing UNC5A-mediated growth cone collapse and converting netrin-1-stimulated chemorepulsion to attraction. To address whether this conversion in axonal response occurs in neurons expressing endogenous levels of UNC5, we show that mouse cerebellar granule axons exhibit chemorepulsion in a netrin-1 gradient and that this chemorepulsion is converted to chemoattraction after PKCalpha activation. We demonstrate that this repulsion depends on UNC5A because Unc5a-/- axons are not repelled and show this conversion depends on PICK1 because PICK1-/- axons are not converted to chemoattraction after PKCalpha activation. Together, these data provide a potential mechanism to explain how developing neurons alter their responsiveness to netrin-1 at intermediate choice points as they navigate to their targets.

Figure 1.

Activated PKCα is recruited to UNC5A at the plasma membrane of hippocampal growth cones. A, Live-labeled MycUNC5A receptors (green) do not colocalize with endogenous PKCα (red). B, Activated PKCα and MycUNC5A are colocalized at the cell surface after a 15 min TPA (100 ng/ml) treatment. C, MycUNC5AΔ3 does not colocalize with PKCα after TPA treatment. The MycUNC5AΔ3C construct encodes a C-terminal deletion of three amino acids that eliminates the PICK1 binding site, preventing PKCα recruitment to UNC5A. The dashed lines represent the outline of the growth cones as visualized in phase contrast. Scale bar, 5 μm.

Figure 2.

UNC5A is internalized, and not shed, after PKCα activation. A, Hippocampal neurons expressing MycUNC5A were TPA treated (100 ng/ml) as indicated to reduce cell surface expression of MycUNC5A. Biotinylation assays demonstrate the loss of MycUNC5A from the cell surface without a concomitant increase in UNC5A ectodomain in the TCA-precipitated cell-culture media (compare top blot with middle blot). Total cellular MycUNC5A was evaluated to control for infection efficiency. B–D, MycUNC5A receptors colocalize with EEA-1 after brief TPA treatment. B, Surface MycUNC5A receptors were live-labeled and treated with TPA to stimulate internalization. Antibody was subsequently stripped from noninternalized MycUNC5A by mild acid treatment. C, Brief (5 min) TPA treatment induces MycUNC5A colocalization with EEA-1. D, After longer (40 min) TPA treatment, there is diminished colocalization of MycUNC5A with EEA-1 likely caused by continued trafficking of MycUNC5A. Scale bar, 10 μm.

Figure 3.

Alanine substitution of all five consensus PKCα phosphorylation sites (Δ5PKC) in UNC5A results in a receptor that fails to internalize in response to PKCα activation. A, Twenty-four hours after infection with MycUNC5A, hippocampal neurons were incubated in the presence or absence of PBS (control) or TPA (100 ng/ml) as indicated to induce PKCα activation and receptor internalization. Bis (100 nm) treatments were performed for 2 h before treatment with PBS or TPA. The graph represents the mean ± SD of at least three separate experiments. For quantitation of the immunoblots, biotinylated receptor was normalized to total receptor expression from the same cultures to control for any variation in expression levels between conditions. The normalized results for each condition were then converted to a percentage of the untreated control condition, which was considered 100%. Graphs represent the mean ± SD of at least three experiments. B, Quantified biotinylation experiments on cells expressing MycUNC5A or MycUNC5A(Δ5PKC), in the presence and absence of TPA stimulation, as indicated. C–J, Quantitative immunofluorescence assays on hippocampal neurons show that TPA treatment results in internalization of MycUNC5A but not MycUNC5AΔ5PKC (compare F, J). Cells were infected with Sindbis viruses and TPA treated (100 ng/ml) as indicated to promote internalization. Intensity of live-labeled UNC5A receptors (C, E, G, I) was compared with the intracellular UNC5A receptor pool (D, F, H, J). The results were quantified by determining the total fluorescence intensity for both the surface-labeled and intracellular-labeled MycUNC5A over the same defined area and graphed as a ratio of S/I (K). The data represent the mean ± SEM S/I ratio. n ≥ 10 cells per condition; *p < 0.001, t test. Scale bar, 20 μm.

Figure 4.

PKCα-mediated internalization of UNC5A is dependent on serines 408 and 587. Hippocampal neurons were infected with either wild-type MycUNC5A or MycUNC5A containing alanine substitutions at the indicated serine or threonine residues and then TPA treated (100 ng/ml) as indicated to induce receptor internalization. A, Results of quantitative immunofluorescence assays, comparing changes in the S/I ratios in response to PKCα activation. The data represent the mean ± SEM S/I ratio from ≥10 cells per condition and are graphed as total surface fluorescence intensity over total internal fluorescence intensity for a fixed area. B, Biotinylation results confirm that MycUNC5A S408A and S587A do not internalize in response to TPA treatment. Graph represents the mean ± SD, where n ≥ 3. *p < 0.001, t test.

Figure 5.

Evaluation of the netrin-1 responsiveness of alanine-substituted UNC5A receptors. Hippocampal neurons (2 DIV), expressing the indicated MycUNC5A receptor constructs, were evaluated for growth cone collapse in the presence of NTN1. Growth cones were treated for 45 min with PBS (A), NTN1 (300 ng/ml; B), or TPA (100 ng/ml; C) followed by NTN1 (300 ng/ml) as indicated and then evaluated for growth cone collapse. The results are reported as the percentage of growth cones collapsed in which ≥50 growth cones were counted from at least three separate experiments. D, Examples of representative MycUNC5A-expressing growth cones before and after netrin-1 treatment. A growth cone was considered collapsed if F-actin staining contained no lamellipodia and two or fewer filopodia. E, Growth cone collapse in uninfected neurons was measured as a control for the treatment conditions. *p < 0.001, one-way ANOVA. Error bars represent SEM.

Figure 6.

Mutation of two dileucine motifs in UNC5A results in a receptor that fails to internalize in response to PKCα activation. Hippocampal neurons were infected with Sindbis viruses encoding wild-type MycUNC5A receptor or a MycUNC5A receptor containing alanine substitutions at leucines 419, 420 and 455, 456 (4L/A). Neurons were TPA treated (100 ng/ml) as indicated, and both biotinylation and immunostaining assays assessed changes in receptor surface expression. A–I, Quantitative immunofluorescence assays. Intensity of live-labeled UNC5A receptors (B, D, F, H) was compared with intracellular UNC5A receptors (A, C, E, G). TPA treatment results in MycUNC5A internalization but no change in cell surface levels of MycUNC5A-4L/A (compare D, H). Scale bar, 20 μm. I, The results were quantified by determining the total fluorescence intensity for both the surface-labeled and intracellular-labeled MycUNC5A over the same defined area and graphed as a ratio of surface-to-intracellular fluorescence. The data represent the mean ± SEM S/I ratio. n ≥ 10 cells per condition; *p < 0.001, t test. J, Biotinylation data from MycUNC5A-4L/A-expressing neurons reveal no significant change in receptor surface expression after TPA treatment (100 ng/ml) compared with wild type. Representative immunoblots from cells treated as indicated are shown. K, Collapse assays demonstrate that PKCα activation does not attenuate netrin-1-induced (300 ng/ml) collapse in UNC5A-4L/A-expressing growth cones compared with wild-type MycUNC5A-expressing controls. *p < 0.001, t test. Error bars represent SEM.

Figure 7.

Turning of hippocampal growth cones in response to a netrin-1 gradient, UNC5A overexpression, and TPA treatment. A, Images of wild-type growth cone at onset (left) and end (right) of a 30 min exposure to a soluble NTN1 gradient (see Materials and Methods for gradient conditions). B, Neuron cultures were incubated with DCC function-blocking antibody 30 min before gradient onset. C, Neurons overexpressing UNC5A at the cell surface are repelled from the netrin-1 gradient. Scale bar, 10 μm. D, Traces of individual growth cone trajectories at 30 min exposure in PBS [control (CTRL)], NTN1, NTN1 with anti-DCC pretreatment, MycUNC5A overexpression in PBS, MycUNC5A overexpression in NTN1, NTN1 with TPA pretreatment. Overexpressed cell surface receptors, antibody preincubations, or TPA pretreatments are indicated at the bottom and gradient condition at the top. E, The average turning angle in degrees (top) and average net neurite extension in micrometers (bottom) for all neurons tested. Overexpressed cell surface receptors, antibody preincubations, or TPA-pretreatments are indicated at the bottom and gradient condition at the top. The asterisk indicates statistically significant difference from control; *p < 0.01, Mann–Whitney test. Scale bar, 10 μm. F, The distribution of all turning angles, plotted at the end of 30 min exposure to netrin-1 gradient. The percentage value refers to the percentage of growth cones with turning angles less than or equal to a given angular value. Positive turning angles correspond to attraction toward the netrin-1 point source. G, Hippocampal growth cone expressing UNC5A on the cell surface at the onset of a netrin-1 gradient and after 30 min (turn 1). After 30 min, the position of netrin-1-containing pipette (5 μg/ml) with respect to the growth cone was reset to time 0 parameters, TPA was added to the bath (100 ng/ml) to induce UNC5A endocytosis, and growth cone was imaged again at 60 min (turn 2). H, Traces of individual growth cone trajectories at 60 min. The large arrows indicate repositioning of the netrin-1 point source at 30 min. The small arrows indicate point along trajectory at which time TPA was added to the bath. I, The average turning angle in degrees (left) and the net neurite extension in micrometers (right). Negative turning angles indicate repulsion, whereas positive turning angles indicate attraction to the netrin-1 point source. **p < 0.05, Mann–Whitney test. Error bars represent SEM.

Figure 8.

Granule cells from P6 cerebellum are repelled by netrin-1 stable gradients. A–D, Representative images of granule cells at time 0 and 30 min after exposure to NTN1 gradient. Wild-type granule cells are repelled from the NTN1 point source (A), and pretreatment with 100 ng/ml TPA for 30 min before NTN1 gradient onset converts the turning response to attraction (B). Granule cells cultured from Unc5a −/− animals are attracted toward the NTN1 point source (C). In contrast, granule cells cultured from PICK1−/− mice are repelled by the NTN1 gradient and this response is not converted to attraction after TPA treatment (D). E, Composite traces from all granule cells subjected to 30 min NTN1 gradients with genotype, TPA pretreatment, and scale bars as indicated. F, The average turning angle in degrees (top) and average net neurite extension in micrometers (bottom) for all granule cells assayed. G, The distribution of granule cell turning angles. Positive turning angles indicate attraction to the netrin-1 point source, and negative turning angles indicate netrin-1 repulsion. *p < 0.01 Mann–Whitney test. Error bars represent SEM. CTRL, Control.