Molecular dissection of the semaphorin 4D receptor plexin-B1-stimulated R-Ras GTPase-activating protein activity and neurite remodeling in hippocampal neurons

Abstract

Plexins serve as receptors for repulsive axonal guidance molecules semaphorins. The cytoplasmic domain of the semaphorin 4D (Sema4D) receptor, Plexin-B1 has two separated Ras GTPase-activating protein (GAP)-homologous domains, C1 and C2. Recently, we reported that the Rho family small GTPase Rnd1 associates with Plexin-B1, and the Plexin-B1-Rnd1 complex stimulates GTPase activity of R-Ras, inducing growth cone collapse in hippocampal neurons in response to Sema4D. However, the molecular mechanisms by which Plexin-B1 exhibits the GAP activity remain unclear. In this report, critical roles of Rnd1 and Sema4D in Plexin-B1-stimulated R-Ras GAP activity and neurite remodeling were examined. The N-terminal region of the cytoplasmic domain of Plexin-B1 containing the C1 domain interacts with the C-terminal region containing the C2 domain, and Rnd1 disrupts this interaction. On the other hand, Sema4D induces clustering of Rnd1-bound Plexin-B1, in parallel with inactivation of R-Ras in cells. Antibody clustering of the recombinant cytoplasmic domain of Plexin-B1 in the presence of Rnd1 triggers the R-Ras GAP activity. Deletion of the extracellular domain of Plexin-B1 causes ligand-independent clustering of the receptor, rendering the receptor constitutively active in the presence of Rnd1, and induces contraction of COS-7 cells and inhibition of neurite outgrowth in hippocampal neurons. These results indicate that Rnd1 opens the two R-Ras GAP domains of Plexin-B1, and Sema4D-induced receptor clustering stimulates R-Ras GAP activity and neurite remodeling in hippocampal neurons.

Figure 1.

Rnd1 disrupts the interaction between the N- and C-terminal regions within the cytoplasmic domain of Plexin-B1. A, Schematic representation of the Plexin-B1 constructs used in this study. The Rnd1-binding region and the R-Ras GAP domain are indicated. Letters indicate specific amino acid residues within domains (A, Ala; F, Phe; G, Gly; L, Leu; P, Pro; R, Arg; V, Val), and numbers indicate amino acid positions within the sequence. B, Lysates from COS-7 cells expressing the Myc-tagged N-terminal portion of the cytoplasmic domain of Plexin-B1 (Myc-N-Cyt) were used in a pull-down assay with the GST-fused C-terminal portion (GST-C-Cyt). Bound proteins and total cell lysate (Lysate) were analyzed by immunoblotting with anti-Myc antibody. C, Myc-tagged C-Cyt and increasing amounts of HA-tagged Rnd1 were expressed in COS-7 cells. Lysates were used in a pull-down assay with GST-N-Cyt. D, Lysates from COS-7 cells expressing Myc-N-Cyt-GGA were used in a pull-down assay with GST-C-Cyt. E, Lysates from COS-7 cells expressing Myc-C-Cyt and HA-tagged Rnd1 were used in a pull-down assay with wild-type (WT) or the GGA mutant of GST-N-Cyt. F, Lysates from COS-7 cells expressing Myc-C-Cyt and wild-type or the A45 mutant of HA-tagged Rnd1 were used in a pull-down assay with GST-N-Cyt. G, Lysates from COS-7 cells expressing Myc-N-Cyt were used in a pull-down assay with GST-Cyt. The results shown are representative of three independent experiments that yielded similar results.

Figure 2.

Sema4D induces clustering of Plexin-B1 in parallel with inactivation of R-Ras. A, COS-7 cells transfected with Myc- and HA-tagged full-length Plexin-B1 and GFP-tagged Rnd1 were stimulated with Sema4D for the indicated times. The cell lysates were immunoprecipitated (IP) with anti-Myc antibody. B, COS-7 cells transfected with Myc-Plexin-B1, GFP-Rnd1, and HA-R-Ras were stimulated with Sema4D for the indicated times. Then the cell lysates were incubated with GST-RBD, and bound R-Ras was detected with anti-HA antibody. The results shown are representative of five independent experiments that yielded similar results. FL, Full length.

Figure 3.

Antibody clustering of the cytoplasmic domain of Plexin-B1 stimulates the GTPase activity of R-Ras. Recombinant R-Ras preloaded with [γ-³²P]GTP was incubated with the recombinant cytoplasmic domain of Plexin-B1 with (filled symbols, solid lines) or without (empty symbols, dashed lines) antibody-based clustering. The remaining radioactivity bound to R-Ras on the indicated times was measured by a nitrocellulose filtration assay. WT, Wild type.

Figure 4.

Deletion of the extracellular domain of Plexin-B1 induces ligand-independent clustering, leading to constitutive activation. A, Schematic representation of Plexin-B1 deletion constructs used in this study. B, The membrane fraction from COS-7 cells transfected with Plexin-B1 mutant receptors was analyzed by SDS-PAGE and immunoblotting both under reducing [with β-mercaptoethanol (β-ME)] and nonreducing (without β-mercaptoethanol) conditions. C, Lysates from COS-7 cells expressing Myc-tagged Plexin-B1 mutants, GFP-Rnd1, and HA-R-Ras were incubated with GST-RBD, and bound R-Ras was detected with anti-HA antibody. D, Relative R-Ras activity was determined by the amount of R-Ras in cell lysates analyzed by NIH Image software. Results are the means ± SEM of three independent experiments. FL, Full length.

Figure 5.

Rnd1 binding is required for the GAP activity mediated by Plexin-B1Δect. A, COS-7 cells were transfected with Myc-tagged Plexin-B1Δect mutants, GFP-Rnd1, and HA-R-Ras. GTP-bound R-Ras isolated with GST-RBD was detected with anti-HA antibody. B, Relative R-Ras activity was determined by the amount of R-Ras in cell lysates analyzed by NIH Image software. Results are the means ± SEM of three independent experiments. WT, Wild type.

Figure 6.

Plexin-B1Δect induces ligand-independent contraction of COS-7 cells. A, COS-7 cells transfected with various deletion mutants of Plexin-B1 together with GFP or GFP-Rnd1 were fixed 14 hr after transfection. Expression of Myc-tagged Plexin-B1 constructs was also detected with anti-Myc antibody (data not shown). Scale bar, 20 μm. B, Quantitative analysis of COS-7 cell contraction induced by Rnd1 and the deletion mutants of Plexin-B1. GFP- and Myc-staining double-positive cells with an area of <350 μm² were scored as a percentage of the total number of transfected cells. Approximately 50 cells were assessed in one experiment, and data are the means ± SEM of three independent experiments. FL, Full length.

Figure 7.

Plexin-B1Δect is constitutively active in rat hippocampal neurons. A, Primary hippocampal neurons from rat embryos were seeded onto laminin-coated coverslips and cotransfected with GFP and Myc-tagged Plexin-B1. Cultures were fixed 16 hr after transfection and stained with anti-Myc antibody. Transfected cells were shown by the fluorescence of GFP. Expression of Myc-tagged Plexin-B1 constructs was also detected with anti-Myc antibody (data not shown). Scale bar, 20 μm. B, Average neurite outgrowth was quantitated. The means ± SEM of three independent experiments are presented. FL, Full length; WT, wild type.