The semaphorin receptor plexin-B1 signals through a direct interaction with the Rho-specific nucleotide exchange factor, LARG

Abstract

Semaphorins are axon guidance molecules that signal through the plexin family of receptors. Semaphorins also play a role in other processes such as immune regulation and tumorigenesis. However, the molecular signaling mechanisms downstream of plexin receptors have not been elucidated. Semaphorin 4D is the ligand for the plexin-B1 receptor and stimulation of the plexin-B1 receptor activates the small GTPase RhoA. Using the intracellular domain of plexin-B1 as an affinity ligand, two Rho-specific guanine nucleotide exchange factors, leukemia-associated Rho GEF (LARG; GEF, guanine nucleotide exchange factors) and PSD-95/Dlg/ZO-1 homology (PDZ)-RhoGEF, were isolated from mouse brain as plexin-B1-specific interacting proteins. LARG and PDZ-RhoGEF contain several functional domains, including a PDZ domain. Biochemical characterizations showed that the PDZ domain of LARG is directly involved in the interaction with the carboxy-terminal sequence of plexin-B1. Mutation of either the PDZ domain in LARG or the PDZ binding site in plexin-B1 eliminates the interaction. The interaction between plexin-B1 and LARG is specific for the PDZ domain of LARG and LARG does not interact with plexin-A1. A LARG-interaction defective mutant of the plexin-B1 receptor was created and was unable to stimulate RhoA activation. The data in this report suggest that LARG plays a critical role in plexin-B1 signaling to stimulate Rho activation and cytoskeletal reorganization.

Fig 1.

Detection of plexin-B1 interacting proteins. (A) Detection of plexin-B1 interacting proteins from biotinylated mouse brain extracts. Biotinylated mouse brain lysates were subjected to affinity purification over either GST or GST-B1_C affinity columns. Bound proteins were detected by streptavidin-HRP Western blot and eluted GST or GST-B1_C was detected by Coomassie blue staining. An asterisk indicates the 200-kDa plexin-B1 interacting protein. (B) Detection of a 200-kDa plexin-B1 interacting protein by silver staining. Purification was performed essentially as described in A. Bound proteins were detected by silver staining. An asterisk indicates the 200-kDa plexin-B1 interacting protein.

Fig 2.

Identification of the 200-kDa plexin-B1 interacting protein as LARG by mass spectrometry analysis. (A) MS–MS spectrum of the 1,040.60-Da peptide. MS–MS spectrum of the 1,040.60-Da peptide predicts the amino acid sequence of (R)GFPSILQPPR. The “(R)” is predicted based on the sequence of human LARG and the specificity of the trypsin digestion. The major Y ions are labeled. Y* indicates the Y ion with loss of an ammonium ion. Mass to charge ratio is denoted, m/z. (B) Domain structures of human LARG and PDZ-RhoGEF. LARG and PDZ-RhoGEF contain PDZ, regulator of G protein signaling (RGS), Dbl homology (DH), and pleckstrin homology (PH) domains as indicated. The three LARG peptides and the one PDZ-RhoGEF peptide identified by MS–MS spectra are indicated.

Fig 3.

LARG and PDZ-RhoGEF interact with plexin-B1. (A) Full-length plexin-B1 and the cytoplasmic domain of plexin-B1 interact with LARG. HEK293 cells were transfected with the indicated plasmids. Immunoprecipitation with HA antibody (Left) or VSV antibody (Right) was performed. Coimmunoprecipitated LARG was detected by AU1 Western blot (Top). HA-A1_C and HA-B1_C are HA-tagged versions of the cytoplasmic domains of plexin-A1 and plexin-B1, respectively. VSV-B1 and VSV-A1 are full-length VSV-tagged versions of plexin-B1 and plexin-A1, respectively. (B) PDZ-RhoGEF interacts with plexin-B1 with lower affinity than with LARG. HEK293 cells were transfected with the indicated plasmids. Glutathione agarose was used to purify GST-B1_C and bound AU1-LARG and AU1-PDZ-RhoGEF were detected by AU1 Western blot.

Fig 4.

Plexin-B1 C terminus binds the PDZ domain of LARG. (A) Schematic representation of various plexin-B1 and LARG constructs used in HEK293 cell expression and yeast two-hybrid studies. HV4 corresponds to amino acids 1612–1910 of plexin-B1. Black boxes in plexin-B1 correspond to the two conserved regions in the cytoplasmic domain. The filled oval corresponds to the Cdc42/Rac interactive binding domain (17). (B) LARG-PDZ directly binds the C terminus of plexin-B1. Yeast two-hybrid assay was performed with the indicated plasmids. Transformed yeast were selected for on leucine and tryptophan-deficient media (SC-LW). Positive interactions were selected for by growth on histidine-, leucine-, and tryptophan-deficient media (SC-HLW). A1_C and B1_C represent the cytosolic domains of plexin-A1 and plexin-B1, respectively. LARG-PDZ denotes the PDZ-domain of LARG. B1_C-HV4 indicates a truncated fragment of the intracellular domain of plexin-B1 lacking the carboxy-terminal sequence. Lamin and smgGDS are negative controls and RacL61 is a positive control. (C) Plexin-B1 does not interact with the Lin-7 type I PDZ domain. HEK293 cells were transfected as indicated and immunoprecipitation with HA antibody was performed. Coimmunoprecipitation of Lin-7 was detected by myc Western blot. No interaction between plexin-B1 and Lin-7 was observed (Left), whereas plexin-B1 interacted with LARG in the same experiment (Right). (D) The K77A mutation in the PDZ domain of LARG abolishes the interaction with plexin-B1. Yeast two-hybrid assay was performed as in B. (E) Mutation of K77A in full-length LARG abrogates interaction with the cytosolic domain of plexin-B1. HEK293 cells were transfected as indicated. Immunoprecipitation with HA antibody was performed. Coimmunoprecipitated LARG was detected by AU1 Western blot. (F) Masking of the plexin-B1 C-terminal PDZ-binding site abolishes the interaction with LARG. HEK293 cells were transfected as indicated. Immunoprecipitation of plexin was performed with VSV antibody and associated LARG was detected by AU1 Western blot.

Fig 5.

Sema4D/plexin-B1 interaction stimulates RhoA through LARG binding. (A) Sema4D stimulates RhoA activation via plexin-B1 and LARG. GST-Rhotekin-RBD pull-down of GTP-RhoA was performed. GTP-RhoA was detected by myc Western blot (Top). Levels of plexin-B1 and RhoA were determined by Western blotting VSV and myc, respectively, in the lysate. (B) Model of plexin-B1 activation in regulation of Rho and Rac. Sema4D binding of plexin-B1 activates RhoA via LARG. The C terminus of plexin-B1 interacts with the PDZ domain of LARG, which in turn activates RhoA. Plexin-B1 also functions to sequester active Rac from downstream effectors and together with active RhoA promote cytoskeletal changes associated with growth cone turning/collapse.