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. 2016 Nov 17;1(19):e86934. doi: 10.1172/jci.insight.86934

Figure 1. ROBO2, SRGAP1, and nonmuscle myosin IIA (NMIIA) form a complex.

Figure 1

(A) Diagrams showing the domain structure of SRGAP1 and ROBO2 proteins. Abbreviations: F-BAR, FES-CIP4 homology and Bin/amphiphysin/Rvs domain; RhoGAP, GTPase-activator protein (GAP) for Rho-like GTPases domain; SH3, Src homolog 3 domain; Ig, immunoglobulin domain; FN3, fibronectin type 3 domain; CC0, CC1, CC2, CC3, cytoplasmic conserved region 0, 1, 2, and 3; TM, transmembrane domain. (B) Yeast 2-hybrid assay shows a positive interaction between the ROBO2 intracellular domain (ROBO2-ICD) and the SH3 domain of SRGAP1. The interaction is mediated by the proline-rich CC3 motif in ROBO2 and the SH3 domain in SRGAP1. LacZ reporter (X-gal): +++, yeast turned dark blue; ++, light blue; –, white in 24 hours. Leucine reporter (-Leu): +, yeast grew; –, yeast did not grow. Numbers indicate residue positions in the full-length ROBO2 protein. (C) Yeast 2-hybrid assay shows a positive interaction between the F-BAR domain of SRGAP1 and the myosin regulatory light chain (MRLC). Deletion of either N-terminal residues 1–33 or C-terminal amino acids 130–172 in MRLC abolishes the interaction. Substitutions of phosphorylation sites Thr18 and Ser19 with Ala-Ala, Ala-Asp, or Asp-Asp do not affect the interaction of MRLC with SRGAP1. EFh, EF-hand calcium-binding motif. Numbers indicate residue positions in the full-length proteins. (D) Full-length SRGAP1, not SRGAP1 with F-BAR deleted, coprecipitates with NMIIA heavy chain (NMHC IIA). His-myc-SRGAP1 or His-myc-SRGAP1-ΔF-BAR was expressed in HEK cells and precipitated with Ni-NTA beads (lanes 1 and 2) or control beads (lane 3) in the presence of SLIT2. (E) Full-length ROBO2, not ROBO2 without CC3 (ROBO2-ΔCC3), coprecipitates with SRGAP1, NMHC IIA, and MRLC. His-myc-ROBO2 or His-myc-ROBO2-ΔCC3 was expressed in HEK cells and precipitated with Ni-NTA beads (lanes 1 and 2) or control beads (lane 3) in the presence of SLIT2. (F) Coimmunoprecipitation of newborn mouse kidney endogenous ROBO2, SRGAP1, MRLC, p-MRLC, and NMHC IIA. Precipitates were prepared using mouse monoclonal anti-ROBO2 antibody and IgG antibody was used as a negative control.