Expression pattern of Grit mRNA and protein. (A) Northern blot analysis. Blots of mRNA from adult human tissues were probed with a fragment of human Grit corresponding to the CT region. Size markers are indicated on the left in kilobases. Abbreviations: s. muscle, skeletal muscle; s. intestine, small intestine. (B) In situ hybridization of Grit mRNA. A sagittal section from an embryonic day 19.5 rat embryo was hybridized with an antisense probe of the internal region of rat Grit. The adjacent section hybridized with a sense control probe gave no specific signals (not shown). (C) Western blot analysis of Grit was carried out by using two kinds of anti-Grit polyclonal antibodies (top, PR205; bottom, CT205) and lysates from various cell lines. Lanes 1 and 6, SH-SY5Y; lane 2, SMS-KCN; lane 3, HeLa; lane 4, 293; lane 5, TIG1; lane 7, PC12. (D) Agarose gel showing Grit RT-PCR products amplified from total RNA isolated from mock- or NGF-treated PC12 cells with (+) or without (−) reverse transcriptase (RTase). (E to G) SH-SY5Y cells incubated with retinoic acid for 2 days were stained with the anti-Grit antibody PR205. Scale bars, 2 μm (E and G) or 10 μm (F). (H to L) PC12 cells transfected (H to J) or not (K and L) with human Grit cDNA were treated with NGF for 2 days, and stained with anti-Grit antibody, PR205 (H to J) or CT205 (K and L). Scale bars: 2 μm (H to J) or 5 μm (K and L). Specificity of anti-Grit staining was checked as follows. (i) Control staining of SH-SY5Y and PC12 cells without primary antibody gave no significant signals at the same dilution of second antibody used here. (ii) Cell staining with a serial dilution of anti-Grit antibodies showed a proportional decrease in specific signals as shown in Fig. 2E-L. (iii) When COS-1 cells were transfected with FLAG-tagged Grit cDNA and costained by anti-Grit PR205 and anti-FLAG antibodies, both staining patterns were identical.

Characterization of in vitro RhoGAP activity of Grit protein. GTPase activation of RhoA, Rac1, and Cdc42 (40 nM each) stimulated by various amounts of recombinant Grit GAP domain (▪, 2 nM; ▴, 20 nM; •, 200 nM RhoGAP) and the intrinsic GTPase activity (○) of the GTPases were measured at 5-min intervals. The percentage γPi associated with the GTPase at each time point was subtracted from 100% to give the percentage of GTP hydrolyzed.

Neurite extension assay of wild-type and mutant Grit proteins. Plasmids expressing FLAG-tagged wild-type or mutants of Grit were transfected into PC12 cells, along with pEGFP-C1. The cells were cultured with NGF for 58 h and fixed for microscopy. The neurite extension assay was carried out as described in Materials and Methods. More than three separate experiments were done for each construct, and at least 50 cells were counted for each experiment. (A) Diagrams of FLAG-tagged wild-type and mutants of Grit. (B) The ratio of neurite length/cell body length was divided into 11 segments as indicated under the horizontal axes. For each construct, a histogram plotting the percentage of cell number in each segment is presented. (C) Cells having neurites whose lengths were twofold longer than their cell body lengths were scored as neurite bearing. Results are expressed as the mean percentage of neurite-bearing cells with the standard deviation (error bars). Symbols indicate the results of t test analysis; *, P < 0.01 compared with the control.

Association of Grit with CrkL/Crk and Cas. (A) PC12 cell lysates were incubated with either 25 μg of GST or GST fusion containing Grit-CT on glutathione-Sepharose. Cell lysates (7% of starting material) and eluates from the affinity beads were immunoblotted with the indicated antibodies. (B) PC12 cells were mock transfected or transfected with FLAG-Grit-CT plasmid, serum starved, and treated as indicated above the panels. Anti-FLAG immunoprecipitates were probed with anti-CrkL (top) or anti-FLAG (bottom) antibodies. (C) Grit-transfected COS-1 cells were serum-starved and lysed for immunoprecipitation with anti-Grit antibodies or control IgG. The immunoprecipitates were probed with anti-Cas, anti-CrkL, or anti-Grit antibodies. (D) COS-1 cells were transfected with FLAG-Grit-CT construct, deprived of serum, and stimulated for various lengths of time with EGF (0 to 30 min). Anti-FLAG immunoprecipitates were immunoblotted with antiphosphotyrosine, anti-FLAG (for Grit-CT detection), anti-EGFR, anti-Cas, or anti-CrkL antibodies. (E) Ligand-induced translocation of Grit in SH-SY5Y cells. SH-SY5Y cells were mock treated or treated with EGF for 5 min, and costained as indicated above the panels. Scale bars, 5 μm.

Analysis of Grit amino acid sequence. (A) Schematic representation of human Grit (KIAA0712). The deposited KIAA0712 amino acid sequence (GenBank AB018255) was inferred to be truncated in the middle of the RhoGAP domain; we thus determined the corresponding nucleotide sequences of Grit cDNA clones obtained by 5′ rapid amplification of cDNA ends and added N-terminally 151 residues of Grit. RhoGAP domain, filled box; TrkA/adapter-binding CT region, hatched box. The amino acid positions of the domain boundaries are shown at top. Potential SH3 binding motifs I to V, predicted by the Scansite program, are shown by bars. (B) Phylogenetic analysis of the GAP domains of Grit and RhoGAP family proteins by use of the GENETYX program. The percent identities to human Grit are shown at the right. H.s., Homo sapiens; D.m., D. melanogaster; C.e., C. elegans. (C) Alignment of the GAP domain of Grit with that of other RhoGAP family members. Grit, residues 37 to 190; CdGAP, residues 35 to 188; p190, residues 1263 to 1409; N-chimerin, residues 282 to 434. Identical residues are indicated by asterisks. The critical glycine and arginine for GAP activity are in boldface type.

Interaction between Grit and TrkA. (A) The cytoplasmic region of human TrkA (not the TrkA/N-Shc chimera used in the initial screening) fused to the GAL4 DNA binding domain or the GAL4 DNA binding domain alone was coexpressed in the yeast two-hybrid assay with the GAL4 transactivation domain alone or with the GAL4 fusion to Shc PTB domain or Grit-CT region. Positive interactions were indicated by the induction of the lacZ reporter (blue color). (B) 293 cells were transfected with the FLAG-tagged Grit-CT construct and the indicated Trk cDNA. Anti-FLAG immunoprecipitates were probed with anti-pan Trk or anti-FLAG (for Grit-CT detection) antibody. Exposure times were adjusted so that comparable levels of signals were obtained in anti-Trk immunoblotting of cell lysates. (C) Lysates from PC12 cells with or without NGF treatment were incubated with either 25 μg of GST or GST fusion containing Grit-CT on glutathione-Sepharose. Whole-cell lysate (7% of starting material) and eluates from the affinity beads were analyzed by anti-Trk immunoblotting (top). Eluates were also subjected to SDS-PAGE and Coomassie staining (bottom right). For evaluation of NGF-induced Trk autophosphorylation, anti-Trk immunoprecipitates from mock- or NGF-treated PC12 cells were probed with antiphosphotyrosine antibody (bottom left). Abbreviations: IP, immunoprecipitation; CBB, Coomassie brilliant blue. (D) ³⁵S-labeled in vitro-translated wild-type TrkA and a series of its mutants (top panel) were incubated with 10 μg of either GST or GST fusion containing Grit-CT protein. Inputs and eluates from the affinity beads were subjected to SDS-PAGE, and the labeled proteins were then detected by autoradiography. Abbreviations: TM, transmembrane domain; TK, tyrosine kinase domain; asterisk, ATP-binding site. (E) PC12 cells were treated with NGF for 2 days and costained as indicated above the panels (merged images at righ show Grit in green and TrkA in red). Scale bar, 5 μm. (F) 293 cells were transfected with FLAG-Grit cDNA, serum starved, and treated with NGF for 5 min. Anti-FLAG immunoprecipitates were probed with antiphosphotyrosine (top) or anti-FLAG (middle) antibody. NGF-dependent Trk autophosphorylation was confirmed as in Fig. 3C (bottom).

Characterization of in vivo RhoGAP activity of Grit protein. (A) Swiss 3T3 cells were transfected with the pIRES-EGFP-Grit GAP domain construct. After 2 days of incubation, phase-contrast (top) and fluorescent (bottom) images were captured with a Zeiss Axiovert microscope. Scale bar, 10 μm. (B and C) Serum-starved Swiss 3T3 cells were microinjected with the purified Grit RhoGAP domain before stimulation with LPA (B) or bradykinin (C). To identify the microinjected cells, we coinjected FITC-dextran along with RhoGAP protein (shown by arrowheads). Actin filaments were visualized by staining with rhodamine-phalloidin. Scale bars, 10 μm. (D) For controls, starved Swiss 3T3 cells were microinjected with FITC-dextran alone (shown by arrowheads) and stimulated with LPA (top) or bradykinin (bottom). Actin filaments were visualized as above. Scale bars, 10 μm.

Association of Grit with N-Shc adapter molecule. (A) COS-1 cells were transfected with the FLAG-Grit-CT construct and the indicated T7-tagged Shc family cDNA, serum-starved, and treated with EGF. Anti-FLAG immunoprecipitates were probed with anti-T7 (top) or anti-FLAG (middle, for Grit-CT detection) antibody. Anti-T7 immunoblotting of cell lysates showed comparable levels of expression of Shc family members (bottom). (B) COS-1 cells were cotransfected with T7-tagged wild-type or mutant N-Shc constructs and FLAG-Grit-CT plasmid. Cell lysates and anti-FLAG immunoprecipitates were probed with anti-T7 antibody. (C) Lysates from COS-1 cells transfected with Grit cDNA were incubated with either 20 μg of GST or GST fusion with N-Shc SH2 domain on glutathione-Sepharose. Cell lysates and eluates from the affinity beads were probed with anti-Grit (top) or anti-GST (bottom) antibodies. (D) FLAG-Grit cDNA with or without N-Shc construct was used to transfect COS-1 cells. The transfected cells were deprived of serum and then mock treated or treated with EGF. Anti-T7 immunoprecipitates were probed with anti-Grit (top) or anti-T7 (middle, for N-Shc detection) antibodies. (E) COS-1 cells were transfected with FLAG-Grit cDNA along with N-Shc or N-Shc PTB-CH1 construct as indicated above the panels. The transfected cells were serum starved and treated or not with EGF. Anti-FLAG immunoprecipitates were probed with antiphosphotyrosine or anti-Grit antibodies. Phosphorylated proteins in the top panel were identified by immunoblotting with their respective antibodies (see Fig. 8). Anti-T7 blot of cell lysates confirmed the expression of N-Shc constructs (bottom). (F) COS-1 cells were mock transfected or transfected with FLAG-Grit and N-Shc constructs or FLAG-tagged Grit GAP domain construct, serum starved, and treated with or without EGF. [γ-³²P]GTP-loaded RhoA or Cdc42 was incubated with anti-FLAG immunoprecipitates from lysates of the transfected cells, and the hydrolyzed GTP was measured as in Fig. 4.