Identification of a novel, putative Rho-specific GDP/GTP exchange factor and a RhoA-binding protein: control of neuronal morphology - PubMed (original) (raw)
Identification of a novel, putative Rho-specific GDP/GTP exchange factor and a RhoA-binding protein: control of neuronal morphology
M F Gebbink et al. J Cell Biol. 1997.
Abstract
The small GTP-binding protein Rho has been implicated in the control of neuronal morphology. In N1E-115 neuronal cells, the Rho-inactivating C3 toxin stimulates neurite outgrowth and prevents actomyosin-based neurite retraction and cell rounding induced by lysophosphatidic acid (LPA), sphingosine-1-phosphate, or thrombin acting on their cognate G protein-coupled receptors. We have identified a novel putative GDP/GTP exchange factor, RhoGEF (190 kD), that interacts with both wild-type and activated RhoA, but not with Rac or Cdc42. RhoGEF, like activated RhoA, mimics receptor stimulation in inducing cell rounding and in preventing neurite outgrowth. Furthermore, we have identified a 116-kD protein, p116(Rip), that interacts with both the GDP- and GTP-bound forms of RhoA in N1E-115 cells. Overexpression of p116(Rip) stimulates cell flattening and neurite outgrowth in a similar way to dominant-negative RhoA and C3 toxin. Cells overexpressing p116(Rip) fail to change their shape in response to LPA, as is observed after Rho inactivation. Our results indicate that (a) RhoGEF may link G protein-coupled receptors to RhoA activation and ensuing neurite retraction and cell rounding; and (b) p116(Rip) inhibits RhoA-stimulated contractility and promotes neurite outgrowth.
Figures
Figure 1
Two-hybrid interaction between RIPs and RhoA. Binding of RIPs, identified in two-hybrid screens, to wild-type (wt) RhoA and activated V14RhoA mutants was analyzed using the two-hybrid system. The C190R mutation in RhoA removes the isoprenylation site to prevent membrane targeting. Rho-GDI, guanine nucleotide dissociation inhibitor (Hancock and Hall, 1993); RIP1 encodes a novel Rho-GDI, termed Rho-GDI2; full-length RIP2 encodes RhoGEF and RIP3 represents p116Rip. The RIP4 isolate encodes a novel protein that remains to be characterized. For further details see text.
Figure 2
Expression pattern of RhoGEF and p116Rip analyzed by Northern blotting. Total RNA (15 μg) of the indicated mouse tissues was analyzed for expression of RhoGEF and p116Rip. In addition, total RNA from proliferating and differentiated (serum-deprived) N1E-115 cells was analyzed. The smaller, ubiquitously expressed transcripts correspond to the cDNAs cloned. As a control for the amount of RNA loaded, the blot was reprobed with a GAPDH probe.
Figure 3
RhoGEF cDNA and predicted amino acid sequence. (A) RhoGEF nucleotide and polypeptide sequence. The DH domain is underlined; the PH domain is doubly underlined. The conserved residues of the zinc finger–like motif are in bold. These sequence data are available from EMBL/GenBank/DDBJ under accession number U73199. (B) Schematic representation of the RhoGEF protein and analogous domains in Lfc and Lbc, with relevent amino acid numbering indicated. Relevant residues are numbered. DH, Dbl-homologous domain; PH, pleckstrin homology domain; COILED, coiled-coil region; L-rich, leucine-rich region; Zn2 +, zinc finger motif. Percentages indicate relative identity of DH/PH or Zn finger domains in pairwise comparison with the corresponding domains in RhoGEF. (C) Alignment of the tandem DH/PH domains of RhoGEF with those of lbc and lfc (GenBank accession numbers U03634 and U28495, respectively). (D) Immunoblot analysis of RhoGEF expression. Lysates of transiently transfected COS cells were analyzed on a 10% SDS-PAGE gel followed by immunoblotting with polyclonal anti-RhoGEF antiserum. Both full-length RhoGEF and an NH2-terminally truncated version of RhoGEF (ΔRhoGEF, lacking amino acids 1–684) were expressed. (E) Lack of two-hybrid interactions between RhoGEF (RIP2) and Rho-family members Rac and Cdc42. Experimental details as in Fig. 1.
Figure 3
RhoGEF cDNA and predicted amino acid sequence. (A) RhoGEF nucleotide and polypeptide sequence. The DH domain is underlined; the PH domain is doubly underlined. The conserved residues of the zinc finger–like motif are in bold. These sequence data are available from EMBL/GenBank/DDBJ under accession number U73199. (B) Schematic representation of the RhoGEF protein and analogous domains in Lfc and Lbc, with relevent amino acid numbering indicated. Relevant residues are numbered. DH, Dbl-homologous domain; PH, pleckstrin homology domain; COILED, coiled-coil region; L-rich, leucine-rich region; Zn2 +, zinc finger motif. Percentages indicate relative identity of DH/PH or Zn finger domains in pairwise comparison with the corresponding domains in RhoGEF. (C) Alignment of the tandem DH/PH domains of RhoGEF with those of lbc and lfc (GenBank accession numbers U03634 and U28495, respectively). (D) Immunoblot analysis of RhoGEF expression. Lysates of transiently transfected COS cells were analyzed on a 10% SDS-PAGE gel followed by immunoblotting with polyclonal anti-RhoGEF antiserum. Both full-length RhoGEF and an NH2-terminally truncated version of RhoGEF (ΔRhoGEF, lacking amino acids 1–684) were expressed. (E) Lack of two-hybrid interactions between RhoGEF (RIP2) and Rho-family members Rac and Cdc42. Experimental details as in Fig. 1.
Figure 3
RhoGEF cDNA and predicted amino acid sequence. (A) RhoGEF nucleotide and polypeptide sequence. The DH domain is underlined; the PH domain is doubly underlined. The conserved residues of the zinc finger–like motif are in bold. These sequence data are available from EMBL/GenBank/DDBJ under accession number U73199. (B) Schematic representation of the RhoGEF protein and analogous domains in Lfc and Lbc, with relevent amino acid numbering indicated. Relevant residues are numbered. DH, Dbl-homologous domain; PH, pleckstrin homology domain; COILED, coiled-coil region; L-rich, leucine-rich region; Zn2 +, zinc finger motif. Percentages indicate relative identity of DH/PH or Zn finger domains in pairwise comparison with the corresponding domains in RhoGEF. (C) Alignment of the tandem DH/PH domains of RhoGEF with those of lbc and lfc (GenBank accession numbers U03634 and U28495, respectively). (D) Immunoblot analysis of RhoGEF expression. Lysates of transiently transfected COS cells were analyzed on a 10% SDS-PAGE gel followed by immunoblotting with polyclonal anti-RhoGEF antiserum. Both full-length RhoGEF and an NH2-terminally truncated version of RhoGEF (ΔRhoGEF, lacking amino acids 1–684) were expressed. (E) Lack of two-hybrid interactions between RhoGEF (RIP2) and Rho-family members Rac and Cdc42. Experimental details as in Fig. 1.
Figure 3
RhoGEF cDNA and predicted amino acid sequence. (A) RhoGEF nucleotide and polypeptide sequence. The DH domain is underlined; the PH domain is doubly underlined. The conserved residues of the zinc finger–like motif are in bold. These sequence data are available from EMBL/GenBank/DDBJ under accession number U73199. (B) Schematic representation of the RhoGEF protein and analogous domains in Lfc and Lbc, with relevent amino acid numbering indicated. Relevant residues are numbered. DH, Dbl-homologous domain; PH, pleckstrin homology domain; COILED, coiled-coil region; L-rich, leucine-rich region; Zn2 +, zinc finger motif. Percentages indicate relative identity of DH/PH or Zn finger domains in pairwise comparison with the corresponding domains in RhoGEF. (C) Alignment of the tandem DH/PH domains of RhoGEF with those of lbc and lfc (GenBank accession numbers U03634 and U28495, respectively). (D) Immunoblot analysis of RhoGEF expression. Lysates of transiently transfected COS cells were analyzed on a 10% SDS-PAGE gel followed by immunoblotting with polyclonal anti-RhoGEF antiserum. Both full-length RhoGEF and an NH2-terminally truncated version of RhoGEF (ΔRhoGEF, lacking amino acids 1–684) were expressed. (E) Lack of two-hybrid interactions between RhoGEF (RIP2) and Rho-family members Rac and Cdc42. Experimental details as in Fig. 1.
Figure 3
RhoGEF cDNA and predicted amino acid sequence. (A) RhoGEF nucleotide and polypeptide sequence. The DH domain is underlined; the PH domain is doubly underlined. The conserved residues of the zinc finger–like motif are in bold. These sequence data are available from EMBL/GenBank/DDBJ under accession number U73199. (B) Schematic representation of the RhoGEF protein and analogous domains in Lfc and Lbc, with relevent amino acid numbering indicated. Relevant residues are numbered. DH, Dbl-homologous domain; PH, pleckstrin homology domain; COILED, coiled-coil region; L-rich, leucine-rich region; Zn2 +, zinc finger motif. Percentages indicate relative identity of DH/PH or Zn finger domains in pairwise comparison with the corresponding domains in RhoGEF. (C) Alignment of the tandem DH/PH domains of RhoGEF with those of lbc and lfc (GenBank accession numbers U03634 and U28495, respectively). (D) Immunoblot analysis of RhoGEF expression. Lysates of transiently transfected COS cells were analyzed on a 10% SDS-PAGE gel followed by immunoblotting with polyclonal anti-RhoGEF antiserum. Both full-length RhoGEF and an NH2-terminally truncated version of RhoGEF (ΔRhoGEF, lacking amino acids 1–684) were expressed. (E) Lack of two-hybrid interactions between RhoGEF (RIP2) and Rho-family members Rac and Cdc42. Experimental details as in Fig. 1.
Figure 4
Regulation of neuronal cell shape by RhoGEF expressed in N1E-115 cells. N1E-115 cells were transiently transfected with the indicated cDNAs, all containing an NH2-terminal epitope tag (HA) and cotransfected with a plasmid containing the gene for β-galactosidase (control, empty vector plasmid). Truncated RhoGEF (ΔRhoGEF) lacks amino acids 1–684. After 48 h, the cells were fixed and stained for β-galactosidase activity. (A) Phenotype of transfected cells (dark cells). Both V14RhoA- and RhoGEF-transfected cells have a rounded morphology without neurites; see also Table I. (B) Analysis of protein expression in immunoblot. Total lysates were prepared from a duplicate transfected culture and analyzed on a 12% SDS-PAGE gel. The blot was probed with anti-HA antibody 12CA5. Bar, 30 μm.
Figure 4
Regulation of neuronal cell shape by RhoGEF expressed in N1E-115 cells. N1E-115 cells were transiently transfected with the indicated cDNAs, all containing an NH2-terminal epitope tag (HA) and cotransfected with a plasmid containing the gene for β-galactosidase (control, empty vector plasmid). Truncated RhoGEF (ΔRhoGEF) lacks amino acids 1–684. After 48 h, the cells were fixed and stained for β-galactosidase activity. (A) Phenotype of transfected cells (dark cells). Both V14RhoA- and RhoGEF-transfected cells have a rounded morphology without neurites; see also Table I. (B) Analysis of protein expression in immunoblot. Total lysates were prepared from a duplicate transfected culture and analyzed on a 12% SDS-PAGE gel. The blot was probed with anti-HA antibody 12CA5. Bar, 30 μm.
Figure 5
p116Rip cDNA, predicted amino acid sequence, and interaction with RhoA in N1E-115 cells. (A) p116Ripnucleotide and polypeptide sequence. The PH domain is underlined. The sequence contains two proline-rich regions (amino acids 164–172: PPTPQEPGP, and amino acids 285–296: PPLLSPPSP) that are putative SH3-binding sites (B). These sequence data for p116Rip are available from EMBL/GenBank/DDBJ under accession number U73200. (B) Schematic representation of domains in p116Rip; pppp, proline-rich putative SH3-binding regions (see above);PH, pleckstrin homology domain; COILED-COIL, coiled-coil domain. The RhoA-interacting region is indicated (amino acids 545–823, black bar). (C) Immunoblot analysis of p116Rip transiently expressed in COS and N1E-115 cells. Cell lysates were analyzed on a 10% SDS-PAGE gel followed by immunoblotting with a polyclonal anti-p116Rip antibody. (Right) Analysis of identical samples, in which the blot was incubated with primary antiserum in the presence of the synthetic peptide used for immunization (see Materials and Methods). (D) Interaction of p116Rip with the GDP- and GTP-bound forms of RhoA in N1E-115 cells. Purified GST–RhoA fusion proteins, preloaded with either GDPβS or GTPγS, were used to fish in lysates of serum-starved N1E-115 cells, as described in Materials and Methods. GST alone served as a control. The GST proteins were isolated on glutathione beads and analyzed by immunoblotting using anti-p116Rip antibody. (Open arrow) Position of p116Rip.
Figure 5
p116Rip cDNA, predicted amino acid sequence, and interaction with RhoA in N1E-115 cells. (A) p116Ripnucleotide and polypeptide sequence. The PH domain is underlined. The sequence contains two proline-rich regions (amino acids 164–172: PPTPQEPGP, and amino acids 285–296: PPLLSPPSP) that are putative SH3-binding sites (B). These sequence data for p116Rip are available from EMBL/GenBank/DDBJ under accession number U73200. (B) Schematic representation of domains in p116Rip; pppp, proline-rich putative SH3-binding regions (see above);PH, pleckstrin homology domain; COILED-COIL, coiled-coil domain. The RhoA-interacting region is indicated (amino acids 545–823, black bar). (C) Immunoblot analysis of p116Rip transiently expressed in COS and N1E-115 cells. Cell lysates were analyzed on a 10% SDS-PAGE gel followed by immunoblotting with a polyclonal anti-p116Rip antibody. (Right) Analysis of identical samples, in which the blot was incubated with primary antiserum in the presence of the synthetic peptide used for immunization (see Materials and Methods). (D) Interaction of p116Rip with the GDP- and GTP-bound forms of RhoA in N1E-115 cells. Purified GST–RhoA fusion proteins, preloaded with either GDPβS or GTPγS, were used to fish in lysates of serum-starved N1E-115 cells, as described in Materials and Methods. GST alone served as a control. The GST proteins were isolated on glutathione beads and analyzed by immunoblotting using anti-p116Rip antibody. (Open arrow) Position of p116Rip.
Figure 5
p116Rip cDNA, predicted amino acid sequence, and interaction with RhoA in N1E-115 cells. (A) p116Ripnucleotide and polypeptide sequence. The PH domain is underlined. The sequence contains two proline-rich regions (amino acids 164–172: PPTPQEPGP, and amino acids 285–296: PPLLSPPSP) that are putative SH3-binding sites (B). These sequence data for p116Rip are available from EMBL/GenBank/DDBJ under accession number U73200. (B) Schematic representation of domains in p116Rip; pppp, proline-rich putative SH3-binding regions (see above);PH, pleckstrin homology domain; COILED-COIL, coiled-coil domain. The RhoA-interacting region is indicated (amino acids 545–823, black bar). (C) Immunoblot analysis of p116Rip transiently expressed in COS and N1E-115 cells. Cell lysates were analyzed on a 10% SDS-PAGE gel followed by immunoblotting with a polyclonal anti-p116Rip antibody. (Right) Analysis of identical samples, in which the blot was incubated with primary antiserum in the presence of the synthetic peptide used for immunization (see Materials and Methods). (D) Interaction of p116Rip with the GDP- and GTP-bound forms of RhoA in N1E-115 cells. Purified GST–RhoA fusion proteins, preloaded with either GDPβS or GTPγS, were used to fish in lysates of serum-starved N1E-115 cells, as described in Materials and Methods. GST alone served as a control. The GST proteins were isolated on glutathione beads and analyzed by immunoblotting using anti-p116Rip antibody. (Open arrow) Position of p116Rip.
Figure 5
p116Rip cDNA, predicted amino acid sequence, and interaction with RhoA in N1E-115 cells. (A) p116Ripnucleotide and polypeptide sequence. The PH domain is underlined. The sequence contains two proline-rich regions (amino acids 164–172: PPTPQEPGP, and amino acids 285–296: PPLLSPPSP) that are putative SH3-binding sites (B). These sequence data for p116Rip are available from EMBL/GenBank/DDBJ under accession number U73200. (B) Schematic representation of domains in p116Rip; pppp, proline-rich putative SH3-binding regions (see above);PH, pleckstrin homology domain; COILED-COIL, coiled-coil domain. The RhoA-interacting region is indicated (amino acids 545–823, black bar). (C) Immunoblot analysis of p116Rip transiently expressed in COS and N1E-115 cells. Cell lysates were analyzed on a 10% SDS-PAGE gel followed by immunoblotting with a polyclonal anti-p116Rip antibody. (Right) Analysis of identical samples, in which the blot was incubated with primary antiserum in the presence of the synthetic peptide used for immunization (see Materials and Methods). (D) Interaction of p116Rip with the GDP- and GTP-bound forms of RhoA in N1E-115 cells. Purified GST–RhoA fusion proteins, preloaded with either GDPβS or GTPγS, were used to fish in lysates of serum-starved N1E-115 cells, as described in Materials and Methods. GST alone served as a control. The GST proteins were isolated on glutathione beads and analyzed by immunoblotting using anti-p116Rip antibody. (Open arrow) Position of p116Rip.
Figure 6
Induction of neurite outgrowth by p116Rip and dominant-negative N19RhoA expressed in N1E-115 cells. (A) N1E-115 cells were transiently transfected with the indicated cDNAs, as in Fig. 4. (B) Analysis of protein expression in immunoblot, as in Fig. 4_B._ (Left) HA-N19RhoA expression visualized in an anti-HA blot; (right) expression of p116Rip and Δp116Rip (anti-p116Rip blot); _a_and b refer to two distinct truncation mutants (amino acids 545–1,024 and 545–823, respectively; see Materials and Methods). Note the presence of endogenous p116Rip in all lanes. This signal is markedly increased in the cells transfected with full-length p116Rip (lane 4). Bar, 30 μm.
Figure 6
Induction of neurite outgrowth by p116Rip and dominant-negative N19RhoA expressed in N1E-115 cells. (A) N1E-115 cells were transiently transfected with the indicated cDNAs, as in Fig. 4. (B) Analysis of protein expression in immunoblot, as in Fig. 4_B._ (Left) HA-N19RhoA expression visualized in an anti-HA blot; (right) expression of p116Rip and Δp116Rip (anti-p116Rip blot); _a_and b refer to two distinct truncation mutants (amino acids 545–1,024 and 545–823, respectively; see Materials and Methods). Note the presence of endogenous p116Rip in all lanes. This signal is markedly increased in the cells transfected with full-length p116Rip (lane 4). Bar, 30 μm.
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