Epitope-tagged VZG-1 is expressed and functionally increases [³H]-LPA binding in plasma membranes of RH7777 hepatoma expressing no vzg-1. (A) Cytoplasmic RNAs (20 μg) from RH7777 hepatoma or B103 neuroblastoma cell line serum-starved for 24 h were subjected to Northern blot analysis (24) along with vzg-1 containing RNAs from brain and TSM, a cortical neuroblast cell line. Loading control, 28S RNA. (B) [³H]-LPA binding in plasma membranes of RH7777 cells transiently transfected with pflag or pflag/vzg-1. Specific binding in pflag/vzg-1-transfected cells was 6.4 ± 0.9 cpm/μg protein, 60% of total binding. Data are the mean ± SEM (n = 3). ∗, P < 0.05 (using Student’s t test) vs. pflag-transfected cells. (Inset) Immunoprecipitation with anti-flag antibody after [³⁵S]-methionine/cysteine labeling of pflag or pflag/vzg-1-transfected cells reveals a protein band of predicted size only in pflag/vzg-1-transfected cells.

VZG-1 directly couples to G_i and other G proteins in plasma membranes after LPA stimulation. (A) Effect of LPA on [³⁵S]-GTPγS binding to membranes from RH7777 cells transiently transfected with pflag or pflag/vzg-1. Basal [³⁵S]-GTPγS binding (no LPA) was 64.8 ± 5.3 (pflag) and 100.7 ± 6.8 (pflag/vzg-1) cpm/μg protein. Data are expressed as a percentage of control (no LPA) and represent the mean ± SEM (n = 3–4). ∗, P < 0.05 vs. no LPA (using Student’s t test). (B) Effect of LPA on [³⁵S]-GTPγS binding to membranes of control and experimental B103 stable cell lines and cortical neuroblast cell line TSM. Basal [³⁵S]-GTPγS binding was 45.7 ± 2.5 (B103), 61.9 ± 2.1 (E-7), 57.2 ± 4.2 (A-5), 72.6 ± 3.7 (S-3), 73.9 ± 5.5 (S-7), and 83.3 ± 5.8 (TSM) cpm/μg protein. Data are expressed as described in A (n ≥ 3). ∗, P < 0.05 (using Student’s t test) vs. no LPA. (C) Effect of PTX treatment on LPA-induced [³⁵S]-GTPγS binding in clone S-3. Cells were treated without or with PTX. Basal [³⁵S]-GTPγS binding was 72.6 ± 3.4 (no PTX) and 71.1 ± 1.3 (PTX) cpm/μg protein. (Inset) Autoradiogram of PTX-catalyzed [³²P]-ADP ribosylation in membranes of S-3 cells pretreated without (Left) or with PTX (Right) demonstrates PTX functionality. Data are expressed as described in A (n = 3). ∗, P < 0.05 (using Student’s ttest) vs. no LPA. #, P < 0.05 vs. no PTX. (D) Effect of structurally related phospholipids on [³⁵S]-GTPγS binding in clone S-3. Basal [³⁵S]-GTPγS binding was 91.9 ± 20.2 cpm/μg protein. Data are expressed as described in A (n = 3). ∗, P < 0.05 (using Student’s t test) vs. no LPA. (E) VZG-1 couples to G_i after LPA stimulation. A-5 or S-3 cell membranes were labeled with [³⁵S]-GTPγS, and G_αi protein was immunoprecipitated as described in Experimental Procedures. Basal binding in the immunoprecipitates was 15.2 ± 4.7 (A-5) or 20.0 ± 6.9 cpm/μg protein (S-3). Data are expressed as described in A (n = 3). ∗, P < 0.05 (using Student’s t test) vs. no LPA. (F) Western blot demonstrates expression of G proteins thought to mediate LPA signaling in mouse brain, B103, and RH7777 cell lines. Isolated membranes (50 μg) were subjected to Western blotting, as described (13). See Table 1 and Experimental Procedures for abbreviations.

VZG-1 mediates stress fiber formation by LPA in RH7777 cells through Rho activation. Immunofluorescent microscopy of RH7777 cells transfected with pflag (A and B) or pflag/vzg-1 (C to E) and then exposed to LPA at 0 nM (A, C, and D) or 100 nM (B, E, and F) for 15 min. Cells were fixed and double-labeled for flag-epitope (using M2 antibody visualized by indirect immunofluorescence using fluorescein isothiocyanate; C and E) and actin (visualized with TRITC-phalloidin; A, B, D, and F). Note that stress fibers are observed only with VZG-1 expression and LPA exposure (E and F). Arrowheads, nontransfected cells. (Bar = 10 μm.) (G) Effect of his-C3 exoenzyme on LPA-induced stress fiber formation in VZG-1-expressing cells. RH7777 cells transfected with pflag/vzg-1 were treated without or with his-C3 exoenzyme, exposed to varying concentrations of LPA, and double-labeled for flag epitope and actin. The change in the number of double-labeled cells with stress fibers after LPA exposure compared with control (no LPA) was expressed as “fold increase.” Data are the mean ± SEM (n = 4). ∗, P < 0.05 (using Student’s t test) vs. no LPA. #, P < 0.05 vs. no C3 exoenzyme. (H) Effect of structurally related phospholipids on VZG-1-mediated stress fiber formation in pflag/vzg-1-transfected RH7777 cells. Data are the mean ± SEM (n = 3). ∗, P < 0.05 (using Student’s t test) vs. no LPA. See Experimental Procedures for abbreviations.

VZG-1 is required for LPA-dependent neurite retraction/cell rounding through Rho activation. Phase contrast microscopy of parental B103 cells (A and B), stable clone for antisense vzg-1 (clone A-5; C and D) or a stable clone for sense vzg-1 (clone S-3; E and F). Cells were treated with control vehicle buffer (A, C, and E) or with 1 μM LPA (B, D, and F) for 30 min. Only the sense-transfected cells exposed to LPA retracted neurites (F). (G) Dose–response relationship of LPA to neurite retraction in control and experimental B103 stable cell lines. Three independently derived, sense-expressing cell lines (S-3, S-4, and S-7) were compared with control cell lines (B103, parental line; E-7, empty vector-transfected line; and A-5, antisense-transfected line). Cells were treated with varying concentrations of LPA and fixed, and the percentages of neurite-retracted cells were determined. Data are the mean ± SEM (n ≥ 3). Only vzg-1 sense-transfected lines respond to LPA exposure. (H) Effect of functional PTX or his-C3 exoenzyme on LPA-induced neurite retraction. Sense-expressing clone S-3 was incubated with or without PTX or his-C3 exoenzyme and then analyzed for neurite retraction/cell rounding. Data are the mean ± SEM (n = 4). ∗, P < 0.05 (using Student’s t test) vs. no LPA. #, P < 0.05 vs. 1 μM LPA in nontreated cultures (None). See Experimental Procedures for abbreviations.

VZG-1 expression and LPA exposure activate SREs and increase DNA synthesis through both PTX- and C3-sensitive signaling pathways. (A) Effect of PTX or his-C3 exoenzyme on SRE activation by LPA through VZG-1 expression. Stable B103 cells containing CAT driven by c-fos SREs (clone C-1) were transfected transiently with pHook2 or pHook2/vzg-1, treated without or with PTX or his-C3 exoenzyme, and treated with varying LPA concentrations, and SRE activation determined. Basal CAT expression was 229 ± 36 (pHook2) or 191 ± 20 pg/mg protein (pHook2/vzg-1). Data are expressed as a percentage of maximal response at 1 μM LPA over control (no LPA), the mean ± SEM (n ≥ 4). LPA at 0.1–10 μM significantly increased CAT expression in pHook2/vzg-1-transfected cells (P < 0.05 vs. no LPA using Student’s t test). Both PTX and his-C3 exoenzyme significantly blocked LPA-induced increase in CAT ex-pression (P < 0.05 vs. no treatment with toxins). (B) Effect of PTX or his-C3 exoenzyme on DNA synthesis stimulated by LPA through VZG-1 expression. The S-3 cells were treated without or with PTX or his-C3 exoenzyme and DNA synthesis assayed after 24 h by a 1-h BrdU pulse. Basal percentages of BrdU-incorporated cells were 19.6 ± 1.9 (no treatment), 22.7 ± 2.0 (PTX), or 24.8 ± 1.6% (his-C3). Data are expressed as a percentage of control (no LPA), mean ± SEM (n = 3–6). ∗, P < 0.05 vs. no LPA. #, P < 0.05 (using Student’s t test) vs. 1 μM LPA in nontreated culture.

VZG-1-Mediated responses after LPA stimulation, based on the presented heterologous expression studies.