Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Feb 2;293(5):1570-1578.
doi: 10.1074/jbc.RA117.000964. Epub 2017 Dec 19.

Activated Heterotrimeric G Protein α i Subunits Inhibit Rap-dependent Cell Adhesion and Promote Cell Migration

Affiliations
Free PMC article

Activated Heterotrimeric G Protein α i Subunits Inhibit Rap-dependent Cell Adhesion and Promote Cell Migration

Jesi Y To et al. J Biol Chem. .
Free PMC article

Abstract

Our recent work uncovered novel roles for activated Gαi signaling in the regulation of neutrophil polarity and adhesion. GαiGTP-mediated enhancement of neutrophil polarization was dependent on inhibition of cAMP/PKA signaling, whereas reversal of Gβγ-stimulated adhesion was cAMP/PKA independent. To uncover the mechanism for Gαi regulation of adhesion, we analyzed the effects of constitutively active Gαi1(Q204L) expression on adhesion driven by constitutively active Rap1a(G12V) or its downstream effector Radil in neutrophil-like HL-60 cells, or in HT-1080 fibrosarcoma cells. In HT-1080 cells, Rap1a(G12V) or Radil cause an increase in cell spreading and adhesion to fibronectin, which are both reversed by Gαi1(Q204L) but not WT Gαi1 In contrast, Gαi1(Q204L) did not reverse Rap1-GTP-interacting adaptor molecule (RIAM)-dependent increases in cell adhesion. This indicates that adhesion regulation by Gαi-GTP occurs downstream of Rap1a and Radil, but is upstream of components such as integrins and talin that are regulated by both Radil and RIAM. HL-60 neutrophil-like cells expressing Rap1a(G12V) or Radil have an elongated phenotype because of enhanced uropod adhesion as they attempt to migrate on fibronectin. This elongated phenotype driven by Rap1a(G12V) or Radil is reversed by Gαi1(Q204L), but not by WT Gαi1 expression, suggesting that Gαi-GTP also regulates adhesion in immune cells at the level of, or downstream of, Radil. These data identify a novel role of Gαi-GTP in regulation of cell adhesion and migration. Cell migration involves cycles of adhesion and de-adhesion, and we propose that the dynamic spatiotemporal balance between Gβγ-promoted adhesion and Gαi-GTP reversal of adhesion is important for this process.

Keywords: G protein; G protein–coupled receptor (GPCR); adhesion; cell migration; cell signaling; chemokines; integrin.

Conflict of interest statement

The authors declare that they have no conflicts of interest regarding the contents of this article

Figures

Figure 1.
Figure 1.
i1-GTP inhibits Rap1a(G12V) and Radil-mediated cell spreading. A, diagram showing key components of the pathway involved in integrin activation and adhesion downstream of Gβγ. The role of Gαi-GTP in this pathway is not known. B, expression of constitutively active Gαi1(Q204L) in HT-1080 fibrosarcoma cells inhibited the Rap1a(G12V) and Radil-dependent increase in cell spreading. The degree of cell spreading was quantified by measuring the area of each cell using ImageJ software. At least 15 cells per condition per experiment were analyzed. Data are mean ± S.E. of pooled data from three independent experiments and analyzed by one-way ANOVA with Bonferroni posttest. ***, p < 0.001; **, p < 0.01.
Figure 2.
Figure 2.
Adhesive phenotype driven by Rap1a(G12V) or Radil is reversed by Gαi1(Q204L) expression. A, representative images of HT-1080 cells stained with DAPI in response to overexpression as indicated. Top panels show attached cells remaining after washes and bottom panels show cells' input before washing. Cells were visualized with 10× objective epifluorescence microscope. B, expression of constitutively active Gαi1(Q204L) in HT-1080 cells inhibited Rap1a(G12V) and/or Radil-dependent increase in cell adhesion on fibronectin-coated surface. Cell adhesion was quantified using a Celigo cell imaging cytometer and quantified as -fold over control. C, same as B except both Rap1a and Radil were expressed in the presence or absence of Gαi1(Q204L) or Gαi1(WT). D, same as B except the effects of transfection of Gαi1 or Gαi1(Q204L) on basal adhesion were measured. E, same as B except the effects of inhibition of PKA on Gαi1(Q204L) regulation of adhesion was tested. F, a representative Western blot to show the relative expression of the various constructs in HT-1080 cells. Data are mean ± S.E. of at least three independent experiments and analyzed by one-way ANOVA with Bonferroni posttest. **, p < 0.01; *, p < 0.05.
Figure 3.
Figure 3.
i1-GTP does not regulate RIAM-dependent adhesion and is Rap1GAPII independent. A, Gαi1(Q204L) expression does not inhibit RIAM-dependent adhesion of HT-1080 cells. B, Gαi1(Q204L) expression inhibits Rap1a(F64A)-dependent adhesion of HT-1080 cells. Cell adhesion was measured as indicated in Fig. 2B. Data are mean ± S.E. of at least three independent experiments and analyzed by one-way ANOVA with Bonferroni posttest. ***, p < 0.001; **, p < 0.01.
Figure 4.
Figure 4.
i1(Q204L) does not affect Rap1a-dependent recruitment of Radil to the plasma membrane. A, HT-1080 cells stably expressing Venus-Radil were transiently transfected with HA-Rap1a(G12V) with or without Gαi1(Q204L). Cells were fixed, stained with α-HA monoclonal antibody, followed by secondary detection with goat anti-mouse IgG (H+L) secondary antibody, Alexa Fluor 594. Samples were imaged for Venus (green) and HA (red) by confocal fluorescence microscopy. B, relative membrane fluorescence was quantified using ImageJ software by measuring the membrane over the cytosolic fluorescence intensity. Three cells were analyzed for each condition for each experiment. Data are mean ± S.E. of pooled data from three independent experiments and analyzed by one-way ANOVA with Bonferroni posttest. *, p < 0.05.
Figure 5.
Figure 5.
Inhibition of Rho signaling attenuates Gαi1(Q204L) inhibitory effect on Rap1- and Radil-dependent adhesion. HT-1080 cells overexpressing different proteins as indicated were pretreated with Y-27632 (10 μm) and were then tested in the adhesion assay. Cell adhesion was analyzed as indicated in Fig. 2. Data are mean ± S.E. of at least three independent experiments and analyzed by one-way ANOVA with Bonferroni posttest. ****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05.
Figure 6.
Figure 6.
i1-GTP rescues elongated phenotype and defective tail retraction in differentiated HL-60 cells expressing Rap1a(G12V) and Radil. A, Gαi1(Q204L) expression reverses elongated phenotype of migrating HL-60 cells coexpressing Rap1a(G12V) or Radil. Representative differential interference contrast images of differentiated HL-60 cells transfected by nucleofection with different constructs as indicated. Positively transfected cells were identified by YFP coexpression. B, Gαi1(Q204L) expression rescues the circularity of migrating HL-60 cells overexpressing Rap1a(G12V) or Radil. Ten cells per condition for each experiment were analyzed. Quantitation of circularity from experiments shown in A calculated in ImageJ software (4π × area/perimeter2).
Figure 7.
Figure 7.
i1(Q204L) expression, but not wildtype Gαi1, rescues migration of Rap1a(G12V) and Radil expressing HT-1080 cells. A, spider plots of video-tracked HT-1080 cell transfected with Rap1a(G12V) and the indicated constructs and YFP. YFP-positive cells were tracked and this is a representative experiment. (See Movies S1–S4 for representative videos.) B, data obtained from at least 100 cells were pooled from three independent experiments as in A and are represented as a box and whisker plot. The box shows the interquartile range that contains values between 25th and 75th percentile. The line inside the box shows the median and the two whiskers go down to the smallest value and up to the largest value observed. Data were analyzed by one-way ANOVA with Bonferroni posttest. ****, p < 0.0001; ns, not significant. C, same as A except Radil was used to inhibit migration. D, same as B except Radil was used.

Similar articles

See all similar articles

Cited by 4 articles

Publication types

MeSH terms

LinkOut - more resources

Feedback