Figure 5: GTP-Gα12 bind to EFA6A-C but not to EFA6D to activate Arf6.

(a) Binding of HA-EFA6B with Gα12 on LPA stimulation. EFA6B/shEFA6B cells (786-O/HA-EFA6B) were either stimulated with LPA or left untreated for 5 min, and the co-precipitation of endogenous trimeric G-proteins with HA-EFA6B was then analysed by anti-HA immunoprecipitation (IP) coupled with immunoblotting using the indicated antibodies. An irrelevant mouse IgG (IgG) was used as a control. (b) Endogenous association of Gα12 with EFA6B. Co-precipitation of Gα12, but not Gα13 or Gαq, with anti-EFA6B immunoprecipitants (IP) of 786-O cell lysates was examined, coupled with immunoblotting as indicated. (c) EFA6A-C, but not EFA6D, binds to GTP-Gα12. 293T cells were transfected with plasmids encoding HA-EFA6A-D or with an empty vector (Mock), together with plasmids encoding Gα12QL, Gα13QL, or GαqQL; and co-precipitation of these G-proteins with EFA6 family members was assessed as in a. (d) Schematic representation of the domain structure of EFA6B and its mutants. (e) The primary structures of the RGS domains of RhoGEFs, ARHGEF1, ARHGEF11, and ARHGEF12, and their amino acid alignment with the N-terminal regions of EFA6A-D. Conserved amino acids are marked in red boxes. (f) The RSG-like domain is essential for the binding to GTP-Gα12. Full-length HA-EFA6B (FL) and its mutants were expressed in 293T cells together with Gα12QL, as indicated, and their co-precipitation was assessed as in a. An empty vector (Mock) was included as a control. (g,h) Requirement for the RSG-like domain in the binding to endogenous Gα12 and the activation of Arf6. Full-length HA-EFA6B (FL) and its ΔRGS-like mutant were expressed in 786-O cells, in which endogenous EFA6B was silenced; and co-precipitation of HA-EFA6B proteins with Gα12 (g) and activation of Arf6 (h) were examined on LPA stimulation. (a–c,f–h) Total, total cell lysates.