Interaction of G alpha(12) with G alpha(13) and G alpha(q) signaling pathways

Abstract

The G(12) subfamily of heterotrimeric G-proteins consists of two members, G(12) and G(13). Gene-targeting studies have revealed a role for G(13) in blood vessel development. Mice lacking the alpha subunit of G(13) die around embryonic day 10 as the result of an angiogenic defect. On the other hand, the physiological role of G(12) is still unclear. To address this issue, we generated G alpha(12)-deficient mice. In contrast to the G alpha(13)-deficient mice, G alpha(12)-deficient mice are viable, fertile, and do not show apparent abnormalities. However, G alpha(12) does not seem to be entirely redundant, because in the offspring generated from G alpha(12)+/- G alpha(13)+/- intercrosses, at least one intact G alpha(12) allele is required for the survival of animals with only one G alpha(13) allele. In addition, G alpha(12) and G alpha(13) showed a difference in mediating cell migratory response to lysophosphatidic acid in embryonic fibroblast cells. Furthermore, mice lacking both G alpha(12) and G alpha(q) die in utero at about embryonic day 13. These data indicate that the G alpha(12)-mediated signaling pathway functionally interacts not only with the G alpha(13)- but also with the G alpha(q/11)-mediated signaling systems.

Figure 1

Targeted inactivation of the murine Gα₁₂ gene. (A) Genomic structure of murine Gα₁₂ gene (WT), targeting construct (TC), and replaced mutant allele (Mut.), as described in Materials and Methods. (B) Western analysis of Gα₁₂ mice. Cholate extracts from mouse brain were used for detection by Western analysis. An approximately 45-kDa band representing Gα₁₂ was seen in wild-type tissue with antibodies against the N (N-20, Santa Cruz Biotechnology) and C termini (AS 233). Expression of Gα₁₃ and Gα_q was also examined in both wild-type and Gα₁₂ mutant mice.

Figure 2

Embryos from offspring of Gα₁₂±Gα₁₃± intercrosses. (A) Embryos taken at E8.25. (B) Whole-mount staining of E9.5 embryos with antiplatelet–endothelial cell adhesion molecule-1 antibody (PharMingen).

Figure 3

LPA-induced cell migration. GFP, Gα₁₂, and Gα₁₃ expressing Gα₁₃−/− and Gα₁₂−/−Gα₁₃−/− cell lines were established by using the retroviral expression system as described in Materials and Methods. Migration experiments were performed in triplicate by using 24-well Transwell migration chambers, and migrated cells were quantitated in five random fields and expressed as a percentage of the control cells (−) that are always run at the same time. Two different G13−/− lines and two different G12−/−G13−/− lines were examined and showed similar results in at least two independent experiments.

Figure 4

Embryos from offspring of Gα_q±Gα₁₂± intercrosses. (A) Embryos taken at E12.5. (B) Embryos taken at E13.5.