The heterotrimeric G protein G(alpha i2) mediates lysophosphatidic acid-stimulated induction of the c-fos gene in mouse fibroblasts

Lysophosphatidic acid (LPA) utilizes a heterotrimeric guanine nucleotide regulatory (G) protein-coupled receptor to activate the mitogen-activated protein kinase pathway and induce mitogenesis in fibroblasts and other cells. A single cell assay system was used to examine the functional interaction of the LPA receptor with G proteins in intact mouse fibroblasts, by measuring LPA-stimulated induction of the immediate early gene, c-fos, as read out by a stably expressed fos-lacZ reporter gene. Pretreatment of these cells with pertussis toxin at 100 ng/ml almost completely abolished LPA-stimulated c-fos induction. Western blotting revealed that two pertussis toxin (PTX)-sensitive G proteins, G(alpha i2) and G(alpha i3), were present in membranes prepared from these cells, and Northern blotting confirmed the absence of message for other PTX-sensitive subunits. Microinjection of an alpha(i1)/alpha(i2)-specific antibody into living cells decreased LPA stimulated induction of c-fos by 60%, whereas introduction of antibodies to either alpha(i3) or alpha(16), a subtype not present in these cells but used as a control, decreased LPA-stimulated c-fos induction by only 19%. In contrast, the alpha(i1)/alpha(i2)-specific antibody had no effect on insulin-induced c-fos expression, which is thought to utilize a G protein independent mechanism of signaling. In addition, cellular expression of an epitope-tagged PTX-resistant mutant of G(alpha i2), but not PTX-resistant G(alpha i3), restored LPA-stimulated c-fos induction in cells in which endogenous G protein alpha subunits were uncoupled from the receptor by pretreatment with PTX. Together, these results provide conclusive in vivo evidence that G(alpha i2) is the PTX sensitive G protein alpha subunit which mediates LPA-stimulated c-fos induction and perhaps mitogenesis in these cells.