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 il/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 a 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 a subunit which mediates LPA-stimulated c-fos induction and perhaps mitogenesis in these cells.
Chuprun
J K
JK
Howard Hughes Medical Institute, Durham, North Carolina 27710, USA.
Raymond
J R
JR
Blackshear
P J
PJ
D000818:Animals; D015153:Blotting, Western; D005347:Fibroblasts / Q000378:metabolism; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016762:Genes, fos; D007328:Insulin / Q000494:pharmacology; D008246:Lysophospholipids / Q000494:pharmacology*; D051379:Mice; D008845:Microinjections; D037342:Pertussis Toxin; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled; D049368:Receptors, Lysophosphatidic Acid; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D007328:Insulin; D008246:Lysophospholipids; D011518:Proto-Oncogene Proteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D049368:Receptors, Lysophosphatidic Acid; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; C498016:Gnai2 protein, mouse