Diacylglycerol and ceramide formation induced by dopamine D2S receptors via Gβγ-subunits in Balb/c-3T3 cells
Diacylglycerol (DAG) and ceramide are important second messengers affecting cell growth, differentiation, and apoptosis. Balb/c-3T3 fibroblast cells expressing dopamine-D2S (short) receptors (Balb-D2S cells) provide a model of G protein-mediated cell growth and transformation. In Balb-D2S cells, apomorphine (EC50= 10 nM) stimulated DAG and ceramide formation by 5.6- and 4.3-fold, respectively, maximal at 1 h and persisting over 6 h. These actions were blocked by pretreatment with pertussis toxin (PTX), implicating Gi/Goproteins. To address which G proteins are involved, Balb-D2S clones expressing individual PTX-insensitive Gαiproteins were treated with PTX and tested for apomorphine-induced responses. Neither PTX-insensitive Gαi2nor Gαi3rescued D2S-induced DAG or ceramide formation. Both D2S-induced DAG and ceramide signals required Gβγ-subunits and were blocked by inhibitors of phospholipase C [1-(6-[([17β]-3-methoxyestra-1,2,3[10]-trien- 17yl)amino]hexyl)-1H-pyrrole-2,5-dione (U-73122) and partially by D609]. The similar G protein specificity of D2S-induced calcium mobilization, DAG, and ceramide formation indicates a common Gβγ-dependent phospholipase C-mediated pathway. Both D2 agonists and ceramide specifically induced mitogen-activated protein kinase (ERK1/2), suggesting that ceramide mediates a novel pathway of D2S-induced ERK1/2 activation, leading to cell growth.