Regulation of immediate-early gene transcription following activation of Gα(q)-coupled designer receptors.
G-protein coupled designer receptors that are specifically activated by designer drugs have been developed. Here, we have analyzed the regulation of gene transcription following activation of Gα(q)-coupled designer receptor (Rα(q)). Stimulation of human embryonic kidney (HEK) 293 cells expressing Rα(q) with clozapine-N-oxide (CNO), a pharmacologically inert compound, induced the expression of biologically active Egr-1, a zinc finger transcription factor. Expression of a dominant-negative mutant of the ternary complex factor (TCF) Elk-1, a key transcriptional regulator of serum response element (SRE)-driven gene transcription, prevented Egr-1 expression. Stimulation of Rα(q) with CNO increased the transcriptional activation potential of Elk-1 and enhanced transcription of an SRE regulated reporter gene. In addition, AP-1 transcriptional activity was significantly elevated. AP-1 activity was controlled by TCFs and c-Jun in cells expressing an activated Gα(q)-coupled designer receptor. CNO stimulation did not increase Egr-1 and AP-1 activity in neuroblastoma cells expressing endogenous M3 muscarinic acetylcholine receptors, indicating that CNO did not function as a ligand for these receptors. Rα(q) stimulation also increased the transcriptional activation potential of CREB and cAMP response controlled gene transcription. Pharmacological and genetic experiments revealed that the protein kinases Raf and ERK were essential to connect Rα(q) stimulation with enhanced Egr-1 and AP-1 controlled transcription. In contrast, MAP kinase phosphatase-1 functioned as a nuclear shut-off device of stimulus-transcription coupling. The fact that Rα(q) stimulation activates the transcription factors Egr-1, Elk-1, AP-1, and CREB indicates that regulation of gene transcription is an integral part of Gα(q)-coupled receptor signaling.
Department of Medical Biochemistry and Molecular Biology, University of Saarland Medical Center, D-66421 Homburg, Germany.
Anke
A
Kaufmann
Anja
A
Keim
Gerald
G
Thiel
D050882:CREB-Binding Protein / Q000235:genetics; D045744:Cell Line, Tumor; D003024:Clozapine / Q000031:analogs & derivatives / Q000494:pharmacology; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics; D054638:Dual Specificity Phosphatase 1 / Q000378:metabolism; D051766:Early Growth Response Protein 1 / Q000096:biosynthesis / Q000235:genetics; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D005786:Gene Expression Regulation; D017781:Genes, Immediate-Early / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000235:genetics*; D009154:Mutation; D009447:Neuroblastoma / Q000235:genetics / Q000378:metabolism; D043587:Receptor, Muscarinic M3 / Q000096:biosynthesis; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D027602:Serum Response Element; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D048490:raf Kinases / Q000378:metabolism
D017362:Cyclic AMP Response Element-Binding Protein; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D043587:Receptor, Muscarinic M3; D043562:Receptors, G-Protein-Coupled; D018808:Transcription Factor AP-1; D050882:CREB-Binding Protein; C494364:Crebbp protein, mouse; D048490:raf Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D054638:Dual Specificity Phosphatase 1; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D003024:Clozapine; C079149:clozapine N-oxide