Mutational uncoupling of alpha1A-adrenergic receptors from G proteins also uncouples mitogenic and transcriptional responses in PC12 cells.
Activation of human alpha1A-adrenergic receptors in PC12 cells causes many second messenger, mitogenic, and transcriptional responses. We examined the role of G protein activation in these responses by uncoupling the receptor through deletion of the first three amino acids from the third intracellular loop (Delta208-210). Expression levels of retrovirus-transfected wild-type and Delta(208-210) alpha1A-adrenergic receptors in PC12 cells were similar and showed identical binding affinities for antagonists. However, the potency of the agonist norepinephrine was increased 9-fold by the Delta (208-210) mutation. In PC12 cells expressing the Delta (208-210) construct, calcium and inositol phosphate responses to norepinephrine were essentially abolished. The strong activation of mitogen-activated protein kinase pathways seen upon stimulation of wild-type alpha1A-adrenergic receptors in PC12 cells was abolished by the Delta (208-210) mutation, as was activation of the tyrosine kinase Pyk2. Norepinephrine also activates several transcriptional reporters through alpha1A-adrenergic receptors in PC12 cells, including reporters for activator protein 1, serum response element, cAMP response element, nuclear factor-kappaB, nuclear factor of activated T cells, gamma-interferon-activated sequence, and signal transducer and activator of transcription. All these transcriptional responses were abolished by the Delta (208-210) mutation. Overexpression of Galpha16 did not rescue any of these responses. These data suggest that known second messenger, mitogenic, and transcriptional effects of alpha1A-adrenergic receptors in PC12 cells all require G protein activation.
Department of Pharmacology, Emory University Medical School, Atlanta, GA 30322, USA.
Deborah
D
Lee
Anna
A
Robeva
Zhongjian
Z
Chen
Kenneth P
KP
Minneman
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D051418:Focal Adhesion Kinase 2; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D016716:PC12 Cells; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000276:immunology; D051381:Rats; D018340:Receptors, Adrenergic, alpha-1 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014158:Transcription, Genetic; D014162:Transfection
C547713:ADRA1A protein, human; C547715:Adra1a protein, rat; D007295:Inositol Phosphates; D011506:Proteins; D018340:Receptors, Adrenergic, alpha-1; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2; C496054:Ptk2b protein, rat; D020928:Mitogen-Activated Protein Kinases; D019204:GTP-Binding Proteins; C112631:G protein alpha 16; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D002118:Calcium