Abstract
Activation of human α1A-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 (Δ208–210). Expression levels of retrovirus-transfected wild-type and Δ208–210 α1A-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 Δ208–210 mutation. In PC12 cells expressing the Δ208–210 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 α1A-adrenergic receptors in PC12 cells was abolished by the Δ208–210 mutation, as was activation of the tyrosine kinase Pyk2. Norepinephrine also activates several transcriptional reporters through α1A-adrenergic receptors in PC12 cells, including reporters for activator protein 1, serum response element, cAMP response element, nuclear factor-κB, nuclear factor of activated T cells, γ-interferon-activated sequence, and signal transducer and activator of transcription. All these transcriptional responses were abolished by the Δ208–210 mutation. Overexpression of Gα16 did not rescue any of these responses. These data suggest that known second messenger, mitogenic, and transcriptional effects of α1A-adrenergic receptors in PC12 cells all require G protein activation.
Footnotes
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This study was supported by the National Institutes of Health.
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DOI: 10.1124/jpet.103.050500.
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ABBREVIATIONS: GPCR, G protein-coupled receptor; AR, adrenergic receptor; NE, norepinephrine; MAPK, mitogen-activated protein kinase; NGF, nerve growth factor; AP1, activator protein 1; SER, serum response element; cAMP response element; NF-κB, nuclear factor-κB; NFAT, nuclear factor of activated T cells; Stat, signal transducer and activator of transcription; HEK, human embryonic kidney; 125I-BE, 125I-BE 2254, 2-[β-(4-hydroxyphenyl)-aminomethyl]tetralone; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; GAS, γ-interferon-activated sequence; GFX 203290, bisindolylmaleimide I.
- Received February 14, 2003.
- Accepted April 16, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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