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CELLULAR AND MOLECULAR

Mutational Uncoupling of _1A-Adrenergic Receptors from G Proteins Also Uncouples Mitogenic and Transcriptional Responses in PC12 Cells

Department of Pharmacology, Emory University Medical School, Atlanta, Georgia

Activation of human _1A-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 (^208–210). Expression levels of retrovirus-transfected wild-type and ^208–210 _1A-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 ^208–210 mutation. In PC12 cells expressing the ^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 _1A-adrenergic receptors in PC12 cells was abolished by the ^208–210 mutation, as was activation of the tyrosine kinase Pyk2. Norepinephrine also activates several transcriptional reporters through _1A-adrenergic receptors in PC12 cells, including reporters for activator protein 1, serum response element, cAMP response element, nuclear factor-B, nuclear factor of activated T cells, -interferon-activated sequence, and signal transducer and activator of transcription. All these transcriptional responses were abolished by the ^208–210 mutation. Overexpression of G16 did not rescue any of these responses. These data suggest that known second messenger, mitogenic, and transcriptional effects of _1A-adrenergic receptors in PC12 cells all require G protein activation.

Address correspondence to: Dr. Kenneth P. Minneman, Department of Pharmacology, Emory University Medical School, Atlanta, GA 30322. E-mail: kminneman{at}pharm.emory.edu