Airway smooth muscle prostaglandin-EP1 receptors directly modulate β2–adrenergic receptors within a unique heterodimeric complex
Dennis W. McGraw, Kathryn A. Mihlbachler, Mary Rose Schwarb, Fahema F. Rahman, Kersten M. Small, Khalid F. Almoosa, Stephen B. Liggett
Dennis W. McGraw, Kathryn A. Mihlbachler, Mary Rose Schwarb, Fahema F. Rahman, Kersten M. Small, Khalid F. Almoosa, Stephen B. Liggett
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Research Article Inflammation

Airway smooth muscle prostaglandin-EP1 receptors directly modulate β2–adrenergic receptors within a unique heterodimeric complex

Abstract

Multiple and paradoxical effects of airway smooth muscle (ASM) 7-transmembrane–spanning receptors activated during asthma, or by treatment with bronchodilators such as β2–adrenergic receptor (β2AR) agonists, indicate extensive receptor crosstalk. We examined the signaling of the prostanoid-EP1 receptor, since its endogenous agonist prostaglandin E2 is abundant in the airway, but its functional implications are poorly defined. Activation of EP1 failed to elicit ASM contraction in mouse trachea via this Gαq-coupled receptor. However, EP1 activation markedly reduced the bronchodilatory function of β2AR agonist, but not forskolin, indicating an early pathway interaction. Activation of EP1 reduced β2AR-stimulated cAMP in ASM but did not promote or augment β2AR phosphorylation or alter β2AR trafficking. Bioluminescence resonant energy transfer showed EP1 and β2AR formed heterodimers, which were further modified by EP1 agonist. In cell membrane [35S]GTPγS binding studies, the presence of the EP1 component of the dimer uncoupled β2AR from Gαs, an effect accentuated by EP1 agonist activation. Thus alone, EP1 does not appear to have a significant direct effect on airway tone but acts as a modulator of the β2AR, altering Gαs coupling via steric interactions imposed by the EP1:β2AR heterodimeric signaling complex and ultimately affecting β2AR-mediated bronchial relaxation. This mechanism may contribute to β-agonist resistance found in asthma.

Authors

Dennis W. McGraw, Kathryn A. Mihlbachler, Mary Rose Schwarb, Fahema F. Rahman, Kersten M. Small, Khalid F. Almoosa, Stephen B. Liggett

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Figure 6

EP1 receptor activation uncouples the β2 AR from Gs .

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EP1 receptor activation uncouples t...
β2AR coupling to Gs was assessed by measuring isoproterenol-stimulated [35S]GTPγS binding in membranes from COS-7 cells transfected with Gαs and the β2AR, or with Gαs and β2AR plus EP1 (A) or mouse primary ASM cells (B). Reactions were performed with partially purified cell membranes. The [35S]GTPγS bound to Gαs was recovered by immunoprecipitation with Gαs antibody as described in Methods. (A) The presence of 17-PTP in the reaction significantly reduced [35S]GTPγS binding stimulated by isoproterenol in the transfected COS-7 cells, while the β2AR expressed without the EP1 had the greatest stimulation by isoproterenol. No effect of 17-PTP was observed on cells transfected to only express β2AR. **P = 0.05 versus β2AR only; ##P = 0.02 versus 17-PTP–untreated cells coexpressing β2AR and EP1 receptor. Shown are mean ± SEM from 6 experiments. (B) The presence of 17-PTP in the reaction decreased isoproterenol-stimulated [35S]GTPγS binding in ASM cell membranes. *P < 0.05 versus isoproterenol in the absence of 17-PTP. Shown are mean ± SEM from 6 experiments. In both types of experiments, basal (non-agonist) binding was ~65% of isoproterenol-stimulated binding.