Airway smooth muscle prostaglandin-EP1 receptors directly modulate β2–adrenergic receptors within a unique heterodimeric complex
Dennis W. McGraw, … , Khalid F. Almoosa, Stephen B. Liggett
Dennis W. McGraw, … , Khalid F. Almoosa, Stephen B. Liggett
Published May 1, 2006
Citation Information: J Clin Invest. 2006;116(5):1400-1409. https://doi.org/10.1172/JCI25840.
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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 5

The EP1 receptor and β2 AR form a heterodimer as assessed by BRET and coimmunoprecipitation.

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The EP1 receptor and β2 ...
(A) Immunofluorescence microscopy revealed potential colocalization of EP1 and β2AR in ASM cells. Magnification, ×200. (B) The emission spectra of HEK 293 cells transfected with the β2AR-Rluc fusion alone showed a single peak with a maximum at ~475 nm following addition of coelenterazine h (open symbols). However, a second peak was observed at ~530 nm when cells were cotransfected with the EP1-YFP fusion construct (closed symbols). Shown is a representative experiment of 4. (C) The BRET ratios derived from HEK 293 cells transfected with a fixed amount of the donor β2AR-Rluc reached saturation with increasing expression of the acceptor EP1-YFP. Data shown are compiled from 12 experiments. (D) GABAb R2 and C-C chemokine receptor 5 (CCR5) failed to form heterodimers with β2AR as indicated by low pseudo-BRET signals. (E) The BRET ratios of HEK 293 cells cotransfected with the β2AR-Rluc and EP1-YFP were increased by treatment with either the EP1 agonist 17-PTP or the βAR agonist isoproterenol. *P < 0.05 versus untreated; n = 4. (F) Coimmunoprecipitation of FLAG-EP1:β2AR receptor complexes. HEK 293 cells were transfected with FLAG-β2AR, EP1 receptor, or both; membranes were solubilized; and proteins were immunoprecipitated with anti-FLAG antibody. Western blots of whole-cell lysates and immunoprecipitates were carried out with polyclonal EP1 receptor antisera. Shown is a representative blot from 5 experiments.