Multiple and paradoxical effects of airway smooth muscle (ASM) 7-transmembrane-spanning receptors activated during asthma, or by treatment with bronchodilators such as β 2 -adrenergic receptor (β 2 AR) agonists, indicate extensive receptor crosstalk. We examined the signaling of the prostanoid-EP 1 receptor, since its endogenous agonist prostaglandin E 2 is abundant in the airway, but its functional implications are poorly defined. Activation of EP 1 failed to elicit ASM contraction in mouse trachea via this G αq -coupled receptor. However, EP 1 activation markedly reduced the bronchodilatory function of β 2 AR agonist, but not forskolin, indicating an early pathway interaction. Activation of EP 1 reduced β 2 AR-stimulated cAMP in ASM but did not promote or augment β 2 AR phosphorylation or alter β 2 AR trafficking. Bioluminescence resonant energy transfer showed EP 1 and β 2 AR formed heterodimers, which were further modified by EP 1 agonist. In cell membrane [ 35 S]GTPγS binding studies, the presence of the EP 1 component of the dimer uncoupled β 2 AR from G αs , an effect accentuated by EP 1 agonist activation. Thus alone, EP 1 does not appear to have a significant direct effect on airway tone but acts as a modulator of the β 2 AR, altering G αs coupling via steric interactions imposed by the EP 1 :β 2 AR heterodimeric signaling complex and ultimately affecting β 2 AR-mediated bronchial relaxation. This mechanism may contribute to β-agonist resistance found in asthma. IntroductionThe 7-transmembrane-spanning (7-TM-spanning) receptors represent the largest signaling family in the genome. We estimate that the lung expresses 25-50 7-TM receptors in airway epithelial cells, airway smooth muscle (ASM), pulmonary vasculature, alveolar walls, and resident immune cells (1). In regard to asthma, several 7-TM receptors play established roles in bronchoconstriction (e.g., M 3 -muscarinic receptor) and bronchodilation (e.g., β 2 -adrenergic receptor [β 2 AR]). Despite identification of the endogenous ligands and receptor localization, there are a number of 7-TM receptors expressed in the airway whose functions are unknown, or appear to function paradoxically, based on recombinantly expressed receptors in model cell systems. This lack of understanding of receptor function has impeded our ability to ascertain the role of these ligands (some of which are markedly increased in asthma) in the relaxation/contraction of ASM; thus the mechanistic basis of bronchial hyperreactivity and bronchoconstriction in asthma remains only partially understood. In many cases the basis for incomplete mechanistic information can be attributed to the nature of recombinant expression systems, which may not take

Dimerization of seven transmembrane-spanning receptors diversifies their pharmacologic and physiologic properties. The alpha(2)-adrenergic receptor (alpha(2)AR) subtypes A and C are both expressed on presynaptic nerves and act to inhibit norepinephrine release via negative feedback. However, in vivo and in vitro studies examining the roles of the two individual alpha(2A)- and alpha(2C)AR subtypes are not readily reconciled. We tested the hypothesis that the receptors form homo- and heterodimers and that the alpha(2A)-alpha(2C) heterodimer has unique properties. SDS-PAGE of epitope-tagged receptors revealed potential oligomers including dimers. BRET of live HEK-293 cells transfected with the subtypes fused to Rluc or YFP revealed that both subtypes form dimers and the heterodimer. A lower BRET(50) for the alpha(2A)-alpha(2C) heterodimer (0.79 +/- 0.20) compared to that of the alpha(2A) or alpha(2C) homodimer (2.331 +/- 0.44 or 3.67 +/- 0.69, respectively) suggests that when both subtypes are expressed, there is a greater likelihood that the two receptors will form the heterodimer than homodimers. Co-immunoprecipitation studies confirmed homo- and heterodimer formation. The presence of the alpha(2C)AR within the heterodimer resulted in a marked reduction in the level of GRK2-mediated alpha(2A)AR phosphorylation, which was accompanied by a qualitative attenuation of beta-arrestin recruitment. Signaling of the alpha(2A)-alpha(2C) heterodimer to the beta-arrestin-dependent activation of Akt was decreased compared to that of the alpha(2A)AR homodimer, while p44/p42 MAP kinase activation was unaffected. Thus, the alpha(2C)AR alters alpha(2A)AR signaling by forming oligomers, and these complexes, which appear to be preferred over the homodimers, should be considered a functional signaling unit in cells in which both subtypes are expressed.

Panebra A, Schwarb MR, Glinka CB, Liggett SB. Heterogeneity of transcription factor expression and regulation in human airway epithelial and smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 293: L453-L462, 2007. First published June 8, 2007; doi:10.1152/ajplung.00084.2007.-Transcription factors represent a major mechanism by which cells establish basal and conditional expression of proteins, the latter potentially being adaptive or maladaptive in disease. The complement of transcription factors in two major structural cells of the lung relevant to asthma, airway epithelial and smooth muscle cells, is not known. A plate-based platform using nuclear extracts from these cells was used to assess potential expression by binding to oligonucleotide consensus sequences representing Ͼ300 transcription factors. Four conditions were studied: basal, ␤-agonist exposure, culture under proasthmatic conditions (IL-13, IL-4, TGF-␤, and leukotriene D 4), and the dual setting of ␤-agonist with proasthmatic culture. Airway epithelial cells expressed 70 transcription factors, whereas airway smooth muscle expressed 110. High levels of multiple transcription factors not previously recognized as being expressed in these cells were identified. Moreover, expression/ binding patterns under these conditions revealed extreme discordance in the direction and magnitude of change between the cell types. Singular (one cell type displayed regulation) and antithetic (both cell types underwent expression changes but in opposite directions) regulation dominated these patterns, with concomitant regulation in both cell types being rare (Ͻ10%). ␤-Agonist evoked up-and downregulation of transcription factors, which was highly influenced by the proasthmatic condition, with little overlap of factors regulated by ␤-agonists under both conditions. Together, these results reveal complex, cell type-dependent networks of transcription factors in human airway epithelium and smooth muscle that are dynamically regulated in unique ways by ␤-agonists and inflammation. These factors may represent additional components in asthma pathophysiology or potential new drug targets. transcription; ␤-agonist; asthma; inflammation IN EUKARYOTIC CELLS, TRANSCRIPTION factors represent the major elements by which gene transcription by RNA polymerase II is controlled (18,26). Such factors regulate the basal expression of genes and serve to dynamically regulate gene expression during normal homeostatic conditions, exposures to xenobiotic agents such as therapeutic drugs or toxins, and pathological conditions, where they may be a basis for, or serve to counteract, aberrant cellular physiology (13,18,26). In asthma, two stromal cell types play significant roles in the syndrome: the airway epithelial cell and the airway smooth muscle cell (5, 9). Although some information is known about the regulation and action of selected transcription factors of immune cells of the lung and peripheral circulation (1,11,12,24), there is a paucity of data on the expression of transcription ...

Beta(2)-adrenergic receptors (beta(2)-AR) expressed on airway epithelial and smooth muscle cells regulate mucociliary clearance and relaxation and are the targets for beta-agonists in the treatment of obstructive lung disease. However, the clinical responses display extensive interindividual variability, which is not adequately explained by genetic variability in the 5'-flanking or coding region of the intronless beta(2)-AR gene. The nonsynonymous coding polymorphism most often associated with a bronchodilator phenotype (Arg16) is found within three haplotypes that differ by the number of Cs (11, 12, or 13) within a 3'-untranslated region (UTR) poly-C tract. To examine potential effects of this variability on receptor expression, BEAS-2B cells were transfected with constructs containing the beta(2)-AR (Arg16) coding sequence followed by its 3'-UTR with the various polymorphic poly-C tracts. beta(2)Arg16-11C had 25% lower mRNA expression and 33% lower beta(2)-AR protein expression compared with the other two haplotypes. Consistent with this lower steady-state expression, beta(2)Arg16-11C mRNA displayed more rapid and extensive degradation after actinomycin D treatment compared with beta(2)Arg16-12C and -13C. However, beta(2)Arg16-12C underwent 50% less downregulation of receptor expression during beta-agonist exposure compared with the other two haplotypes. Thus these haplotypes direct a potential low-response phenotype due to decreased steady-state receptor expression combined with wild-type agonist-promoted downregulation (beta(2)Arg16-11C) and a high-response phenotype due to increased baseline expression combined with decreased agonist-promoted downregulation (beta(2)Arg16-12C). This heterogeneity may contribute to the variability of clinical responses to beta-agonist, and genotyping to identify these 3'-UTR polymorphisms may improve predictive power within the context of beta(2)-AR haplotypes in pharmacogenetic studies.