Receptor-mediated airway smooth muscle (ASM) contraction via G αq , and relaxation via G αs , underlie the bronchospastic features of asthma and its treatment. Asthma models show increased ASM G αi expression, considered the basis for the proasthmatic phenotypes of enhanced bronchial hyperreactivity to contraction mediated by M 3 -muscarinic receptors and diminished relaxation mediated by β 2 -adrenergic receptors (β 2 ARs). A causal effect between G i expression and phenotype has not been established, nor have mechanisms whereby G i modulates G q /G s signaling. To delineate isolated effects of altered G i , transgenic mice were generated overexpressing G αi2 or a G αi2 peptide inhibitor in ASM. Unexpectedly, G αi2 overexpression decreased contractility to methacholine, while G αi2 inhibition enhanced contraction. These opposite phenotypes resulted from different crosstalk loci within the G q signaling network: decreased phospholipase C and increased PKCα, respectively. G αi2 overexpression decreased β 2 AR-mediated airway relaxation, while G αi2 inhibition increased this response, consistent with physiologically relevant coupling of this receptor to both G s and G i . IL-13 transgenic mice (a model of asthma), which developed increased ASM G αi , displayed marked increases in airway hyperresponsiveness when G αi function was inhibited. Increased G αi in asthma is therefore a double-edged sword: a compensatory event mitigating against bronchial hyperreactivity, but a mechanism that evokes β-agonist resistance. By selective intervention within these multipronged signaling modules, advantageous G s /G q activities could provide new asthma therapies. IntroductionAirway smooth muscle (ASM) contraction and relaxation are primarily regulated by G protein-coupled receptors, the former mediated by receptors signaling to G q and the latter by those that couple to G s (1, 2). Many inflammatory cascades in asthma evoke bronchoconstriction by promoting local increases of G q receptor agonists such as acetylcholine, cysteinyl leukotrienes, prostaglandins, and histamine, which activate their cognate receptors expressed on ASM. There appear to be fewer G s -coupled receptors that act via endogenous agonists to counteract bronchoconstriction, but the β 2 -adrenergic receptor (β 2 AR) of ASM is the target of pharmacologically administered β-agonists and is typically highly effective in relaxing constricted airways. The molecular events and critical transduction elements for these 2 classes of receptors are well recognized. Agonist binding to receptors such as the M 3 -muscarinic receptor promote disassociation of heterotrimeric G q into G α and G βγ subunits, with the α subunit activating phospholipase C (PLC; which promotes inositol-3 phosphate and diacylglycerol production) and the latter activating PKC. Receptors such as the β 2 AR act via G αs to stimulate the effector adenylyl cyclase, resulting in cAMP production and activation of PKA. Substantial interest has

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 ...

Like other intronless G protein-coupled receptor genes, the ␤ 2 -adrenergic receptor (␤ 2 AR) has minimal genetic space for population variability, and has attained such via multiple coding and noncoding polymorphisms. Yet most clinical studies use the two nonsynonymous polymorphisms of the coding region for association analysis despite low levels of linkage disequilibrium with some promoter and 5UTR polymorphisms. To assess the potential for allele-specific transcription factor binding to ␤ 2 AR 5-flanking sequence, 3-biotin-labeled oligonucleotide duplexes were synthesized. Each was centered on variable sites representing major or minor alleles found in the human population with frequencies of 5% or greater (20 polymorphic sites). Electrophoretic mobility shift assays were performed using human airway smooth muscle or airway epithelial cell nuclear extracts. Many of these polymorphisms resulted in an alteration in binding, and both major allele and minor allele dominance were observed. For example, in airway smooth muscle nuclear extracts, 10 polymorphisms decreased and 2 increased binding, whereas 5 showed no differences. Concordance between airway smooth muscle and epithelial cell nuclear extract binding to polymorphic alleles was found in only ‫ف‬ 50% of cases. There was no tendency for the rare variants to be more likely to have altered nuclear extract binding compared to the more common variants. Taken together, these results provide potential mechanisms by which ␤ 2 AR 5-flanking polymorphisms affect obstructive lung phenotypes.Keywords: asthma; ␤-agonist; polymorphismThe ␤ 2 -adrenergic receptor (␤ 2 AR) gene is highly polymorphic in the human population within both the coding and noncoding regions. Within the coding block, two nonsynonymous polymorphisms are common, occurring at nucleotides 46 and 79 relative to the ATG start site (1). These result in Arg or Gly at amino acid 16 and Glu or Gln at position 27, with three of the four possible two-site haplotypes being prevalent in normal cohorts as well as those with asthma and chronic obstructive lung disease. An infrequent, nonsynonymous polymorphism at nucleotide 491 results in a substitution of Ile for Thr at amino acid 164 (1). These polymorphisms have been studied in recombinant systems whereby constructs (without cognate 5ЈUTR or promoter) were used to stably or transiently transfect model cells, such as fibroblasts (2, 3) and COS cells (4). These studies revealed several phenotypes of the polymorphic receptors, including those related to agonist-promoted receptor trafficking and receptor coupling to G s (see Refs. 5 and 6 for review). Given the interindividual variability in the response to ␤-agonists in asthma, of which ‫ف‬ 50% has been attributed to genetic variation (7), these polymor- CLINICAL RELEVANCEThe variability in the response to ␤-agonists in obstructive lung disease is not fully understood. We show that polymorphisms in the promoter 5Ј-flanking region of the receptor alter transcription factor binding. phisms of the ␤ 2 AR have been...