Targeted transgenesis reveals discrete attenuator functions of GRK and PKA in airway beta2-adrenergic receptor physiologic signaling.

Phosphorylation by protein kinase A (PKA) and G protein-coupled receptor kinases (GRKs) desensitize beta2-adrenergic receptor (beta2AR) signaling, and these are thought to be mechanisms involved with cell and organ homeostasis and tolerance to agonists. However, there is little direct evidence that these events are relevant to beta2AR physiological function, such as airway smooth muscle (ASM) relaxation leading to bronchodilation. To maintain cell- and receptor-specificity without altering the natural complement of kinases/arrestins, transgenic mice were generated expressing the human WT and mutated beta2ARs lacking PKA and/or GRK phosphorylation sites on ASM at approximately 4-fold over background. Functional gains in response to beta-agonist from the selective loss of these mechanisms were determined in mouse airways. Relaxation kinetics were altered in all mutant airways compared with beta2WT. At low receptor occupancy, beta2PKA(-) had enhanced agonist-promoted relaxation, while beta2GRK(-) airways were unaffected. In contrast, at saturating agonist concentrations, the greatest relaxation enhancement was with beta2GRK(-), with no evidence for additivity when PKA sites were also removed. For the full range of responses, the beta2PKA(-)/GRK(-) airways had the greatest relaxation efficiency, indicating a graded effect of GRKs as agonist concentration increased. ASM cAMP levels paralleled relaxation phenotypes. No interaction between PKA phosphorylation of beta2AR and GRK-promoted events was identified by beta-arrestin-2 recruitment. Thus, these two mechanisms indeed impact a relevant beta2AR physiologic function, acting as attenuators of the acute response, and represent specific interfaces where adjunct therapy or biased ligands may improve beta-agonist treatment of obstructive lung disease.

A polymorphism of G-protein coupled receptor kinase5 alters agonist-promoted desensitization of beta2-adrenergic receptors.

Beta-agonist treatment of asthma displays substantial interindividual variation, which has prompted polymorphism discovery and characterization of beta2-adrenergic (beta2AR) signaling genes. beta2AR function undergoes desensitization during persistent agonist exposure because of receptor phosphorylation by G-protein coupled receptor kinases (GRKs). GRK5 was found to be highly expressed in airway smooth muscle, the tissue target for beta-agonists. The coding region is polymorphic at codon 41, where Gln can be substituted by Leu (minor allele), but almost exclusively in those of African descent. In transfected cells, GRK5-Leu41 evoked a greater degree of agonist-promoted desensitization of adenylyl cyclase compared with GRK5-Gln41. Consistent with this functional effect, agonist-promoted beta2AR phosphorylation was greater in cells expressing GRK5-Leu41, as was the rate of agonist-promoted receptor internalization. In studies with mutated beta2AR lacking PKA-phosphorylation sites, this phenotype was confirmed as being GRK-specific. So, GRK5-Leu41 represents a gain-of-function polymorphism that evokes enhanced loss-of-function of beta2AR during persistent agonist exposure, and thus may contribute to beta-agonist variability in asthma treatment of African-Americans.

Bitransgenesis with beta(2)-adrenergic receptors or adenylyl cyclase fails to improve beta(1)-adrenergic receptor cardiomyopathy.

Cardiomyopathic effects of beta-adrenergic receptor (betaAR) signaling are primarily due to the beta(1)AR subtype. beta(1)/beta(2)AR and beta(1)/adenylyl cyclase type 5 (AC5) bitransgenic mice were created to test the hypothesis that beta(2)AR or AC5 co-overexpression has beneficial effects in beta(1)AR-mediated cardiomyopathy. In young mice, beta(1)/beta(2) hearts had a greater increase in basal and isoproterenol-stimulated contractility compared to beta(1)/AC5 and beta(1)AR hearts. By 6 months, beta(1)AR and beta(1)/beta(2) hearts retained elevated basal contractility but were unresponsive to agonist. In contrast, beta(1)/AC5 hearts maintained a small degree of agonist responsiveness, which may be due to a lack of beta(1)AR downregulation that was noted in beta(1)- and beta(1)/beta(2) hearts. However, by 9 -months, beta(1), beta(1)/beta(2), and beta(1)/AC5 mice had all developed severely depressed fractional shortening in vivo and little response to agonist. p38 mitogen activated protein kinase (MAPK) was minimally activated by beta(1)AR, but was markedly enhanced in the bitransgenics. Akt activation was only found with the bitransgenics. The small increase in cystosolic second mitochondria-derived activator of caspase (Smac), indicative of apoptosis in 9-month beta(1)AR hearts, was suppressed in beta(1)/AC5, but not in beta(1)/beta(2), hearts. Taken together, the unique signaling effects of enhanced beta(2)AR and AC5, which have the potential to afford benefit in heart failure, failed to salvage ventricular function in beta(1)AR-mediated cardiomyopathy.

Crosstalk between Gi and Gq/Gs pathways in airway smooth muscle regulates bronchial contractility and relaxation.

Receptor-mediated airway smooth muscle (ASM) contraction via G(alphaq), and relaxation via G(alphas), underlie the bronchospastic features of asthma and its treatment. Asthma models show increased ASM G(alphai) 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 beta(2)-adrenergic receptors (beta(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(alphai2) or a G(alphai2) peptide inhibitor in ASM. Unexpectedly, G(alphai2) overexpression decreased contractility to methacholine, while G(alphai2) inhibition enhanced contraction. These opposite phenotypes resulted from different crosstalk loci within the G(q) signaling network: decreased phospholipase C and increased PKCalpha, respectively. G(alphai2) overexpression decreased beta(2)AR-mediated airway relaxation, while G(alphai2) 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(alphai), displayed marked increases in airway hyperresponsiveness when G(alphai) function was inhibited. Increased G(alphai) in asthma is therefore a double-edged sword: a compensatory event mitigating against bronchial hyperreactivity, but a mechanism that evokes beta-agonist resistance. By selective intervention within these multipronged signaling modules, advantageous G(s)/G(q) activities could provide new asthma therapies.

Pleiotropic beta-agonist-promoted receptor conformations and signals independent of intrinsic activity.

Beta-agonists used for treatment of obstructive lung disease have a variety of different structures but are typically classified by their intrinsic activities for stimulation of cAMP, and predictions are made concerning other downstream signals based on such a classification. We generated modified beta(2)-adrenergic receptors with insertions of energy donor and acceptor moieties to monitor agonist-promoted conformational changes of the receptor using intramolecular bioluminescence resonance energy transfer in live cells. These studies suggested unique conformations stabilized by various agonists that were not based on their classic intrinsic activities. To address the cellular consequences of these differences, G(s)-coupling, G(i)-coupling (p44/p42 activation), G protein-coupled receptor kinase-mediated receptor phosphorylation, internalization, and down-regulation were assessed in response to isoproterenol, albuterol, terbutaline, metaproterenol, salmeterol, formoterol, and fenoterol. In virtually every case, agonists did not maintain the classic rank order, indicating that distinct signaling is evoked by beta-agonists of different structures, which is unrelated to intrinsic activity. The extensive pleiotropy of agonist responses shown here suggests that classification of agonists by cAMP-based intrinsic activity is inadequate as it pertains to other intracellular events and that it may be possible to engineer a beta-agonist that stabilizes conformations that evoke an ideal portfolio of signals for therapeutic purposes.

Airway smooth muscle prostaglandin-EP1 receptors directly modulate beta2-adrenergic receptors within a unique heterodimeric complex.

Multiple and paradoxical effects of airway smooth muscle (ASM) 7-transmembrane-spanning receptors activated during asthma, or by treatment with bronchodilators such as beta(2)-adrenergic receptor (beta(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(alphaq)-coupled receptor. However, EP(1) activation markedly reduced the bronchodilatory function of beta(2)AR agonist, but not forskolin, indicating an early pathway interaction. Activation of EP(1) reduced beta(2)AR-stimulated cAMP in ASM but did not promote or augment beta(2)AR phosphorylation or alter beta(2)AR trafficking. Bioluminescence resonant energy transfer showed EP(1) and beta(2)AR formed heterodimers, which were further modified by EP(1) agonist. In cell membrane [(35)S]GTPgammaS binding studies, the presence of the EP(1) component of the dimer uncoupled beta(2)AR from G(alphas), 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 beta(2)AR, altering G(alphas) coupling via steric interactions imposed by the EP(1):beta(2)AR heterodimeric signaling complex and ultimately affecting beta(2)AR-mediated bronchial relaxation. This mechanism may contribute to beta-agonist resistance found in asthma.