Available drugs are unable to effectively rescue the folding defects in vitro and ameliorate the clinical-phenotype of cystic fibrosis (CF), caused by deletion of F508 (ΔF508 or F508del) and some point mutations in the CF transmembrane conductance regulator (CFTR), a plasma membrane (PM) anion channel. To overcome the corrector efficacy ceiling, here we show that compounds targeting distinct structural defects of CFTR can synergistically rescue mutants expression and function at the PM. High throughput cell-based screens and mechanistic analysis identified three small-molecule series that target defects at the nucleotide binding domain (NBD1), NBD2 and their membrane spanning domains (MSDs) interfaces. While individually these compounds marginally improve ΔF508-CFTR folding efficiency, function, and stability, their combinations lead to ~50–100% of wild type-level correction in immortalized and primary human airway epithelia, and in mouse nasal epithelia. Likewise, corrector combinations were effective for rare missense mutations in various CFTR domains, probably acting via structural allostery, suggesting a mechanistic framework for their broad application.

␤-Arrestins were initially shown, in conjunction with G protein-coupled receptor kinases, to be involved in the desensitization and internalization of activated seven-transmembrane receptors. Recently, ␤-arrestin 2 has been shown to act as a signal mediator in mitogen-activated protein kinase cascades and to play a positive regulatory role in chemotaxis. We now show that ␤-arrestin 1 is required to activate the small GTPase RhoA leading to the re-organization of stress fibers following the activation of the angiotensin II type 1A receptor. This angiotensin II type 1A receptor-directed RhoA activation and stress fiber formation also require the activation of the heterotrimeric G protein G ␣q/11 . Whereas neither ␤-arrestin 1 nor G ␣q/11 activation alone is sufficient to robustly activate RhoA, the concurrent recruitment of ␤-arrestin 1 and activation of G ␣q/11 leads to full activation of RhoA and to the subsequent formation of stress fibers.

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Receptor desensitization progressively limits responsiveness of cells to chronically applied stimuli. Desensitization in the continuous presence of agonist has been difficult to study with available assay methods. Here, we used a fluorescence resonance energy transfer-based live cell assay for the second messenger diacylglycerol to measure desensitization of a model seventransmembrane receptor, the G q -coupled angiotensin II type 1 A receptor, expressed in human embryonic kidney 293 cells. In response to angiotensin II, we observed a transient diacylglycerol response reflecting activation and complete desensitization of the receptor within 2-5 min. By utilizing a variety of approaches including graded tetracycline-inducible receptor expression, mutated receptors, and overexpression or short interfering RNA-mediated silencing of putative components of the cellular desensitization machinery, we conclude that the rate and extent of receptor desensitization are critically determined by the following: receptor concentration in the plasma membrane; the presence of phosphorylation sites on the carboxyl terminus of the receptor; kinase activity of G protein-coupled receptor kinase 2, but not of G protein-coupled receptor kinases 3, 5, or 6; and stoichiometric expression of ␤-arrestin. The findings introduce the use of the biosensor diacylglycerol reporter as a powerful means for studying G q -coupled receptor desensitization and document that, at the levels of receptor overexpression commonly used in such studies, the properties of the desensitization process are markedly perturbed and do not reflect normal cellular physiology.The family of heptahelical receptors, also called seven-transmembrane receptors (7TMRs) 4 or G protein-coupled receptors, regulates myriad physiological and pathological signal transduction pathways. Despite a large diversity of ligands, including catecholamines, chemokines, lipids, peptides, odorants, and photons, these receptors share remarkable similarity in their intracellular regulatory mechanisms. Classically, receptor function is initiated by ligand-induced activation of heterotrimeric G proteins. This is followed by receptor inactivation, mediated by receptor phosphorylation by G protein-coupled receptor kinases (GRKs) and other kinases, ␤-arrestin recruitment, and receptor internalization (1, 2). This inactivation results in desensitization, the loss of agonist efficacy following sustained stimulation. Until recently our appreciation of the kinetics of 7TMR molecular pharmacology has been limited to extrapolation from either complex in vivo readouts such as changes in blood pressure or from biochemical techniques such as phosphoinositide hydrolysis assays (3), which cannot be performed in live, unperturbed cells. Thus, it has not been possible to observe receptor level desensitization in the continuous presence of agonist, the situation that most closely parallels many physiological circumstances. Consequently, despite a vast literature on 7TMR desensitization, uncertainty remains regard...