GPCRs have diverse signaling capabilities, based on their ability to assume various conformations. Moreover, it is now appreciated that certain ligands can promote distinct receptor conformations and thereby bias signaling toward a specific pathway to differentially affect cell function. The recently deorphanized G protein-coupled receptor OGR1 [ovarian cancer G protein-coupled receptor 1 ( GPR68)] exhibits diverse signaling events when stimulated by reductions in extracellular pH. We recently demonstrated airway smooth muscle cells transduce multiple signaling events, reflecting a diverse capacity to couple to multiple G proteins. Moreover, we recently discovered that the benzodiazepine lorazepam, more commonly recognized as an agonist of the γ-aminobutyric acid A (GABA) receptor, can function as an allosteric modulator of OGR1 and, similarly, can promote multiple signaling events. In this study, we demonstrated that different benzodiazepines exhibit a range of biases for OGR1, with sulazepam selectively activating the canonical Gs of the G protein signaling pathway, in heterologous expression systems, as well as in several primary cell types. These findings highlight the potential power of biased ligand pharmacology for manipulating receptor signaling qualitatively, to preferentially activate pathways that are therapeutically beneficial.-Pera, T., Deshpande, D. A., Ippolito, M., Wang, B., Gavrila, A., Michael, J. V., Nayak, A. P., Tompkins, E., Farrell, E., Kroeze, W. K., Roth, B. L., Panettieri, R. A. Jr Benovic, J. L., An, S. S., Dulin, N. O., Penn, R. B. Biased signaling of the proton-sensing receptor OGR1 by benzodiazepines.

Ovarian cancer G protein-coupled receptor 1 (OGR1) is a recently deorphanized G protein-coupled receptor shown to signal in response to low extracellular pH (↓pHo) or certain benzodiazepines. The pleiotropic nature of OGR1 signaling in human airway smooth muscle (HASM) cells suggests that OGR1 is a potential therapeutic target for the management of obstructive lung diseases. However, the basic pharmacological and regulatory features of OGR1 remain poorly understood. We employed model systems of heterologously expressed [human embryonic kidney 293 (HEK293) cells] or endogenous (HASM) OGR1 to assess changes in expression, subcellular localization, and signaling capabilities following acute or chronic treatment with ↓pHo or the benzodiazepines lorazepam and sulazepam. In HEK293 cells expressing OGR1, treatment with ↓pHo and/or lorazepam, but not sulazepam, caused rapid OGR1 internalization. In HASM cells, acute treatment with ↓pHo or benzodiazepines did not alter abundance of OGR1 mRNA; however, significant downregulation was observed following chronic treatment. Acute and chronic pretreatment of HASM cells with sulazepam or lorazepam resulted in receptor desensitization as demonstrated by reduced phosphorylation of vasodilator-stimulated phosphoprotein (VASP) or p42/p44 upon rechallenge. Acid (acute but not chronic) pretreatment of HASM cells induced desensitization of OGR1-mediated VASP (but not p42/p44) phosphorylation. In contrast to a recent study reporting OGR1 upregulation and sensitization in cardiac tissue subject to ischemic/acidic insult, chronic OGR1 activation in multiple model systems did not increase OGR1 expression or signaling capacity. The ability to induce OGR1 internalization and desensitization was activator dependent, reflecting the ability of different activators to induce specific receptor confirmations and engagement of specific heterotrimeric G proteins.

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Na+/H+ exchanger regulatory factor 1 (NHERF1; also known as ezrin‐radixin‐moesin–binding phosphoprotein 50) is a PSD‐95, disc large, zona occludens‐1 adapter that acts as a scaffold for signaling complexes and cytoskeletal‐plasma membrane interactions. NHERF1 is crucial to β‐2‐adrenoceptor (β2AR)‐mediated activation of cystic fibrosis transmembrane conductance regulator (CFTR) in epithelial cells, and NHERF1 has been proposed to mediate the recycling of internalized β2AR back to the cell membrane. In the current study, we assessed the role of NHERF1 in regulating cAMP‐mediated signaling and immunomodulatory functions in airway smooth muscle (ASM). NHERF1 knockdown attenuated the induction of (protein kinase A) phospho–vasodilator‐stimulated phosphoprotein (p‐VASP) by isoproterenol (ISO), prostaglandin E2 (PGE2), or forskolin (FSK) as well as the induction of p‐heat shock protein 20 after 4 h of stimulation with ISO and FSK. NHERF1 knockdown fully abrogated the ISO‐, PGE2‐, and FSK‐induced IL‐6 gene expression and cytokine production without affecting cAMP‐mediated phosphodiesterase 4D (PDE4D) gene expression, phospho–cAMP response element–binding protein (p‐CREB), and cAMP response element (CRE)‐Luc, or PDGF‐induced cyclin D1 expression. Interestingly, NHERF1 knockdown prevented ISO‐induced chromatin‐binding of the transcription factor CCAAT‐enhancer–binding protein‐β (c/EBPβ). c/EBPβ knockdown almost completely abrogated the cAMP‐mediated IL‐6 but not PDE4D gene expression. The differential regulation of cAMP‐induced signaling and gene expression in our study indicates a role for NHERF1 in the compartmentalization of cAMP signaling in ASM.—Pera, T., Tompkins, E., Katz, M., Wang, B., Deshpande, D. A., Weinman, E. J., Penn, R. B. Specificity of NHERF1 regulation of GPCR signaling and function in human airway smooth muscle. FASEB J. 33, 9008–9016 (2019). http://www.fasebj.org