Mapping physiological G protein-coupled receptor signaling pathways reveals a role for receptor phosphorylation in airway contraction

Abstract

G protein-coupled receptors (GPCRs) are known to initiate a plethora of signaling pathways in vitro. However, it is unclear which of these pathways are engaged to mediate physiological responses. Here, we examine the distinct roles of Gq/11-dependent signaling and receptor phosphorylation-dependent signaling in bronchial airway contraction and lung function regulated through the M3-muscarinic acetylcholine receptor (M3-mAChR). By using a genetically engineered mouse expressing a G protein-biased M3-mAChR mutant, we reveal the first evidence, to our knowledge, of a role for M3-mAChR phosphorylation in bronchial smooth muscle contraction in health and in a disease state with relevance to human asthma. Furthermore, this mouse model can be used to distinguish the physiological responses that are regulated by M3-mAChR phosphorylation (which include control of lung function) from those responses that are downstream of G protein signaling. In this way, we present an approach by which to predict the physiological/therapeutic outcome of M3-mAChR–biased ligands with important implications for drug discovery.

Targeting acetylcholine receptor M3 prevents the progression of airway hyperreactivity in a mouse model of childhood asthma

“…For example, b2AR activation triggers bronchodilation through the cAMP and PKA pathway and in turn inhibits Ca 2+ signaling and sensitization of the contractile apparatus (28,29). However, stimulation of ChRM3 by its agonists not only activates these cAMP-regulated pathways but also triggers a noncanonical RhoA-Rho-associated protein kinase pathway via receptor phosphorylation and subsequently increases Ca 2+ sensitivity and actin filament assembly (30,31). In addition, ChRM3 activation may augment TGF-b1-induced expression of contractile proteins in ASM (32).…”

Updates in the function and regulation of α1‐adrenoceptors

“…Considerable effort has been made during recent years to define this for a variety of receptors, mainly using partial affinity purification and MS (see some recent examples: Alfonzo-Méndez, Alcántara-Hernández, & García-Sáinz, 2017;Alvarez-Curto et al, 2016;Bouzo-Lorenzo et al, 2016;Bradley et al, 2016;Butcher et al, 2011;Butcher et al, 2014;Prihandoko et al, 2016;Zindel et al, 2016). Alfonzo .…”

Barcoding of GPCR trafficking and signaling through the various trafficking roadmaps by compartmentalized signaling networks

“…Mutagenesis of the phosphorylatable sites of the M3-mACHR, abolished arrestin-mediated internalization of the agonist-activated M3-mACHR, without interfering with downstream signaling by Gq [43]. These mutant mice manifested impairments in insulin secretion [43], hippocampal learning and memory [44] and bronchial contractions [45]. These block mutations provide a role for GRK-mediated phosphorylation in driving specific physiological responses, but mutant mice with deletions in specific phosphorylation sites will be required to dissect the role of specific kinases and their associated barcodes in regulating specific physiological outcomes.…”