β2-Adrenergic Receptor Signaling and Desensitization Elucidated by Quantitative Modeling of Real Time cAMP Dynamics

Abstract

G protein-coupled receptor signaling is dynamically regulated by multiple feedback mechanisms, which rapidly attenuate signals elicited by ligand stimulation, causing desensitization. The individual contributions of these mechanisms, however, are poorly understood. Here, we use an improved fluorescent biosensor for cAMP to measure second messenger dynamics stimulated by endogenous ␤ 2 -adrenergic receptor (␤ 2 AR) in living cells. ␤ 2 AR stimulation with isoproterenol results in a transient pulse of cAMP, reaching a maximal concentration of ϳ10 M and persisting for less than 5 min. We investigated the contributions of cAMP-dependent kinase, G protein-coupled receptor kinases, and ␤-arrestin to the regulation of ␤ 2 AR signal kinetics by using small molecule inhibitors, small interfering RNAs, and mouse embryonic fibroblasts. We found that the cAMP response is restricted in duration by two distinct mechanisms in HEK-293 cells: G protein-coupled receptor kinase (GRK6)-mediated receptor phosphorylation leading to ␤-arrestin mediated receptor inactivation and cAMP-dependent kinase-mediated induction of cAMP metabolism by phosphodiesterases. A mathematical model of ␤ 2 AR signal kinetics, fit to these data, revealed that direct receptor inactivation by cAMPdependent kinase is insignificant but that GRK6/␤-arrestin-mediated inactivation is rapid and profound, occurring with a halftime of 70 s. This quantitative system analysis represents an important advance toward quantifying mechanisms contributing to the physiological regulation of receptor signaling.Tachyphylaxis, or desensitization, denoting the attenuation of a biological response to sustained or repeated intervention, is a pervasive phenomenon in physiological systems. For G protein-coupled receptors (GPCRs) 7 (or, more broadly, seventransmembrane receptors), desensitization occurs through molecular mechanisms that can profoundly limit further stimulation of downstream signals, either through direct receptor inactivation or inhibition of downstream signaling. At the physiological level, we refer to this general loss of responsiveness as desensitization; we refer to the more specific case of direct inhibition of receptor molecules as "receptor inactivation." At the level of the receptor, GPCR signals represent a dynamic balance between ligand-stimulated activities, such as G protein coupling, and negative feedback mechanisms, such as receptor phosphorylation and ␤-arrestin recruitment (1). An agonist's efficacy is determined by the balance between these activities and is limited by the kinetics of receptor inactivation. Despite intensive research into the molecular mechanisms of desensitization by GPCR inactivation, the relative contributions of these mechanisms are largely unknown. One reason for this is that, until recently, there have been few techniques that directly measure receptor signaling in real time. Rather, data showing desensitization and receptor inactivation are usually based on either physiological assessments, such as hemodynamic parameters, whi...

Dendritic cell chemotaxis in 3D under defined chemokine gradients reveals differential response to ligands CCL21 and CCL19

“…3A), where receptor desensitization would occur with CCL19. Furthermore, there are some indications that receptor desensitization could take place in a ligand specific manner (8) as described for other seven-transmembrane receptors (47). As mature DCs are secreting CCL19, CCL19-specific desensitization might be also responsible for the observed CCL21 preference (Fig.…”

Distinct conformations of GPCR–β-arrestin complexes mediate desensitization, signaling, and endocytosis

“…The mechanism underlying βarr1-mediated desensitization is thought to involve the interaction between βarr and the receptor core; this core conformation, presumably, sterically blocks the G protein-binding site in the receptor core (21). To assess the importance of the FLR of βarr1 for receptor desensitization directly, we monitored the attenuation of agonist-stimulated heterotrimeric Gs protein signaling, measured here as cAMP accumulation, in either the DKO (for the β 2 AR) or a βarr1/βarr2/β 2 AR triple-knockout (for the β 2 V 2 R and V 2 R) HEK293 cell line expressing ICUE2, a fluorescence resonance energy transfer biosensor-detecting cytoplasmic cAMP (22). This ICUE2 biosensor measures cAMP concentration in real time, and thus represents equilibrium between production and degradation of cAMP.…”

β2 Adrenergic Receptor, Protein Kinase A (PKA) and c-Jun N-terminal Kinase (JNK) Signaling Pathways Mediate Tau Pathology in Alzheimer Disease Models

“…In a recent epidemiological study, it was found that antihypertensive medication, including ␤ blockers, may reduce the risk of AD (12). Another survey in AD patients indicates that ␤ blockers may be associated with a delay of functional decline in the patients (13).…”