β2-Adrenergic receptor signaling and desensitization elucidated by quantitative modeling of real time cAMP dynamics

Jonathan D. Violin, Lisa M. DiPilato, Necmettin Yildirim, Timothy C. Elston, Jin Zhang, Robert J. Lefkowitz

Research output: Contribution to journalArticlepeer-review

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 β2-adrenergic receptor (β2AR) in living cells. β2AR stimulation with isoproterenol results in a transient pulse of cAMP, reaching a maximal concentration of ∼10 μM and persisting for less than 5 min. We investigated the contributions of cAMP-dependent kinase, G protein-coupled receptor kinases, and β-arrestin to the regulation of β2AR 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 β-arrestin mediated receptor inactivation and cAMP-dependent kinase-mediated induction of cAMP metabolism by phosphodiesterases. A mathematical model of β2AR signal kinetics, fit to these data, revealed that direct receptor inactivation by cAMP-dependent kinase is insignificant but that GRK6/β-arrestin-mediated inactivation is rapid and profound, occurring with a half-time of 70 s. This quantitative system analysis represents an important advance toward quantifying mechanisms contributing to the physiological regulation of receptor signaling.

Original languageEnglish (US)
Pages (from-to)2949-2961
Number of pages13
JournalJournal of Biological Chemistry
Volume283
Issue number5
DOIs
StatePublished - Feb 1 2008

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

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