期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 283, 期 5, 页码 2949-2961出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M707009200
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资金
- NHLBI NIH HHS [R01 HL 16037, R01 HL 70631] Funding Source: Medline
- NIDDK NIH HHS [F31 DK 074831, DK 073368] Funding Source: Medline
- NIGMS NIH HHS [GM 079271] Funding Source: Medline
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 beta(2)-adrenergic receptor (beta(2)AR) in living cells. beta(2)AR stimulation with isoproterenol results in a transient pulse of cAMP, reaching a maximal concentration of similar to 10 mu M and persisting for less than 5 min. We investigated the contributions of cAMP-dependent kinase, G protein-coupled receptor kinases, and beta-arrestin to the regulation of beta(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 beta-arrestin mediated receptor inactivation and cAMP-dependent kinase-mediated induction of cAMP metabolism by phosphodiesterases. A mathematical model of beta(2)AR signal kinetics, fit to these data, revealed that direct receptor inactivation by cAMP-dependent kinase is insignificant but that GRK6/beta-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.
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