Arrestin specificity for G protein-coupled receptors in human airway smooth muscle

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Despite a widely accepted role of arrestins as "uncouplers" of G protein-coupled receptor (GPCR) signaling, few studies have demonstrated the ability of arrestins to affect second messenger generation by endogenously expressed receptors in intact cells. In this study we demonstrate arrestin specificity for endogenous GPCRs in primary cultures of human airway smooth muscle (HA,SM). Expression of arrestin-green fluorescent protein (ARR2-GFP or ARR3-GFP) chimeras in HASM significantly attenuated isoproterenol (beta (2)-adrenergic receptor (beta (2)AR)-mediated)- and 5'-(N-ethylcarboxamido)adenosine (A2b adenosine receptor-mediated)stimulated cAMP production, with fluorescent microscopy demonstrating agonist-promoted redistribution of cellular ARR2-GFP into a punctate formation. Conversely, prostaglandin E-2 (PGE(2))-mediated cAMP production was unaffected by arrestin-GFP, and PGE(2) had little effect on arrestin-GFP distribution. The pharmacological profile of various selective EP receptor ligands suggested a predominantly EP2 receptor population in HASM. Further analysis in COS-1 cells revealed that ARR2-GFP expression increased agonist-promoted internalization of wild type beta (2)AR and EP4 receptors, whereas EP2 receptors remained resistant to internalization. However, expression of an arrestin whose binding to GPCRs is largely independent of receptor phosphorylation (ARR2(R169E)-GFP) enabled substantial agonist-promoted EP2 receptor internalization, increased beta (2)AR internalization to a greater extent than did ARR2-GFP, yet promoted EP4 receptor internalization to the same degree as did ARR2-GFP. Signaling via endogenous EP4 receptors in CHO-KI cells was attenuated by ARR2-GFP expression, whereas ARR2(R169E)-GFP expression in HASM inhibited EP2 receptor-mediated cAMP production. These findings demonstrate differential effects of arrestins in altering endogenous GPCR signaling in a physiologically relevant cell type and reveal a variable dependence on receptor phosphorylation in dictating arrestin-receptor interaction.
Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Issue Date
2001-08
Language
English
Article Type
Article
Keywords

EP4 PROSTAGLANDIN RECEPTORS; GREEN FLUORESCENT PROTEIN; BETA-ARRESTIN; BETA(2)-ADRENERGIC RECEPTOR; DEPENDENT ENDOCYTOSIS; ACTIVATED RECEPTOR-2; SELECTIVE REGULATION; KINASE ACTIVATION; OPIOID RECEPTORS; TERMINAL DOMAIN

Citation

JOURNAL OF BIOLOGICAL CHEMISTRY, v.276, no.35, pp.32648 - 32656

ISSN
0021-9258
DOI
10.1074/jbc.M104143200
URI
http://hdl.handle.net/10203/240873
Appears in Collection
MSE-Journal Papers(저널논문)
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