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J. Biol. Chem., Vol. 276, Issue 35, 32648-32656, August 31, 2001
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From the 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 (HASM). Expression of arrestin-green fluorescent protein (ARR2-GFP or ARR3-GFP) chimeras in HASM significantly attenuated
isoproterenol (
Arrestin Specificity for G Protein-coupled Receptors in Human
Airway Smooth Muscle*,
§,
,**,,,, and
Department of Microbiology and Immunology,
Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia,
Pennsylvania 19107, the ¶ Division of Critical Care, Pulmonary,
Allergic and Immunological Diseases, Department of Medicine, Jefferson
Medical College, Philadelphia, Pennsylvania 19107, and the
Division of Pulmonary and Critical
Care, Department of Medicine, University of Pennsylvania School of
Medicine, Philadelphia, Pennsylvania 19104
2-adrenergic receptor
(2AR)-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 E2 (PGE2)-mediated cAMP production was
unaffected by arrestin-GFP, and PGE2 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 2AR 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
2AR 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-K1 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.
*
This work was supported by Grants HL58506 and GM47417 from
the National Institutes of Health.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains supplemental material and three videos.
§
To whom correspondence should be addressed: Thomas Jefferson
University, Kimmel Cancer Institute, Rm. 930 B.L.S.B., 233 S. 10th St.,
Philadelphia, PA 19107. E-mail: rpenn@lac.jci.tju.edu.
Recipient of a Glaxo Wellcome Pulmonary Fellowship, a Merck
Young Investigator Award, and a Parker B. Francis Fellowship.
**
Recipient of an American Heart Association Predoctoral Fellowship.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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