Arrestin Specificity for G Protein-coupled Receptors in Human Airway Smooth Muscle

Abstract

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 (␤ 2 -adrenergic receptor (␤ 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 ␤ 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 ␤ 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-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.

The structural basis of arrestin-mediated regulation of G-protein-coupled receptors

“…In human airway smooth muscle, prostaglandin receptor EP2 does not recruit arrestins upon stimulation, does not internalize, and does not demonstrate any effects of arrestins on signaling (Penn et al, 2001). Nonetheless, the phosphorylation-independent arrestin2 mutant R169E induced marked desensitization of EP2 signaling and enabled substantial receptor internalization (Penn et al, 2001). The same arrestin2(R169E) mutant was found to induce agonist-independent internalization of the serotonin 5-HT2A receptor, which was previously considered arrestin insensitive (Gray et al, 2003).…”

“…In human airway smooth muscle, prostaglandin receptor EP2 does not recruit arrestins upon stimulation, does not internalize, and does not demonstrate any effects of arrestins on signaling (Penn et al, 2001). Nonetheless, the phosphorylation-independent arrestin2 mutant R169E induced marked desensitization of EP2 signaling and enabled substantial receptor internalization (Penn et al, 2001).…”

“…Every cell type that has been carefully studied has multiple GPCR subtypes (human airway smooth muscle is one well-characterized example; Penn et al, 2001). Yet the pool of GRKs and arrestins in the cell appears to be common and shared by all.…”

Oligomerization of Wild Type and Nonfunctional Mutant Angiotensin II Type I Receptors Inhibits Gαq Protein Signaling but Not ERK Activation

“…cDNA Constructs-The EGFP-tagged bovine ␤-arrestin 2, as described previously (26), was kindly provided by Jeffery Benovic. The AT 1 -luc and GFP 2 and CaR-Luc are described in Ref.…”

Structure of an Arrestin2-Clathrin Complex Reveals a Novel Clathrin Binding Domain That Modulates Receptor Trafficking

“…Total Internal Reflection Fluorescence (TIRF) Microscopy and Image Analysis-Arrestin constructs were C-terminal-tagged with enhanced GFP as described previously (39) and expressed by transient transfection using Effectene (Qiagen) of a previously characterized clone of stably transfected HEK293 cells expressing FLAG-tagged ␤ 2 AR (40). Cells were imaged 72-96 h after transfection, and GFP fluorescence was used to select cells expressing the indicated arrestin-GFP constructs at similar levels for subsequent analysis.…”