Arrestin specificity for G protein-coupled receptors in human airway smooth muscle.
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 (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-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.
Penn
R B
RB
Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA. rpenn@lac.jci.tju.edu
Pascual
R M
RM
Kim
Y M
YM
Mundell
S J
SJ
Krymskaya
V P
VP
Panettieri
R A
RA
Benovic
J L
JL
D019830:Adenosine-5'-(N-ethylcarboxamide) / Q000494:pharmacology; D000818:Animals; D019390:Arrestins / Q000235:genetics / Q000502:physiology*; D016466:CHO Cells; D019556:COS Cells; D002460:Cell Line; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D015232:Dinoprostone / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics; D009130:Muscle, Smooth / Q000166:cytology / Q000502:physiology*; D010750:Phosphoproteins / Q000235:genetics / Q000502:physiology*; D010766:Phosphorylation; D021381:Protein Transport; D018343:Receptors, Adrenergic, beta-2 / Q000187:drug effects / Q000502:physiology*; D018078:Receptors, Prostaglandin E / Q000187:drug effects / Q000502:physiology*; D058307:Receptors, Prostaglandin E, EP2 Subtype; D018047:Receptors, Purinergic P1 / Q000187:drug effects / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D014132:Trachea / Q000166:cytology / Q000502:physiology*; D014162:Transfection
D019390:Arrestins; D008164:Luminescent Proteins; C545644:PTGER2 protein, human; D010750:Phosphoproteins; D018343:Receptors, Adrenergic, beta-2; D018078:Receptors, Prostaglandin E; D058307:Receptors, Prostaglandin E, EP2 Subtype; D018047:Receptors, Purinergic P1; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019830:Adenosine-5'-(N-ethylcarboxamide); D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D015232:Dinoprostone; D007545:Isoproterenol