Multiple independent functions of arrestins in the regulation of protease-activated receptor-2 signaling and trafficking.
The irreversible proteolytic nature of protease-activated receptor-2 (PAR2) activation suggests that mechanism(s) responsible for termination of receptor signaling are critical determinants of the magnitude and duration of PAR2-elicited cellular responses. Rapid desensitization of activated G-protein-coupled receptors (GPCRs) involves both phosphorylation and binding of arrestins. Arrestins also function as scaffolds and transducers of mitogen-activated protein (MAP) kinase signaling cascades. The PAR2 cytoplasmic tail (C-tail) contains multiple sites of phosphorylation and may be an important determinant for arrestin interaction. Desensitization and internalization of activated PAR2 were markedly impaired in arrestin-deficient cells compared with wild-type control cells. PAR2 C-tail truncation mutants displayed normal agonist-induced internalization, caused rapid distribution of betaarr2-GFP to the plasma membrane, and desensitized in an arrestin-dependent manner similar to that of wild-type PAR2. It is interesting that PAR2 C-tail mutants lost the capacity to stably associate with arrestins and consequently, redistributed to endocytic vesicles without betaarr2-GFP, whereas internalized wild-type PAR2 remained stably associated with betaarr2-GFP in endosomes. Moreover, activated PAR2 caused rapid and prolonged activation of endogenous extracellular signal-regulated kinase (ERK1/2). It was striking that in arrestin-deficient cells, activated PAR2 induced an initial peak in ERK1/2 activity that rapidly declined. The inability of internalized PAR2 C-tail mutants to stably associate with arrestins also resulted in loss of prolonged ERK2 activation. Thus, the PAR2 C-tail regulates the stability of arrestin interaction and kinetics of ERK1/2 activation but is not essential for desensitization or internalization. These findings further suggest that the diverse functions of arrestins in regulating PAR2 signaling and trafficking are controlled by multiple independent interactions involving both the intracellular loops and the C-tail.
Stalheim
Lisa
L
Department of Pharmacology, University of North Carolina at Chapel Hill, 1106 Mary Ellen Jones Bldg., Chapel Hill, NC 27599-7365, USA. joann_trejo@med.unc.edu
Ding
Yu
Y
Gullapalli
Anuradha
A
Paing
May M
MM
Wolfe
Breann L
BL
Morris
Dionne R
DR
Trejo
JoAnn
J
D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000494:pharmacology*; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D002522:Chlorocebus aethiops; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D044464:Receptor, PAR-2 / Q000819:agonists / Q000187:drug effects / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014162:Transfection
D019390:Arrestins; D009842:Oligopeptides; D010716:Phosphatidylinositols; D034741:RNA, Small Interfering; D044464:Receptor, PAR-2; D011994:Recombinant Proteins