Arrestins 2 and 3 differentially regulate ET_A and P2Y₂ receptor-mediated cell signaling and migration in arterial smooth muscle

Abstract

Morris GE, Nelson CP, Brighton PJ, Standen NB, Challiss RA, Willets JM. Arrestins 2 and 3 differentially regulate ETA and P2Y2 receptor-mediated cell signaling and migration in arterial smooth muscle. Am J Physiol Cell Physiol 302: C723-C734, 2012. First published December 7, 2011; doi:10.1152/ajpcell.00202.2011.-Overstimulation of endothelin type A (ETA) and nucleotide (P2Y) G␣q-coupled receptors in vascular smooth muscle causes vasoconstriction, hypertension, and, eventually, hypertrophy and vascular occlusion. G protein-coupled receptor kinases (GRKs) and arrestin proteins are sequentially recruited by agonist-occupied G␣q-coupled receptors to terminate phospholipase C signaling, preventing prolonged/inappropriate contractile signaling. However, these proteins also play roles in the regulation of several mitogen-activated protein kinase (MAPK) signaling cascades known to be essential for vascular remodeling. Here we investigated whether different arrestin isoforms regulate endothelin and nucleotide receptor MAPK signaling in rat aortic smooth muscle cells (ASMCs). When intracellular Ca 2ϩ levels were assessed in isolated ASMCs loaded with Ca 2ϩ -sensitive dyes, P2Y 2 and ETA receptor desensitization was attenuated by selective small-interfering (si)RNA-mediated depletion of G protein-coupled receptor kinase 2 (GRK2). Using similar siRNA techniques, knockdown of arrestin2 prevented P2Y 2 receptor desensitization and enhanced and prolonged p38 and ERK MAPK signals, while arrestin3 depletion was ineffective. Conversely, arrestin3 knockdown prevented ET A receptor desensitization and attenuated ET1-stimulated p38 and ERK signals, while arrestin2 depletion had no effect. Using Transwell assays to assess agonist-stimulated ASMC migration, we found that UTP-stimulated migration was markedly attenuated following arrestin2 depletion, while ET1-stimulated migration was attenuated following knockdown of either arrestin. These data highlight a differential arrestin-dependent regulation of ET A and P2Y2 receptorstimulated MAPK signaling. GRK2 and arrestin expression are essential for agonist-stimulated ASMC migration, which, as a key process in vascular remodeling, highlights the potential roles of GRK2 and arrestin proteins in the progression of vascular disease.G protein-coupled receptor kinase; arrestin; extracellular signal-regulated kinase; p38 mitogen-activated protein kinase; rat aortic smooth muscle; endothelin type A INCREASING EVIDENCE SUGGESTS that overstimulation of G␣ qcoupled receptor signaling pathways leads to vascular hypertrophy and hypertension (7), implying that understanding the mechanisms that regulate G␣ q -mediated signaling is of potential importance in understanding and therapeutically treating vascular diseases. Continual or repeated agonist stimulation of G protein-coupled receptors (GPCRs) usually leads to reduced responsiveness to further agonist stimulation (36, 45), protecting cells from the adverse effects of overstimulation or inappropriate signaling. Phosphorylation at key serine or thre...

Small-Molecule G Protein–Coupled Receptor Kinase Inhibitors Attenuate G Protein–Coupled Receptor Kinase 2–Mediated Desensitization of Vasoconstrictor-Induced Arterial Contractions

“…Previous in vitro studies identified GRK2 as the key regulator of contractile GPCR-mediated PLC signalling in arterial smooth muscle cells (Cohn et al, 2008;Morris et al, 2012;Morris et al, 2011;Morris et al, 2010), however, the process of interrogating the role that GRK2…”

“…We have previously shown, in smooth muscle cells isolated from 3 rd order rat mesenteric arteries, that GRK2 is the key regulator of phospholipase C/Ca 2+ signalling induced by the vasoconstrictors endothelin and UTP, via the endothelin A (ETA) and purinergic (P2Y2) receptors (Morris et al, 2012;Morris et al, 2011;Morris et al, 2010). GRK2 is also reported to regulate signalling by two further vasoconstrictors, AngII and noradrenaline, through negative regulation of AngII type 1 receptor (AT1) (Kim et al, 2009;Oppermann et al, 1996) and 1D-adrenoceptor (Cohn et al, 2008) signalling.…”

“…10 histamine receptor desensitization we applied a similar R1/Rmax/R2 protocol to that utilised above for the P2Y2 receptor, however, in this case ULTR cells were transfected with the IP3 biosensor (eGFP-tagged PH domain of PLC1; 0.5 µg, for 48 h) (Morris et al, 2011;Morris et al, 2010;Willets et al, 2009;Willets et al, 2008). We assessed receptor/PLC activity by assessing IP3 biosensor translocation, rather than intracellular Ca 2+ changes; nevertheless, we have previously shown that both outputs are valid and essentially interchangeable readouts of GPCR/PLC activity and receptor desensitization (Morris et al, 2012;Morris et al, 2011;Morris et al, 2010;Willets et al, 2009;Willets et al, 2008). Following a 30 min pre-incubation with fluoxetine, the reduction in the R2 relative to R1 was similar to that seen in vehicle-treated cells (R2 expressed as a % of R1: vehicle-control, 43.8 ± 6.3, n=7 versus fluoxetine, 47.4 ± 7.9, n=5; mean ± SEM), suggesting that fluoxetine has no effect on H1 histamine receptor desensitization ( Fig …”

P2X1 receptor-mediated inhibition of the proliferation of human coronary smooth muscle cells involving the transcription factor NR4A1

“…α,β-methylene-ATP inhibited with a halfmaximal concentration of about 0.1 μM the migration of the cells. P2Y receptors including the P2Y 2 , P2Y 6 , and P2Y 12 subtypes mediate the opposite effect-an increase in migration [54][55][56][57][58][59]. Migration facilitated by the activation of P2Y 2 receptors involved the ERK1/2 as well as the arrestin2 pathway [54,58].…”

Purinergic Signaling and Blood Vessels in Health and Disease

“…Cytokines Purinergic Signaling and Blood Vessels induce upregulation of P2Y 2 receptors on vascular smooth muscle cells, resulting in increased mitogenic responses to UTP and ATP (Hou et al, 2000). Arrestindependent regulation of P2Y 2 receptor-stimulated MAPK signaling is essential for migration of aortic smooth muscle cells, a key event in vascular remodeling (Morris et al, 2012).…”