G protein-coupled receptor kinase 2 and arrestin2 regulate arterial smooth muscle P2Y-purinoceptor signalling

Morris, George E.; Nelson, Carl P.; Everitt, Diane E.; Brighton, Paul J.; Standen, N B; Challiss, R. A. John; Willets, Jonathon M.

doi:10.1093/cvr/cvq249

Cited by 35 publications

(72 citation statements)

References 28 publications

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“…Our finding supports this possibility that both small-molecule inhibition and knockdown of GRK2 enhance photorelaxation. Consistent with our results, GRK2 seems to be the principal GRK in regulating vascular smooth muscle-associated GPCRs (28), including α 1 -adrenergic (29), endothelin (30), purinergic (31), and β-adrenergic receptors (32).…”

Section: Discussionsupporting

confidence: 85%

Melanopsin mediates light-dependent relaxation in blood vessels

Sikka

Hussmann

Pandey

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Melanopsin (opsin4; Opn4), a non-image-forming opsin, has been linked to a number of behavioral responses to light, including circadian photo-entrainment, light suppression of activity in nocturnal animals, and alertness in diurnal animals. We report a physiological role for Opn4 in regulating blood vessel function, particularly in the context of photorelaxation. Using PCR, we demonstrate that Opn4 (a classic G protein-coupled receptor) is expressed in blood vessels. Force-tension myography demonstrates that vessels from Opn4 −/− mice fail to display photorelaxation, which is also inhibited by an Opn4-specific small-molecule inhibitor. The vasorelaxation is wavelength-specific, with a maximal response at ∼430-460 nm. Photorelaxation does not involve endothelial-, nitric oxide-, carbon monoxide-, or cytochrome p450-derived vasoactive prostanoid signaling but is associated with vascular hyperpolarization, as shown by intracellular membrane potential measurements. Signaling is both soluble guanylyl cyclase-and phosphodiesterase 6-dependent but protein kinase G-independent. β-Adrenergic receptor kinase 1 (βARK 1 or GRK2) mediates desensitization of photorelaxation, which is greatly reduced by GRK2 inhibitors. Blue light (455 nM) regulates tail artery vasoreactivity ex vivo and tail blood blood flow in vivo, supporting a potential physiological role for this signaling system. This endogenous opsin-mediated, lightactivated molecular switch for vasorelaxation might be harnessed for therapy in diseases in which altered vasoreactivity is a significant pathophysiologic contributor.hotorelaxation, the reversible relaxation of blood vessels to cold light, was initially described by Furchgott et al. in 1955 (1). Subsequent studies have attempted to define the signal transduction mechanisms responsible for this phenomenon. The process seems to be cGMP-dependent but endothelialindependent. The role of nitric oxide (NO) in photorelaxation has been controversial (2-7), with some studies showing that NOS inhibition with L-NAME not only fails to inhibit the response (2) but in some cases enhances and prolongs it (3). Moreover, several published reports examining photorelaxation demonstrate an attenuation of the response with each subsequent light stimulation. A number of investigators have proposed that NO dependence results from the photo-release of NO stores from nitrosothiols and that the endothelium and NOS are important for the repriming of these stores (stores that become depleted with each photo-stimulation); however, the source of those nitrosothiols has not as yet been clearly identified (6). Importantly, photo-release of NO occurs in the UV-A spectrum at 366 nm (4-6), a wavelength at which intravascular nitrosospecies and nitrite have the potential to release substantial quantities of NO (7). However, this wavelength is very different from that at which others have observed vascular responses. Given the controversy surrounding the photorelaxation mechanism, we postulated an entirely new mechanism: that photorelaxation i...

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Section: Discussionsupporting

confidence: 85%

Melanopsin mediates light-dependent relaxation in blood vessels

Sikka

Hussmann

Pandey

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…The expression plasmid encoding FLAGtagged b-arrestin1 was a generous gift from Robert Lefkowitz (Luttrell and Lefkowitz, 2002). The expression plasmid encoding bovine GRK2 was a generous gift from Jon Willets (Morris et al, 2011), and the expression plasmid encoding bovine GRK2-K220R was from Addgene (plasmid number 14691; Cambridge, MA). Either 10 27 M SDF-1a (R&D Systems, Minneapolis, MN) or 10 ng/ml PMA (EMD Millipore, Billerica, MA) was used for cell stimulation.…”

Section: Methodsmentioning

confidence: 99%

“…Since several reports suggest that b-arrestins associate with GRKs, and that these proteins function together to regulate GPCRs (Willets et al, 2001;Busillo et al, 2010;Morris et al, 2011), we asked if GRK2 also participates in regulating b-arrestin1-dependent CXCR4 internalization in neuroblastoma cells. GRK2 was endogenously expressed in both SH-SY5Y and SK-N-SH cells (Fig.…”

Section: Sdf-1 Uses B-arrestin1 and Cxcr4 Structures In Neuroblastomamentioning

confidence: 99%

β-Arrestin1 and Distinct CXCR4 Structures Are Required for Stromal Derived Factor-1 to Downregulate CXCR4 Cell-Surface Levels in Neuroblastoma

et al. 2014

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CXC chemokine receptor 4 (CXCR4) is a G protein-coupled receptor (GPCR) located on the cell surface that signals upon binding the chemokine stromal derived factor-1 (SDF-1; also called CXCL 12). CXCR4 promotes neuroblastoma proliferation and chemotaxis. CXCR4 expression negatively correlates with prognosis and drives neuroblastoma growth and metastasis in mouse models. All functions of CXCR4 require its expression on the cell surface, yet the molecular mechanisms that regulate CXCR4 cell-surface levels in neuroblastoma are poorly understood. We characterized CXCR4 cell-surface regulation in the related SH-SY5Y and SK-N-SH human neuroblastoma cell lines. SDF-1 treatment caused rapid down-modulation of CXCR4 in SH-SY5Y cells. Pharmacologic activation of protein kinase C similarly reduced CXCR4, but via a distinct mechanism. Analysis of CXCR4 mutants delineated two CXCR4 regions required for SDF-1 treatment to decrease cell-surface CXCR4 in neuroblastoma cells: the isoleucine-leucine motif at residues 328 and 329 and residues 343-352. In contrast, and unlike CXCR4 regulation in other cell types, serines 324, 325, 338, and 339 were not required. Arrestin proteins can bind and regulate GPCR cell-surface expression, often functioning together with kinases such as G protein-coupled receptor kinase 2 (GRK2). Using SK-N-SH cells which are naturally deficient in b-arrestin1, we showed that b-arrestin1 is required for the CXCR4 343-352 region to modulate CXCR4 cell-surface expression following treatment with SDF-1. Moreover, GRK2 overexpression enhanced CXCR4 internalization, via a mechanism requiring both b-arrestin1 expression and the 343-352 region. Together, these results characterize CXCR4 structural domains and b-arrestin1 as critical regulators of CXCR4 cell-surface expression in neuroblastoma. b-Arrestin1 levels may therefore influence the CXCR4-driven metastasis of neuroblastoma as well as prognosis.

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“…Similarly, in mouse mesenteric arteries, there was evidence for contractile responses mediated by both P2Y 6 and P2X 1 receptors, because responses to UTP and UDP were reduced by a selective P2Y 6 antagonist, and a contractile response to a,b-meATP was blocked by the P2X 1 receptor antagonist NF023 (Koltsova et al, 2009). G proteincoupled receptor kinase 2 and arrestin-2 regulate UTPstimulated P2Y 2 receptors mediating constriction of rat mesenteric artery smooth muscle (Morris et al, 2011). Whole-cell voltage clamp studies of guinea pig mesenteric terminal arterioles have suggested that ATP activation of Ca 2+ currents was mediated by P2Y 1 -and P2Y 11 -like receptors (Morita et al, 2002).…”

Section: Mesenteric Vesselsmentioning

confidence: 99%

“…Similarly, the use of knockouts and antisense oligonucleotides showed the functional expression of vasocontractile P2Y 4 and P2Y 6 but not P2Y 2 receptors in mouse mesenteric arteries and cerebral arterioles (Vial and Evans, 2002;Bar et al, 2008;Kauffenstein et al, 2010a;Brayden et al, 2013). In contrast, in rat mesenteric arteries, small interfering RNA (siRNA) knockdown of P2Y 2 mRNA demonstrated the almost exclusive involvement of P2Y 2 receptors in contraction to UTP (Morris et al, 2011). This demonstrates pronounced species differences in the expression of vasocontractile P2Y receptors, which were not detected through the use of pharmacology alone.…”

Section: Introductionmentioning

confidence: 99%