GRK2 regulates endothelin signalling 1. Introduction Endothelin (ET)-1 is a potent vasoconstrictor controlling vascular tone with an established role in vascular smooth muscle hyper- trophy1,2 and hyperplasia,3,4 ﬁbrosis and inﬂammatory responses, leading to vascular remodelling and hypertension.4,5 ET-1 plays a crucial role in a number of experimental models of hyperten- sion,5,6 and increased plasma ET-1 levels have been reported in hypertensive patients.7 Endothelins can signal through two receptor subtypes ETA and ETB, however in arterial smooth muscle ETA receptors (ETAR) mediate constrictor responses.8 ETARs couple through Gaq/11 to activate phospholipase C (PLC), generating inositol 1,4,5-trisphosphate (IP3) and diacylgly- cerol (DAG), and leading to the release of intracellular Ca2þ stores and the activation of protein kinase C.2,8 ETAR-mediated intracellular Ca2þ increases underlie a major component of ET-1-induced vasoconstriction. ET-1 also promotes PKC-induced inhibition of voltage-gated Kþ channels to further enhance vasoconstriction.9 Previous studies have shown that antagonizing ETAR signalling can reverse hypertrophy and hypertension in animal models,10 indicating a potentially important role for ETAR over-activation in the development of vascular disease. Over-stimulation of ETAR signalling clearly has adverse effects on vascular smooth muscle cells, and understanding the mechanisms that regulate Gaq/11 signalling is of potential importance in understanding and treating vascular diseases. Continual or repeated stimulation of receptors by agonists usually leads to reduced responsiveness to further agonist challenge.11 This process, known as receptor desensitization, has been shown to protect cells from the adverse effects of over-stimulation or inappropriate signalling. Phosphorylation of key serine and/or threonine residues within the third intracellular loop and/or C-terminal tail of the G protein-coupled receptor is thought to be the primary event initi- ating desensitization and is often mediated by one or more members of a family of seven serine/threonine kinases, the G protein-coupled receptor kinases (GRKs).11 Phosphorylation by GRKs enhances receptor afﬁnity for the non-visual arrestins 2 and 3, which can sterically suppress further interaction between receptor and G proteins.12 Although previous studies have shown that recombinantly expressed ETARs are phosphorylated and desensitized in a GRK-dependent manner in model cells, such as HEK293 cells,13 little evidence is currently available to indicate which, if any, GRKs regulate endogenous ETAR signalling in vascular smooth muscle. This is an important issue as recombinant receptors are often regulated very differently to those expressed endogenously. Indeed, a growing number of studies are revealing that precise deﬁ- nition of the GRKs involved in receptor regulation can only be truly deﬁned in a system endogenously expressing the receptor of interest and through manipulation of endogenous GRK popu- lations.11,14 – 16 Here, we have combined confocal imaging tech- niques and speciﬁc inhibition of endogenous GRK isoenzymic activities to examine the interaction between GRKs and the native ETAR population in isolated, cultured mesenteric smooth muscle cells (MSMCs), a widely studied model of systemic resist- ance arteries.9,17 425 2. Methods 2.1 Isolation and culture of mesenteric arterial smooth muscle cells Adult male Wistar rats were sacriﬁced by stunning and cervical dislo- cation, a method approved under the UK Animals (Scientiﬁc Pro- cedures) Act 1986. Smooth muscle cells were isolated from small branches of mesenteric artery by enzymatic dissociation as previously described.17 Smooth muscle cells were separated by gentle trituration in 231 medium (Cascade Biologics, Nottingham, UK), supplemented with smooth muscle growth supplement, 100 IU21 penicillin, 100 mg/mL streptomycin, and 2.5 mg/mL amphotercin B. Cells plated onto cover- slips were maintained at 378C, 5% CO2 in humidiﬁed conditions. MSMCs were characterized by immunocytochemistry and immunoblot- ting for the presence of smooth muscle-speciﬁc a-actin and calponin (data not shown). The investigation conforms to Guide for Care and Use of Laboratory Animals US (NIH Publication No. 85-23, revised 1996). 2.2 Immunocytochemistry MSMCs were cultured for 72 h before ﬁxation and permeabilization in 100% methanol (10 min at 2208C). GRK expression was identiﬁed through addition of speciﬁc anti-rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) against GRK2, GRK3, GRK5, and GRK6. Protein expression was visualized with the addition of ﬂuorescein isothiocyanate-conjugated secondary antibodies, before viewing on an Olympus FV500 laser scanning confocal IX70 inverted microscope. 2.3 Small interfering RNA knockdown of endogenous GRK2 levels and western blotting Expression of GRKs and dominant-negative GRKs (see below) was determined using standard immunoblotting protocols as described previously14 using speciﬁc anti-rabbit polyclonal antibodies against GRK2, GRK3, GRK5, and GRK6. MSMCs (1 106) were transfected with 10 or 100 nM negative-control or anti-GRK2 (50-GCAGGUACCUCCAGAUCUCtt-30) (Applied Biosystems, UK) small interfering (si)RNAs. Cells were lysed after 48 h and GRK expression assessed by immunoblotting (see above). GRK expression was quantiﬁed using the GeneGnome image analysis system (Syngene, Cambridge, UK). 2.4 Manipulating cellular GRK activity GRK2/3 constructs were mutated in both the N-terminal RGS-like domain (D110A) (to prevent binding to Gaq/11 proteins11) and the catalytic-domain (K220R) to allow potential receptor/GRK phosphorylation-dependent interactions to be examined.11,19 In MSMCs transfected with D110A,K220RGRK2 or D110A,K220RGRK3 con- structs, normal PLC signalling was unaffected. In contrast, expression of K220RGRK2 or K220RGRK3 markedly attenuated PLC signalling (data not shown), most likely through the previously reported phosphorylation-independent inhibition of Gaq/11 transduction through an interaction with the GRK2/3 RGS-homology (RH) domain.19 GRK activity was manipulated using the dominant-negative con- structs D110A,K220RGRK2, D110A,K220RGRK3, K215RGRK5, or K215RGRK6 (0.5 mg), negative-control plasmid (pcDNA3), or through addition of anti-GRK2, or negative-control siRNAs (10 nM). Cells were transfected using LipofectAMINE2000 (Invitrogen, Paisley, UK) according to the manufacturer’s instructions.