432 which is mutated to prevent both kinase activity and Gaq/ 11-binding,19 markedly attenuated ETAR desensitization. In contrast, over-expression of D110A,K220RGRK3, K215RGRK5, or K215RGRK6 mutants had no effect on the extent or time course of recovery of ETAR responsiveness to ET-1. A potential criticism of the dominant-negative GRK over- expression approach is the possibility of ‘off-target’ effects or lack of sufﬁcient speciﬁcity towards the GRK isoenzyme being targeted. This clearly was not the case here given the marked contrast between effects of the GRK2 and 3 constructs, nevertheless, to provide a complementary experimental approach we used speciﬁc siRNAs to deplete (by 75%) endogenous GRKs in MSMCs. In agreement with the D110A,K220RGRK2 experiments, siRNA-induced depletion of GRK2 also markedly attenuated the ET-1-induced ETAR desensitization with respect to IP3, Ca2þ, and DAG/PKC signal readouts. Furthermore, redistribution of endogenous GRK2 from the cytoplasm to the membrane could be detected after a brief ET-1 stimulation, most likely indicating recruitment to agonist-occupied ETARs. Interestingly, despite previous reports of the plasma membrane association of GRK5, this isoenzyme is predo- minantly located within the nuclei of MSMCs. Nuclear accumulation of GRK5 has been reported previously in cardiomyocytes,26 pre- sumably as a consequence of its nuclear localization sequence.27 Our study also represents a ﬁrst report of the nuclear GRK5 local- ization in MSMCs, although the signiﬁcance of this ﬁnding for GPCR signalling and vascular physiology remains to be established. Collectively, these data indicate that GRK2 is the key endogen- ous GRK subtype initiating ETAR desensitization in MSMCs, with either GRK2 knockdown or disruption of the normal GRK2- ETAR interactions causing an 50% attenuation of ETAR desensi- tization. This partial blockade may arise, because the experimental strategy is incompletely effective, or may indicate that other, so far undeﬁned mechanisms are involved in regulating ETAR responsive- ness. In MSMCs, ETAR activation is known to recruit several PKC isoenzymes9,20 and PKC activation is known to phosphorylate ETAR, at least in a HEK293 cell background,13 suggesting a poten- tial role for this protein kinase family in ETAR desensitization. In addition, PKCs are also known to phosphorylate GRK2 enhancing its membrane recruitment28 and ability to desensitize GPCRs.29 Unfortunately, most of the available PKC inhibitors are ﬂuorescent molecules precluding their use with the translocating, ﬂuorescent biosensors. Consequently, we used the complementary approaches of inhibiting PKC activity with either the broad- speciﬁcity PKC inhibitor staurosporine, or down-regulating con- ventional and novel PKC isoenzymes through chronic treatment of MSMCs with the phorbol ester, PDBu. Neither manipulation of MSMCs suggested that PKCs play a role in the ETAR desensiti- zation process nor alter the ability of GRK2 to exert its effect on the ETAR. Nevertheless, it remains a possibility that PKC-mediated ETAR phosphorylation may have as yet undeﬁned roles in directing the signalling outputs of this receptor subtype (e.g. ET-1-mediated inhibition of Kþ channels to alter vasoconstrictor responses9,17). Nonetheless, since GRK2 inhibition appears only partially to prevent ETAR desensitization, it is possible that other mechanisms are involved, perhaps involving more distal regulatory processes such as b-arrestin binding,25 receptor internalization and/or recep- tor down-regulation.24 G.E. Morris et al. Accumulating evidence indicates that GRKs play an important role in the pathogenesis of vascular disease. In experimental systems, adenovirus-mediated over-expression of GRK2 in cul- tured rabbit aortic smooth muscle cells attenuated ET-1-induced proliferation,30 whereas a two- to three-fold over-expression of GRK2 in vascular smooth muscle signiﬁcantly reduced functional (increases in mean arterial pressure) responses to angiotensin II in mice.31 These ﬁndings suggest a protective role for GRK2 expression, presumably by enhancing the desensitization of these pro-hypertensive signals. However, in apparent contradiction to these ﬁndings, GRK2 expression has been reported to be enhanced in both hypertensive patients32 and rat models of hyper- tension. A possible explanation for this discrepancy is that over- expression of GRK2 not only attenuates vasoconstrictor (e.g. angiotensin II) signalling, but also vasodilator signalling. Speciﬁcally, signalling through the b2-adrenoceptor is substantially reduced in vascular smooth muscle over-expressing GRK2.31 In addition, since GRK2 is able to suppress Gaq-signalling through its N- terminal RGS-like domain,11 it is conceivable that elevated GRK2 expression is initially an adaptive response to over-stimulation of Gaq-signalling pathways, preventing inappropriate vasoconstriction and resultant hypertension.33 It is therefore clear that GRK2 is linked to hypertensive adaptations, but its exact role in the devel- opment of hypertension merits further investigation. In summary, here we have provided important new evidence highlighting the role played by GRK2 in regulating ET-1/ETAR/Gaq-mediated vaso- constriction and suggest that this GRK isoenzyme might protect against ET-1-induced vascular dysfunction.1,3 – 6,34 Supplementary material Supplementary Material is available at Cardiovascular Research online. Acknowledgements We thank Tobias Meyer (Stanford University, USA) for generously providing the eGFP-PH biosensor, and Stephen Ferguson (Univer- sity of Western Ontario, Canada) for the gift of eGFP-tagged PKC isoenzymes. Conﬂict of interest: none declared. Funding This work was generously supported by the British Heart Foundation [PG06/161/22136 and RG06/008/22062]. Funding to pay the Open Access publication charges for this article was provided by the British Heart Foundation. References 1. Mayet J, Hughes A. Cardiac and vascular pathophysiology in hypertension. Heart 2003;89:1104–1109. 2. Berk BC. Vascular smooth muscle growth: autocrine growth mechanisms. Physiol Rev 2001;81:999–1030. 3. Hirata Y, Takagi Y, Fukuda Y, Marumo F. Endothelin is a potent mitogen for rat vascular smooth muscle cells. Atherosclerosis 1989;78:225–228. 4. Bousette N, Giaid A. Endothelin-1 in atherosclerosis and other vasculopathies. Can J Physiol Pharmacol 2003;81:578–587. 5. Schiffrin EL. Endothelin: potential role in hypertension and vascular hypertrophy. Hypertension 1995;25:1135–1143.