Characterising the Roles That GRK and Arrestin Proteins Play in The Regulation of Gαs-Coupled Receptor Signalling in Arterial Smooth Muscle Cell

Hypertension and vascular remodelling play central roles in the pathophysiology of heart disease, stroke, peripheral atherosclerosis, and renal failure. Gαs-coupled G protein-coupled receptor (GPCR) activation [e.g. β2-adrenoceptor (β2AR), adenosine A2B receptor (A2BR)] causes vascular smooth muscle cell (VSMC) relaxation, counteracting effects of vasoconstrictors. G protein-coupled receptor kinases (GRK) and arrestins regulate GPCR signalling, and dysregulated Gαs/GPCR signalling is key in the development of hypertension, probably via increased GRK2/arrestin VSMC expression. Evidence indicates β2AR and A2BR are GRK/arrestin substrates, however similar potential interactions have not been fully investigated in VSMC. β2AR and A2BR signalling promotes cAMP production, activating multiple downstream signalling pathways, including CREB, mediating vaso-relaxatory effects of Gαs-coupled GPCRs. Characterisation of β2AR-induced CREB signalling revealed PKA is essential for the early stages, and EPAC the later stages of β2AR-mediated CREB-phosphorylation, implying that cAMP-driven processes are required to produce β2AR-stimulated CREB-signalling. A2BR-induced CREB signalling relied on p38/Src-kinase, and not cAMP/PKA/EPAC. GRK2 siRNA-targeted knockdown/or chemical inhibition, blocked β2AR-stimulated CREB-phosphorylation. Similar data were obtained following arrestin2 depletion. A2BR-stimulated CREB-phosphorylation required GRK5/arrestin3 expression. β2AR-stimulated p38 signals were PKA/EPCA independent, but required GRK2/arrestin2 expression. A2BR-induced p38 signalling was GRK5-dependent, but arrestin/PKA /EPAC-independent. VSMC migration is central to hypertension-induced vascular remodelling, and here β2AR and A2BR-stimulated Gαs/GPCR signalling blocks VSMC migration through cAMP-dependent signalling pathways including CREB. CREB, GRK2 or GRK5 inhibition attenuated vasoconstrictor-stimulated VSMC migration, but failed to affect the ability of β2AR and A2BR signalling to prevent vasoconstrictor-stimulated VSMC migration. Activation EPAC was equally as effective as β2AR and A2BR agonists at blocking vasoconstrictor-stimulated VSMC migration. This research indicates that β2AR and A2BR activate CREB and p38 signalling in RASM, and these signalling pathways are selectively regulated by GRK and arrestin proteins. β2AR and A2BR activity prevents vasoconstrictor-promoted VSMC migration, however, it is unclear whether this blockade relies on agonist-stimulated CREB and/or p38 activation.