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 (A_2BR)] 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 A_2BR are GRK/arrestin substrates, however similar potential interactions have not been fully investigated in VSMC.

β₂AR and A_2BR 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 A_2BR-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 A_2BR signalling to prevent vasoconstrictor-stimulated VSMC migration. Activation EPAC was equally as effective as β₂AR and A_2BR agonists at blocking vasoconstrictor-stimulated VSMC migration.

This research indicates that β₂AR and A_2BR activate CREB and p38 signalling in RASM, and these signalling pathways are selectively regulated by GRK and arrestin proteins. β₂AR and A_2BR activity prevents vasoconstrictor-promoted VSMC migration, however, it is unclear whether this blockade relies on agonist-stimulated CREB and/or p38 activation.