Reciprocal regulation of β2-adrenoceptor-activated cAMP response-element binding protein signalling by arrestin2 and arrestin3

Activation of Gs coupled receptors (e.g. beta(2)-adrenoreceptor (beta(2)AR)) expressed within the uterine muscle layer (myometrium), promotes intracellular cAMP generation, inducing muscle relaxation through short-term inhibition of contractile proteins, and longer-term modulation of cellular phenotype to promote quiescence. In the myometrium cAMP-driven modulation of cell phenotype is facilitated by CREB activity, however despite the importance of CREB signalling in the promotion of myometrial quiescence during pregnancy, little is currently known regarding the molecular mechanisms involved. Thus, we have characterised beta-adrenoceptor-stimulated CREB signalling in the immortalised ULTR human myometrial cell line. The non-selective beta-adrenoceptor agonist isoprenaline induced time-and concentration-dependent CREB phosphorylation, which was abolished by the beta 2AR selective antagonist IC1118,551. beta(2)AR-stimulated CREB phosphorylation was mediated through a short-term PICA-dependent phase, and longer-term Src/p38 MAPKdependent/PICA-independent phase. Since in model cells, arrestin2 can facilitate beta(2)AR-mediated Src/p38 recruitment, we examined whether CREB signalling was activated through a similar process in myometrial cells. Depletion of arrestin2 attenuated p38 phosphorylation, whilst arrestin3 depletion enhanced and prolonged isoprenaline-stimulated p38 signals, which was reversed following inhibition of Src. Knockdown of arrestin2 led to enhanced short-term (up to 10 min), and attenuated longer-term (> 10 min) isoprenaline-stimulated CREB phosphorylation. Contrastingly, removal of arrestin3 enhanced and prolonged isoprenaline-stimulated CREB phosphorylation, whilst depletion of both arrestins abolished CREB signals at time points > 5 min. In summary, we have delineated the molecular mechanisms coupling beta(2)AR activity to CREB signalling in ULTR myometrial cells, revealing a biphasic activation process encompassing short-term PKA-dependent, and prolonged Src/arrestin2/p38-dependent components. Indeed, our data highlight a novel arrestin-mediated modulation of CREB signalling, suggesting a reciprocal relationship between arrestin2 and arrestin3, wherein recruitment of arrestin3 restricts the ability of beta(2)AR to activate prolonged CREB phosphorylation by precluding recruitment of an arrestin2/Src/p38 complex.