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

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posted on 24.08.2017, 10:16 by Alexander Pearce, Lucy Sanders, Paul J. Brighton, Shashi Rana, Justin C. Konje, Jonathon M. Willets
Activation of Gs coupled receptors (e.g. β2-adrenoreceptor (β2AR)) 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 β-adrenoceptor-stimulated CREB signalling in the immortalised ULTR human myometrial cell line. The non-selective β-adrenoceptor agonist isoprenaline induced time- and concentration-dependent CREB phosphorylation, which was abolished by the β2AR selective antagonist ICI118,551. β2AR-stimulated CREB phosphorylation was mediated through a short-term PKA-dependent phase, and longer-term Src/p38 MAPK-dependent/PKA-independent phase. Since in model cells, arrestin2 can facilitate β2AR-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 β2AR 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 β2AR to activate prolonged CREB phosphorylation by precluding recruitment of an arrestin2/Src/p38 complex.

History

Citation

Cellular Signalling, 2017. 38, pp. 182-191

Author affiliation

/Organisation/COLLEGE OF MEDICINE, BIOLOGICAL SCIENCES AND PSYCHOLOGY/MBSP Non-Medical Departments/Molecular & Cell Biology

Version

AM (Accepted Manuscript)

Published in

Cellular Signalling

Publisher

Elsevier

issn

0898-6568

eissn

1873-3913

Acceptance date

16/07/2017

Copyright date

2017

Available date

18/07/2018

Publisher version

http://www.sciencedirect.com/science/article/pii/S0898656817301894?via=ihub

Notes

The file associated with this record is under embargo until 12 months after publication, in accordance with the publisher's self-archiving policy. The full text may be available through the publisher links provided above.

Language

en