Phosphodiesterase type 4 anchoring regulates cAMP signaling to Popeye domain-containing proteins

Amy J Tibbo,Delphine Mika,Sara Dobi,Jiayue Ling,Aisling Mcfall,Gonzalo S Tejeda,Connor Blair,Ruth Macleod,Niall Macquaide,Caglar Gök,William Fuller,Brian O Smith,Godfrey L Smith,Grégoire Vandecasteele,Thomas Brand,George S Baillie

doi:10.1016/j.yjmcc.2022.01.001

Abstract

Cyclic AMP is a ubiquitous second messenger used to transduce intracellular signals from a variety of Gs-coupled receptors. Compartmentalisation of protein intermediates within the cAMP signaling pathway underpins receptor-specific responses. The cAMP effector proteins protein-kinase A and EPAC are found in complexes that also contain phosphodiesterases whose presence ensures a coordinated cellular response to receptor activation events. Popeye domain containing (POPDC) proteins are the most recent class of cAMP effectors to be identified and have crucial roles in cardiac pacemaking and conduction. We report the first observation that POPDC proteins exist in complexes with members of the PDE4 family in cardiac myocytes. We show that POPDC1 preferentially binds the PDE4A sub-family via a specificity motif in the PDE4 UCR1 region and that PDE4s bind to the Popeye domain of POPDC1 in a region known to be susceptible to a mutation that causes human disease. Using a cell-permeable disruptor peptide that displaces the POPDC1-PDE4 complex we show that PDE4 activity localized to POPDC1 modulates cycle length of spontaneous Ca2+ transients firing in intact mouse sinoatrial nodes.

Highlights

Phosphodiesterases (PDEs) are the only known family of enzymes that can desensitize cAMP signaling via their ability to degrade cAMP
Initial studies in HEK293 cells that were transiently transfected with POPDC1-FLAG and PDE4A-VSV suggested that the proteins co-localized (Fig. 1A), overexpression of POPDC1 did cause it to be expressed throughout the cell rather than just in the plasma membrane compartment
Previously reported for adult mouse cardiomyocytes [26], POPDC1 was located at the plasma mem brane and t-tubules in adult rabbit ventricular myocytes (ARVMs) and showed some co-localisation with PDE4A (Supplementary Fig. 1).The PDE4A5 enzyme is known to have both membrane and cytosolic pools conferred by different targeting cassettes within its sequence [27], in agreement with the observed staining pattern in neonatal rat ventricular myocytes (NRVMs) (Fig. 1A) and ARVMs (Supplementary Fig. 1)

Summary

Introduction

Phosphodiesterases (PDEs) are the only known family of enzymes that can desensitize cAMP signaling via their ability to degrade cAMP. It can be translocated to the vicinity of Gs-coupled re ceptors by the signaling scaffold protein beta-arrestin in order to hy drolyze cAMP, while beta-arrestin concomitantly hinders G-protein signaling [2]. This dual functionality is essential for effective desensi tization of β-adrenoceptor signaling [3]. PDE4D5 can anchor to another scaffold protein, RACK1, to localize the enzyme at the leading edge of moving cells to allow direction sensing [4]. Specific disruption of the RACK-PDE4D5 complex hinders cell movement via an exchange factor directly activated by cAMP (EPAC)-mediated signaling mecha nism but does not affect β-adrenergic signaling [5]. Disas sembly of the β-arrestin-PDE4D5 complex allows prolonged cAMP signaling following β-adrenergic stimulation that results in hyperphosphorylation of the β-adrenergic receptor by protein kinase A (PKA) but this action does not affect PDE4D5 located in focal adhesions [6]

Methods

Results

Conclusion