The Unique Amino-terminal Region of the PDE4D5 cAMP Phosphodiesterase Isoform Confers Preferential Interaction with β-Arrestins

Abstract

Isoproterenol challenge of Hek-B2 cells causes a transient recruitment of the endogenous PDE4D isoforms found in these cells, namely PDE4D3 and PDE4D5, to the membrane fraction. PDE4D5 provides around 80% of the total PDE4D protein so recruited, although it only comprises about 40% of the total PDE4D protein in Hek-B2 cells. PDE4D5 provides about 80% of the total PDE4D protein found associated with ␤-arrestins immunopurified from Hek-B2, COS1, and A549 cells as well as cardiac myocytes, whereas its overall level in these cells is between 15 and 50% of the total PDE4D protein. Truncation analyses indicate that two sites in PDE4D5 are involved in mediating its interaction with ␤-arrestins, one associated with the common PDE4 catalytic region and the other located within its unique amino-terminal region. Truncation analyses indicate that two sites in ␤-arrestin 2 are involved in mediating its interaction with PDE4D5, one associated with its extreme amino-terminal region and the other located within the carboxylterminal domain of the protein. We suggest that the unique amino-terminal region of PDE4D5 allows it to preferentially interact with ␤-arrestins. This specificity appears likely to account for the preferential recruitment of PDE4D5, compared with PDE4D3, to membranes of Hek-B2 cells and cardiac myocytes upon challenge with isoproterenol.The heptahelical ␤-adrenergic receptors (␤ 2 ARs) 1 act by coupling through the G-protein G s to stimulate adenylyl cyclase and thereby increase intracellular cAMP concentrations (1-4). It is now well established that the rapid uncoupling of this response is achieved by the action of the G-protein receptorcoupled kinase 2 (2). This phosphorylates the carboxyl-terminal tail of the plasma membrane-associated ␤ 2 AR, allowing the recruitment of ␤-arrestins from the cytosol (5-7). It is this recruitment of ␤-arrestin, rather than phosphorylation per se, that elicits uncoupling, presumably by sterically blocking coupling of the ␤ 2 AR to G s . The importance of ␤-arrestin interaction to G-protein-coupled receptors (GPCRs) in vivo has been clearly established in knockout mouse models (8 -10).The attenuation of cAMP signaling is also achieved through the action of phosphodiesterases (PDEs) that are able to hydrolyze cAMP to 5Ј-AMP (11-17). Since they provide the sole route for degradation of cAMP in cells, they are poised to play a key role in controlling cAMP signaling. Multiple genes encode a large superfamily of PDEs, which differ in their regulatory and kinetic properties. Of these, the PDE4 cAMP-specific phosphodiesterase family (13-16) has recently attracted much interest, since PDE4-selective inhibitors are currently being developed as potential therapeutic agents for various inflammatory diseases of the respiratory system, such as asthma and chronic obstructive pulmonary disease (18 -21). The PDE4 enzyme family is encoded by four genes (PDE4A, -B, -C, and -D), which generate over 16 different isoforms through the use of distinct promoters and alternative mRNA splicing (12,13,...

Beyond the dogma: novel β2-adrenoceptor signalling in the airways

“…ENGELS et al [266] have also reported induction of PDE4 isogenes in U937 and Jurkat T-cells in response to prolonged exposure to dibutyryl cAMP, and, more recently, the same phenomenon was documented in guinea pig macrophages [266], human T-lymphocytes [156], human neutrophils [256] and human ASM cells [157]. In the latter study, upregulation of the PDE4D5 splice variant was described, and this may be of particular significance given that this isoform interacts preferentially with b-arrestins and may play a role in b 2 -adrenoceptor desensitisation (see above) [237,238].…”

“…Thus, following agonism of b 2 -adrenoceptors expressed in HEK 293 cells, a b-arrestin/PDE4D complex forms and is recruited to the receptor, where it limits activation of the cAMP/PKA cascade by simultaneously suppressing AC activity, through receptor desensitisation, and accelerating the removal of cAMP, through enhanced degradation [236]. It appears that the unique amino-terminal region of PDE4D5 confers preferential interaction with barrestins and may represent the normal binding partner of this scaffold in intact cells [237]. This targeting of PDE4D to the b 2 -adrenoceptor complex may have highly discrete functional implications for the cell as it will selectively regulate the activity of a pool of PKA that is co-localised to the same subcellular microdomain via an interaction with an AKAP [238].…”

“…In addition, the ability of GRKs to disrupt b 2 -adrenoceptor signalling through phosphorylation is enhanced ,10-fold following the binding of scaffold proteins called barrestins [234,235]. Recently, elegant studies have extended the scaffold functions of b-arrestin to include certain members of the PDE4D family of enzymes, including PDE4D3 and, in particular, PDE4D5 [236,237]. Thus, following agonism of b 2 -adrenoceptors expressed in HEK 293 cells, a b-arrestin/PDE4D complex forms and is recruited to the receptor, where it limits activation of the cAMP/PKA cascade by simultaneously suppressing AC activity, through receptor desensitisation, and accelerating the removal of cAMP, through enhanced degradation [236].…”

Regulation of amygdalar PKA by β-arrestin-2/phosphodiesterase-4 complex is critical for fear conditioning

“…Houslay and colleges demonstrated that inhibition of PDE-4 affects CNS responses including depression, schizophrenia and memory (22)(23)(24)(25)(26). ␤-arrestin-2 can form stable complex with PDE-4s (27,28), and PDE-4D5 is the preferred binding partner of ␤-arrestin-2 (29). In this study, we reveal a role of ␤-arrestin-2/PDE-4D complex in feedback regulation of amygdalar PKA activity in response to fear conditioning and demonstrate ␤-arrestin-2 is critical for the formation of associative fear memory.…”

Intracellular Membrane Association of the Aplysia cAMP Phosphodiesterase Long and Short Forms via Different Targeting Mechanisms

“…For example, the NTR of PDE4A4/5 and PDE4D4 interacts with the SH3 domain of the Src family of tyrosine kinases such as Lyn, Fyn, and Src (16,17). The NTR of PDE4D5 interacts with ␤-arrestin (18) or with the receptor for activated protein kinase C (RACK1) (19), and the NTR of PDE4D4/5 interacts with Aryl hydrocarbon receptorinteracting protein (AIP) (20). The NTR of PDE4A1, a brainspecific isoform, is known to be the only PDE4 isoform that interacts with the membrane directly and is targeted to the Golgi complex and its associated vesicles through the two helices (helices 1 and 2) within the NTR (21,22).…”