938 Biochemical Society Transactions (2007) Volume 35, part 5 cAMP-specific phosphodiesterase-4D5 (PDE4D5) provides a paradigm for understanding the unique non-redundant roles that PDE4 isoforms play in shaping compartmentalized cAMP cell signalling M.J. Lynch, G.S. Baillie and M.D. Houslay 1 Division of Biochemistry and Molecular Biology, IBLS (Institute of Biomedical and Life Sciences), Wolfson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K. Abstract The PDE4 (phosphodiesterase-4) enzyme family consists of a distinct array of N-terminal splice variant isoforms arising from four subfamily genes (4A, 4B, 4C and 4D). These all hydrolyse specifically the intracellular second messenger cAMP. Although identical in catalytic function, each isoform appears to serve a non-superfluous regulatory role. For example, a β -arrestin-sequestered subpopulation of the PDE4D5 isoform specifically regulates the phosphorylation of the β 2 -AR (β 2 -adrenergic receptor) by PKA (protein kinase A; also called cAMP-dependent protein kinase). This was elucidated by the use of novel technologies, including dominant-negative approaches, siRNA (small interfering RNA) knockdown and spot-immobilized peptide array analyses. Functional phenotypes uncovered using these methodologies have shown that β -arrestin- sequestered PDE4D5 shapes the spatial cAMP gradient around the membrane-bound β 2 -AR, regulating its phosphorylation by PKA and its ability to activate ERK (extracellular-signal-regulated kinase) through G i in cardiomyocytes and HEK-293 (human embryonic kidney)-B2 cells. This approach has provided the very first identification of a non-redundant and specific role for a PDE isoform. The fact that phenotypes can be uncovered by displacing PDE4 isoforms from specific anchor sites using dominant-negative constructs and cell-permeable peptides points to novel means for developing therapeutics aimed at disrupting specifically sequestered PDE isoforms and even specifically sequestered subpopulations of individual isoforms. Introduction cAMP is a critical second messenger that can influence and regulate a vast array of cellular events vital for the response and control of signalling pathways to environmental cues [1– 3]. It is now apparent that cAMP is spatially and temporarily localized within the cell, leading to compartmentalized signalling. Vital to the generation, preservation and regulation of these spatially distinct zones is the hydrolysis of cAMP to 5  -AMP by specific members of the cyclic nucleotide PDE (phosphodiesterase) superfamily, where members of the PDE4 family play a crucial regulatory role in many cell types and provide the paradigm for intracellular targeting [4–6]. Four genes comprise the PDE4 ensemble (4A, 4B, 4C and 4D), allowing the manufacture of over 20 variant PDE4 isoforms [5,7]. These share the ability to hydrolyse cAMP specifically, while containing domains and motifs allowing for regulation by multisite phosphorylation, involving, for ex- Key words: β2-adrenergic receptor, β-arrestin, cAMP phosphodiesterase, cardiomyocyte, phosphodiesterase-4D5 (PDE4D5), protein kinase A (PKA). Abbreviations used: AKAP, A-kinase-anchoring protein; β2-AR, β2-adrenergic receptor; ERK, extracellular-signal-regulated kinase; GPCR, G-protein-coupled receptor; HEK-293 cells, human embryonic kidney cells; PDE, phosphodiesterase; PKA, protein kinase A; RACK1, receptors for activated C-kinase 1; siRNA, small interfering RNA. 1 To whom correspondence should be addressed (email m.houslay@bio.gla.ac.uk). ample, PKA (protein kinase A; also called cAMP-dependent protein kinase), ERK (extracellular-signal-regulated kinase) and a kinase downstream of PI3K (phosphoinositide 3- kinase) [7–11], and the targeting of specific PDE4 isoforms to various signalling hubs, membranes and cytoskeletal structures within particular intracellular locales [5,6,12]. In this, the N-terminal region unique to each PDE4 isoform invariably plays a pivotal role [5,6,13]. PDE4 targeting to specific intracellular locales forms ‘sinks’ of cAMP [14] where the localized cAMP gradient can be shaped through the action of immobilized PDE4 [14–17]. This will be dynamically regulated by changes in PDE4 activity achieved through phosphorylation and the recruitment or release of PDE4 isoforms from spatially restricted sites within the cell. The first insight into compartmentalized cAMP signalling came from studies performed in cardiomyocytes [17,18] where observations were made showing that PKA isoforms could be selectively activated by stimulation of different GPCRs (G-protein-coupled receptors) [19]. The field then opened up following pioneering work showing that PKA iso- forms were targeted to discrete intracellular locales by a large family of anchor proteins [AKAPs (A-kinase-anchoring pro- teins; also called PKA-anchoring protein)] able to sense and act upon gradients of cAMP [1,2,12,20] and, subsequently, C  The Authors Journal compilation C  2007 Biochemical Society