Phosphorylation of Isolated Human Phosphodiesterase-5 Regulatory Domain Induces an Apparent Conformational Change and Increases cGMP Binding Affinity

Abstract

The superfamily of cyclic nucleotide phosphodiesterases (PDEs) 1 is comprised of eleven known families of PDEs that vary in substrate specificity, regulatory properties, and tissue distribution (1, 2). The regulatory domains of five of the known PDE families (PDEs 2, 5, 6, 10, and 11) contain either one or two sequences known as GAF domains, and these are homologous among the five families. In three of these families (PDEs 2, 5, and 6), at least one of these sequences in each monomer forms an allosteric site for cGMP binding (3-10). These latter PDEs belong to a subgroup of PDE families known as cGMP binding PDEs. In PDE2, cGMP binding to the cGMP binding allosteric sites increases catalytic activity of the enzyme toward cAMP and cGMP by an unknown mechanism (11, 12). In PDE5, the effect of allosteric cGMP binding is still not clear, but cGMP binding to the PDE5 R domain controls phosphorylation of a specific serine that is near the amino terminus of the R domain (13,14). Phosphorylation at this serine activates both PDE5 catalytic and allosteric cGMP-binding activities (15). Phosphorylation of PDE5 occurs in intact vascular smooth muscle cells after cGMP elevation, and this phosphorylation is associated with increased catalytic activity of this enzyme (16 -18). Mechanism(s) by which cGMP binding and phosphorylation of the R domain of PDE5 alter enzyme properties are not understood.PDE5 exists in at least two conformational states. In absence of cGMP, the enzyme assumes an apparently more compact structure (19). Occupation of the PDE5 catalytic site by either cGMP or inhibitors such as 3-isobuty-1-methylxanthine, zaprinast, or sildenafil stimulates cGMP binding to the allosteric sites, and when both catalytic and binding sites are occupied PDE5 undergoes an apparent elongation (19). However, little is known about specific structural events that occur in PDEs upon interaction with regulatory agents or after post-translational modification by phosphorylation. Regulation of PDE5 involves cGMP interaction with the R domain, which is required for efficient post-translational modification through phosphorylation (13,15).Models of allosteric regulation of many enzymes include modulation of the equilibrium of distribution of proteins between two conformers, i.e. a relaxed (R) state that is active and has higher affinity for substrate and a taut (T) state that is less active and has lower affinity for substrate (20,21). Phosphorylase is an example of this model wherein either binding of an allosteric activator, 5ЈAMP, or phosphorylation by phosphorylase kinase alters equilibrium between the two states to produce a more active enzyme (21). PDE5 may conform to that model, because cGMP binding to allosteric binding sites induces an apparent conformational change in PDE5, perhaps converting it from a less active state to a more active state (15,19). However, phosphorylation of Ser 92 in bovine PDE5 and activation of PDE5 occur efficiently only when cGMP is elevated, and the enzyme is already in the cGMP-bound (R) conformat...

Characterization of the Tandem GAF Domain of Human Phosphodiesterase 5 Using a Cyanobacterial Adenylyl Cyclase as a Reporter Enzyme

“…Those fast k on and k off values for hPDE5 GAF-cyaB1 AC are at variance with results for cGMP binding to PDE5, where on and off kinetics were about 15 min for the on-reaction (11) and up to 7 h for the off-reaction (10,24). Similarly, binding assays with the isolated PDE5 GAF domains have demonstrated that cGMP binding and release are slow (15,17,18,24). We examined cGMP binding to hPDE5 GAF-cyaB1 AC chimera following standard protocols (10, 27).…”

“…Effect of the N Terminus on PDE 5 GAF-cyaB1 AC ActivationUsing various PDE5 GAF domain constructs, cGMP binding constants between 0.027 and 1.9 M have been reported in the past (15,17,18,22,24,27). Possibly different N-and C-terminal truncations have contributed.…”

“…In view of the rapid on and off rates of hPDE5 GAF-cyaB1 AC activation, we assumed that bound cyclic nucleotides were washed off during the washing step. The differences may be rationalized by different interactions between the GAF domain and the PDE5 or cyaB1 AC catalytic domains and/or the use of native protein isolated from lung (28) or recombinant proteins expressed in COS-7 cells (10), in Sf9 and High Five insect cells (22), and in E. coli (17).…”

“…Yet, considerable discrepancies have been reported in cGMP binding studies, and the role of Ser-102 phosphorylation (hPDE5 numbering) in cGMP binding to the GAF domain is contentious (15)(16)(17)(18)(19)(20)(21)(22)(23)(24). We characterized the contributions of the GAF A and B domains as well as the potential phosphorylation site at Ser-102 to the allosteric cGMP regulation of the cyanobacterial cyaB1 AC by the hPDE5 tandem GAF domain.…”

Characteristics of Photoreceptor PDE (PDE6): similarities and differences to PDE5

“…20,35,36 The g subunit of PDE6 also acts as a substrate for phosphorylation at several distinct sites within the central region of this 10 kDa protein. Phosphorylation of g at Thr (22) or Thr (35) has little effect on the PDE6 holoenzyme itself, but greatly diminishes the ability of activated transducin to bind the g subunit and relieve inhibition of catalysis. 37 Phosphorylation of g at Thr(62) in nonretinal tissue has been reported to regulate mitogenic signaling via interactions with proteins other than PDE6 catalytic subunits (whose expression is confined to the retina and pineal gland).…”