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

Abstract

We analyzed cGMP signaling by the human phosphodiesterase 5 (hPDE5) tandem GAF domain based on a functional activation assay. The C-terminal catalytic domain of the cyanobacterial adenylyl cyclase (AC) cyaB1 was used as a reporter enzyme. We demonstrate functional coupling between the hPDE5 GAF ensemble and the AC resulting in a chimera stimulated 10-fold by cGMP. The hPDE5 GAF domain has an inhibitory effect on AC activity, which is released upon cGMP activation. Removal of 109 amino acids from the N terminus resulted in partial disengagement of the GAF domain and AC, i.e. in a 10-fold increase in basal activity, and affected cGMP affinity. The Ser-102 phosphorylation site of hPDE5 increased cGMP affinity, as shown by a 5-fold lower K D for cGMP in a S102D mutant, which mimicked complete modification. The function of the NKFDE motif, which is a signature of all GAF domains with known cyclic nucleotide binding capacity, was elucidated by targeted mutations. Data with either single and double mutants in either GAF A or GAF B or a quadruple mutant affecting both subdomains simultaneously indicated that it is impossible to functionally assign cGMP binding and intramolecular signaling to either GAF A or B of hPDE5. Both subdomains are structurally and functionally interdependent and act in concert in regulating cycaB1 AC and, most likely, also hPDE5.In essentially all eukaryotic cells, cAMP and cGMP act as second messengers. Therefore the concentration of these nucleotides is meticulously regulated by the rates of biosynthesis and breakdown (1, 2). Although for years most studies have focused on mammalian ACs, 2 more recently PDEs have come into focus. Based on biochemical properties and sequence alignments, mammalian PDEs have been grouped into 11 families (PDE1-PDE11). All catalytic domains are similar (20 -45% identity (3)), and peculiar regulatory features are mediated by differing N-terminal domains. PDEs 2, 5, 6, 10, and 11 contain N-terminal tandem GAF domains (the acronym derives from proteins of initial identification: mammalian cGMP-binding PDEs, Anabaena adenylyl cyclases, and Escherichia coli transcription factor FhlA (4, 5)). To date, GAF domains have been identified in more than 3000 proteins. They bind a variety of small ligands and promote protein dimerization (2, 6 -8).The tandem GAF domains in mammalian PDEs 2, 5, and 6, which bind cGMP, have been analyzed intensively (2, 7-12). The studies have been hampered by the fact that cGMP concurrently serves as a substrate and as an allosteric regulator creating an unsolvable kinetic conundrum. Because the tandem GAF domains of mammalian PDEs are closely related to those of cyanobacterial ACs (6, 13, 14), we have replaced the cyanobacterial tandem GAF domain in the cyaB1 AC, which imparts cAMP regulation, with that of rPDE2a. The chimera is regulated by cGMP acting via the GAF B domain and uses ATP as a substrate (6).Here, we successfully replaced the tandem GAF domain of the cyaB1 AC with that of the hPDE5, again and surprisingly generating a cGMP-regulat...

Multiple Affinity States of cGMP-Specific Phosphodiesterase for Sildenafil Inhibition Defined by cGMP-Dependent and cGMP-Independent Mechanisms

“…The advantage of these constructs was that they allowed easy monitoring of cGMP/GAF domain-induced signaling through changes in adenylate cyclase activity (Kanacher et al, 2002). Human PDE5 GAF-AB domain chimeras showed significant activation by cGMP (Bruder et al, 2006). However, constructs containing either GAF-A or GAF-B PDE5 were unable to respond to cGMP stimulation (Hofbauer et al, 2008), suggesting that both GAF-A and GAF-B domains are necessary for cGMP/GAF domain PDE5 signaling.…”

Characterization of Conformational Changes and Protein-Protein Interactions of Rod Photoreceptor Phosphodiesterase (PDE6)

“…7B). In this context, the N-terminal half of P␥ may play a role in PDE6 regulation not unlike the N-terminal regions of other GAF-containing cyclic nucleotide phosphodiesterase catalytic subunits, which exert regulatory control over catalytic activity (61,62).…”

Characterization of tandem GAF domains of phosphodiesterases

“…We show that the N-terminal domains which precede the PDE GAF domains in PDE5 and PDE11 greatly affect GAF domain signalling and, thus, likely participate in intramolecular signalling [2,3]. For example, the GAF tandem of PDE11 has a low cGMP affinity (EC50 = 72 μM) i.e.…”

“…This cyclase has an N-terminal GAF tandem which is similar to those in mammalian PDEs and regulates cyclase activity in a feed-forward manner using the product cAMP as an activator. Surprisingly, the GAF tandem domains of PDE 2, 5, 10, and 11 functionally couple to the cyclase and regulate it in a manner consistent with their function in the respective PDEs [1][2][3].…”

“…This cyclase has an N-terminal GAF tandem which is similar to those in mammalian PDEs and regulates cyclase activity in a feed-forward manner using the product cAMP as an activator. Surprisingly, the GAF tandem domains of PDE 2, 5, 10, and 11 functionally couple to the cyclase and regulate it in a manner consistent with their function in the respective PDEs [1][2][3].We show that the N-terminal domains which precede the PDE GAF domains in PDE5 and PDE11 greatly affect GAF domain signalling and, thus, likely participate in intramolecular signalling [2,3]. For example, the GAF tandem of PDE11 has a low cGMP affinity (EC50 = 72 μM) i.e.…”