cAMP Is a Ligand for the Tandem GAF Domain of Human Phosphodiesterase 10 and cGMP for the Tandem GAF Domain of Phosphodiesterase 11

Gross-Langenhoff, Marco; Hofbauer, Karina; Weber, Jost; Schultz, Anita; Schultz, Joachim E.

doi:10.1074/jbc.m511468200

Cited by 115 publications

(111 citation statements)

References 39 publications

(34 reference statements)

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“…Possibly different N-and C-terminal truncations have contributed. Recently, we have demonstrated that the N-terminal domain of the tandem GAF of PDE114A is critical for cGMP stimulation, because activation was observed only with the complete N terminus (26). Therefore, we examined the role of the N terminus of hPDE5 of 147 amino acids that contains a potentially important phosphorylation site (Ser-102).…”

Section: Biochemical Characterization Of Hpde5 Gaf-cyab1 Ac-mentioning

confidence: 99%

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

Bruder

Schultz

2006

Journal of Biological Chemistry

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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...

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Section: Biochemical Characterization Of Hpde5 Gaf-cyab1 Ac-mentioning

confidence: 99%

“…Formerly we had replaced the cyanobacterial tandem GAF domain in cyaB1 with those of rPDE2a and hPDEs 10 and 11 (6,26). This enabled us to use the cyaB1 AC as a reporter enzyme to characterize the GAF domains from mammalian PDEs 2, 5, 6, 10, and 11.…”

Section: Biochemical Characterization Of Hpde5 Gaf-cyab1 Ac-mentioning

confidence: 99%

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

Bruder

Schultz

2006

Journal of Biological Chemistry

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“…PDE6 is also regulated by cGMP binding to GAF-A, in the unique context of interaction with the P␥ subunit (19,20). The physiological role of GAF domain regulation for PDE10 and 11 is still to be determined (21). The molecular mechanism(s) whereby binding of signaling molecules in the N-terminal domains of different PDEs regulates activity of the catalytic domain is not yet understood.…”

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confidence: 99%

Mechanism for the allosteric regulation of phosphodiesterase 2A deduced from the X-ray structure of a near full-length construct

Pandit

Forman

Fennell

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

150

159

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We report the X-ray crystal structure of a phosphodiesterase (PDE) that includes both catalytic and regulatory domains. PDE2A (215-900) crystallized as a dimer in which each subunit had an extended organization of regulatory GAF-A and GAF-B and catalytic domains connected by long ␣-helices. The subunits cross at the GAF-B/ catalytic domain linker, and each side of the dimer contains in series the GAF-A and GAF-B of one subunit and the catalytic domain of the other subunit. A dimer interface extends over the entire length of the molecule. The substrate binding pocket of each catalytic domain is occluded by the H-loop. We deduced from comparisons with structures of isolated, ligand-bound catalytic subunits that the H-loop swings out to allow substrate access. However, in dimeric PDE2A (215-900), the H-loops of the two catalytic subunits pack against each other at the dimer interface, necessitating movement of the catalytic subunits to allow for H-loop movement. Comparison of the unliganded GAF-B of PDE2A (215-900) with previous structures of isolated, cGMP-bound GAF domains indicates that cGMP binding induces a significant shift in the GAF-B/catalytic domain linker. We propose that cGMP binding to GAF-B causes movement, through this linker region, of the catalytic domains, such that the H-loops no longer pack at the dimer interface and are, instead, free to swing out to allow substrate access. This increase in substrate access is proposed as the basis for PDE2A activation by cGMP and may be a general mechanism for regulation of all PDEs.cGMP activation ͉ GAF domains ͉ PDE-2A T he cyclic nucleotides cAMP and cGMP are ubiquitous intracellular signaling molecules that mediate a vast array of biological processes throughout the body. The means by which these two molecules participate in diverse functions, in different cell types and within single cells, involves tight regulation of the spatial and temporal residence of their concentrations. The phosphodiesterases (PDEs) are a superfamily of enzymes that metabolically inactivate cAMP and cGMP (1) to play key roles in both these aspects of regulation. The PDEs are modular enzymes characterized by a relatively conserved C-terminal catalytic domain and a more variable N-terminal domain involved in regulation of activity, subcellular localization, and interactions with other proteins. There are 11 PDE gene families, with different families encoded by one to four genes, and further diversity derived from alternative splicing. The PDE families differ broadly in specificity and affinity for cAMP and cGMP. Much has been learned about the molecular bases for these differences from studies of X-ray crystal structures of the catalytic domains of

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“…In mammalian cells there are at least three known types of cAMP effector proteins: protein kinase A (PKA), exchange proteins activated by cAMP (EPACs), and cyclic nucleotide gated ion channels (CNGs and HCNs), 3 plus a potential fourth target recently identified, Phosphodiesterase type 10. 4 The second messenger is universally generated by adenylyl cyclases (AC; EC 4.6.1.1), enzymes that catalyze the cyclization of ATP to generate cAMP and inorganic pyrophosphate. Nucleotidyl cyclases (i.e.…”

Section: Introductionmentioning

confidence: 99%

Molecular Details of cAMP Generation in Mammalian Cells: A Tale of Two Systems

Kamenetsky

Middelhaufe

Bank

et al. 2006

Journal of Molecular Biology

292

340

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The second messenger cAMP has been extensively studied for half a century, but the plethora of regulatory mechanisms controlling cAMP synthesis in mammalian cells is just beginning to be revealed. In mammalian cells, cAMP is produced by two evolutionary related families of adenylyl cyclases, soluble adenylyl cyclases (sAC) and transmembrane adenylyl cyclases (tmAC). These two enzyme families serve distinct physiological functions. They share a conserved overall architecture in their catalytic domains and a common catalytic mechanism, but they differ in their sub-cellular localizations and responses to various regulators. The major regulators of tmACs are heterotrimeric G proteins, which transduce extracellular signals via G protein-coupled receptors. sAC enzymes, in contrast, are regulated by the intracellular signaling molecules bicarbonate and calcium. Here, we discuss and compare the biochemical, structural and regulatory characteristics of the two mammalian AC families. This comparison reveals the mechanisms underlying their different properties but also illustrates many unifying themes for these evolutionary related signaling enzymes.

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cAMP Is a Ligand for the Tandem GAF Domain of Human Phosphodiesterase 10 and cGMP for the Tandem GAF Domain of Phosphodiesterase 11

Cited by 115 publications

References 39 publications

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

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

Mechanism for the allosteric regulation of phosphodiesterase 2A deduced from the X-ray structure of a near full-length construct

Molecular Details of cAMP Generation in Mammalian Cells: A Tale of Two Systems

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