Intramolecular signaling in tandem-GAF domains from PDE5 and PDE10 studied with a cyanobacterial adenylyl cyclase reporter

• Published in: Cellular signalling Vol. 24; no. 3; pp. 629 - 634
• Main Authors: Banjac, Ana, Zimmermann, Markus O., Boeckler, Frank M., Kurz, Ursula, Schultz, Anita, Schultz, Joachim E.
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 01.03.2012
• Subjects: Adenylyl cyclase; Adenylyl Cyclases - genetics; Adenylyl Cyclases - metabolism; Amino Acid Sequence; Amino Acid Substitution; Chimeras; Cyanobacteria - enzymology; Cyclic Nucleotide Phosphodiesterases, Type 5 - chemistry; Cyclic Nucleotide Phosphodiesterases, Type 5 - genetics; Cyclic Nucleotide Phosphodiesterases, Type 5 - metabolism; GAF tandem domain; Genes, Reporter; Humans; Molecular Sequence Data; Phosphodiesterases; Phosphoric Diester Hydrolases - chemistry; Phosphoric Diester Hydrolases - genetics; Phosphoric Diester Hydrolases - metabolism; Protein Structure, Tertiary; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Signal Transduction; Signaltransduction; AC; Adenylyl cyclase; hPDE; GAF tandem domain; Chimeras; Phosphodiesterases; Signaltransduction
• ISSN: 0898-6568; 1873-3913
• DOI: 10.1016/j.cellsig.2011.10.013

The dimeric mammalian phosphodiesterases (PDEs) are regulated by N-terminal domains. In PDE5, the GAF-A subdomain of a GAF-tandem (GAF-A and -B) binds the activator cGMP and in PDE10 GAF-B binds cAMP. GAF-tandem chimeras of PDE5 and 10 in which the 36 aa linker helix between GAF-A and -B was swapped lost allosteric regulation of a reporter adenylyl cyclase. In 16 consecutive constructs we substituted the PDE10 linker with that from PDE5. An initial stretch of 10 amino acids coded for isoform specificity. A C240Y substitution uncoupled cyclase activity from regulation, whereas C240F, L or G did not. The C240Y substitution increased basal activity to stimulated levels. Notably, over the next 12 substitutions basal cyclase activity decreased linearly. Further targeted substitutions were based on homology modeling using the PDE2 structure. No combination of substitutions within the initial 10 linker residues caused loss of regulation. The full 10 aa stretch was required. Modeling indicated a potential interaction of the linker with a loop from GAF-A. To interrupt H-bonding a glycine substitution of the loop segment was generated. Despite reduction of basal activity, loss of regulation was maintained. Possibly, the orientation of the linker helix is determined by formation of the dimer at the initial linker segment. Downstream deflections of the linker helix may have caused loss of regulation. ►Sequence specificity of linker between GAF-A and B in phosphodiesterases 5 and 10. ►Modeling of linker region along structure of phosphodiesterase 2. ►Linker mutations identify structural requirements for intramolecular signaling.