Functional chimeras of the phosphodiesterase 5 and 10 tandem GAF domains.
The tandem GAF domain of hPDE10A uses cAMP as an allosteric ligand (Gross-Langenhoff, M., Hofbauer, K., Weber, J., Schultz, A., and Schultz, J. E. (2006) J. Biol. Chem. 281, 2841-2846). We used a two-pronged approach to study how discrimination of ligand is achieved in human (h)PDE10A and how domain selection in the phosphodiesterase GAF tandems is determined. First, we examined which functional groups of cAMP are responsible for purine ring discrimination. Changes at the C-6 ring position (removal of the amino group; chloride substitution) and at the N-1 ring position reduced stimulation efficacy by 80%, i.e. marking those positions as decisive for nucleotide discrimination. Second, we generated a GAF tandem chimera that consisted of the cGMP-binding GAF-A unit from hPDE5A1, which signals through cGMP in PDE5, and the GAF-B from hPDE10A1, which signals through cAMP in PDE10. Stimulation of the reporter enzyme exclusively was through the GAF-B domain of hPDE10A1 (EC(50) = 7 microm cAMP) as shown by respective point mutations. The PDE5 GAF-A domain in the chimera did not signal, and its function was reduced to a strictly structural role. Signaling was independent of the origin of the N terminus. Generating 10 additional PDE5/10 tandem GAF chimeras surprisingly demonstrated that the length-conserved linker in GAF tandems between GAF-A and GAF-B played an unforeseen decisive role in intramolecular signaling. Swapping the linker sections between PDE5 and PDE10 GAF tandem domains abrogated signaling completely pointing to specific domain interactions within GAF tandems, which are not visible in the available crystal structures with bound ligands.
Hofbauer
Karina
K
Pharmazeutisches Institut, Fakultät für Chemie und Pharmazie, Morgenstelle, Universität Tübingen, 72076 Tübingen, Germany.
Schultz
Anita
A
Pharmazeutisches Institut, Fakultät für Chemie und Pharmazie, Morgenstelle, Universität Tübingen, 72076 Tübingen, Germany.
Schultz
Joachim E
JE
Pharmazeutisches Institut, Fakultät für Chemie und Pharmazie, Morgenstelle, Universität Tübingen, 72076 Tübingen, Germany. Electronic address: joachim.schultz@uni-tuebingen.de.
D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D000242:Cyclic AMP / Q000737:chemistry; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5 / Q000737:chemistry*; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry*; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000242:Cyclic AMP; C119843:PDE10A protein, human; D010727:Phosphoric Diester Hydrolases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5