N-terminal tandem GAF domains are present in 5 out of 11 mammalian phosphodiesterase (PDE) families. The ligand for the GAF domains of PDEs 2, 5, and 6 is cGMP, whereas those for PDEs 10 and 11 remained enigmatic for years. Here we used the cyanobacterial cyaB1 adenylyl cyclase, which has an N-terminal tandem GAF domain closely related to those of the mammalian PDEs, as an assay system to identify the ligands for the human PDEs 10 and 11 GAF domains. We report that a chimera between the PDE10 GAF domain and the cyanobacterial cyclase was 9-fold stimulated by cAMP (EC 50 ؍ 19.8 M), whereas cGMP had only low activity. cAMP increased V max in a non-cooperative manner and did not affect the K m for ATP of 27 M. In an analogous chimeric construct with the tandem GAF domain of human PDE11A4, cGMP was identified as an allosteric activator (EC 50 ؍ 72.5 M) that increased V max of the cyclase non-cooperatively 4-fold. GAF-B of PDE10 and GAF-A of PDE11A4 contain an invariant NKFDE motif present in all mammalian PDE GAF ensembles. We mutated the aspartates within this motif in both regions and found that intramolecular signaling was considerably reduced or abolished. This was in line with all data concerning GAF domains with an NKFDE motif as far as they have been tested. The data appeared to define those GAF domains as a distinct subclass within the >3100 annotated GAF domains for which we propose a tentative classification scheme.
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 M 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.
Research in the field of innovation is very important, because innovations are necessary prerequisites for the success of companies. Innovation management has to be executed in consideration of external influences (environment) and internal influences (company). The research topic is about optimizing the budget allocation for innovation management. The main research question is how to decide between the two basic options: exploitation and exploration. A promising way is to balance these two antipodes. One extent is to do research and development to explore new knowledge and create basic innovations as well as to use new technologies to invent new products (exploration). The other extent is to fully utilize and exploit existing possibilities, potential, and technologies (exploitation). It is a crucial management task to find the right balance between these two proceedings. To answer the research question is how to balance exploration and exploitation in an adequate way. The applied method is literature research and analytical development of a tool, which should help to find the right management decision about innovation strategy and budget allocation. This article is a conceptual paper with a deductive approach, in which the framework of variables influencing the decision about exploration and exploitation is introduced and in which the Innovation-Alignment-Portfolio (IAP) as a tool to substantiate the decision between the two antipodes is presented. As a result, the specific contribution to academic discussion, based on critical literature review, is twofold: firstly, deployment of a systematic framework of antecendents and tradeoffs influencing the management decision. Secondly, the development and introduction of the Innovation-Alignment-Portfolio. The structure of the paper consists of three main parts. The first part is about the introduction and problem formulation. In the second part, a critical literature review is done and leads to the research results, which are summarized in the framework of innovation management. Factors of influence are analyzed and discussed. In the third part, the Innovation-Alignment-Portfolio is introduced as a tool to balance exploration and exploitation. The conclusion of this paper is that decisions about innovation strategies can be made in a more differentiated way by using the Innovation-Alignment-Portfolio.
In mammals, cGMP is generated by soluble and membrane-bound guanylyl cyclases and can be degraded by altogether eight out of 11 mammalian PDE families. Apart from cGMP-activated protein kinases and from cGMPgated cation channels 5 of the 11 PDE families are also subject to regulation by cNMP via their N-terminal tandem GAF ensembles. Four, PDE 2, 5, 6, and 11 appear to be regulated by cGMP; PDE10 is similarly regulated by cAMP. Thus, the cyclic nucleotides act as allosteric activators which enhance their own degradation at the catalytic site, i.e. they concomitantly serve as modulators of enzyme activity and as substrates. This creates a biochemical conundrum which cannot be disentangled kinetically. We use a cyanobacterial adenylyl cyclase, cyaB1, as a reporter enzyme to characterize intramolecular GAF domain signalling. 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. in a non-physiological concentration range. However, shortening the 196 aa long N-terminus by 177 aa lowers the EC50 for cGMP to 3.5 μM, well in a physiologically meaningful range. A cysteine protease present in human tissues such as prostate, kidney, bladder, skeletal muscle, heart, and uterus restricts the N-terminus of PDE11 and may represent an additional layer of PDE regulation.In PDE10 the allosteric activator is cAMP whereas in all other mammalian PDEs of this sub-family cGMP serves as an activator. Mutational studies indicated that the PDE 10 GAF tandem domain signals via its GAF-B region like PDE2 whereas the GAF tandem of PDE5 signals via its GAF-A region. We generated hybrid chimeras between the GAF tandems of PDE 5, binding cGMP, and PDE 10, binding cAMP, in order to investigate whether we would obtain a GAF tandem capable of signalling by cGMP via GAF A and by cAMP via GAF B. This was not unequivocally accomplished. However, we demonstrate that the mode of signalling appears to be strongly affected by the source of the α-helix which connects the GAF-A and GAF-B regions. A hybrid construct comprised of the N-terminal and GAF-A from PDE5 and the linker and GAF-B from PDE10 could be stimulated by cAMP and, to a lesser extent, by cGMP. However, a similar construct in which the linker between GAF-A (from PDE5) and B (from PDE10) was from the PDE 5 was unresponsive to both, cGMP and cAMP. Apart from the unresolved question how in the GAF domains cNMP specificity is encoded this highlights the importance of the linker region for the formation of a functional ...
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