Parallel Allostery by cAMP and PDE Coordinates Activation and Termination Phases in cAMP Signaling

Abstract

The second messenger molecule cAMP regulates the activation phase of the cAMP signaling pathway through high-affinity interactions with the cytosolic cAMP receptor, the protein kinase A regulatory subunit (PKAR). Phosphodiesterases (PDEs) are enzymes responsible for catalyzing hydrolysis of cAMP to 5' AMP. It was recently shown that PDEs interact with PKAR to initiate the termination phase of the cAMP signaling pathway. While the steps in the activation phase are well understood, steps in the termination pathway are unknown. Specifically, the binding and allosteric networks that regulate the dynamic interplay between PKAR, PDE, and cAMP are unclear. In this study, PKAR and PDE from Dictyostelium discoideum (RD and RegA, respectively) were used as a model system to monitor complex formation in the presence and absence of cAMP. Amide hydrogen/deuterium exchange mass spectrometry was used to monitor slow conformational transitions in RD, using disordered regions as conformational probes. Our results reveal that RD regulates its interactions with cAMP and RegA at distinct loci by undergoing slow conformational transitions between two metastable states. In the presence of cAMP, RD and RegA form a stable ternary complex, while in the absence of cAMP they maintain transient interactions. RegA and cAMP each bind at orthogonal sites on RD with resultant contrasting effects on its dynamics through parallel allosteric relays at multiple important loci. RD thus serves as an integrative node in cAMP termination by coordinating multiple allosteric relays and governing the output signal response.

The Role of Allostery in the Termination of Second Messenger Signaling

“…This remarkable scenario fits well the HDXMS data (6). Importantly, the substrate channeling model of Krishnamurthy et al (3,6) implies that PDE alone cannot efficiently hydrolyze cAMP.…”

“…This remarkable scenario fits well the HDXMS data (6). Importantly, the substrate channeling model of Krishnamurthy et al (3,6) implies that PDE alone cannot efficiently hydrolyze cAMP. Measurements of the catalytic k cat and K m of PDE (9) in the presence and absence of R D could verify the critical role of allostery in cAMP hydrolysis.…”

“…Kinetic data might indicate the possibility of an alternative allosteric activation taking place through a conformational change in PDE elicited by R D binding. The data in Krishnamurthy et al (6) clearly illustrate that in the absence of cAMP, PDE does not bind R D . This fact tells us that one function of the allosteric interaction between PDE and R D binding is to ensure that PDE (even if it is already expressed in the cell) is precisely activated only when the cAMP level is high.…”

“…In this issue of the Biophysical Journal, Krishnamurthy et al (6) focus on the significant question of how R D :PDE complex formation and dissociation take place, illustrating for the first time, to our knowledge, a mechanism through which cAMP signaling can be terminated in a timely manner through allostery. Amide hydrogen/ deuterium exchange mass spectrometry (HDXMS) (7,8) is a powerful conformational probe tool for mapping allosteric signaling, identifying flexible and disordered regions.…”

“…In this issue of the Biophysical Journal, Krishnamurthy et al (6) provide a unique, in-depth view of the termination step for cAMP signaling by the cytosolic cAMP receptor, protein kinase A (PKA) regulatory subunit, carried out by phosphodiesterase (PDE). PKA regulatory subunit and PDE from Dictyostelium discoideum (R D and RegA, respectively) were used as a model system.…”