Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that directs a plethora of elementary physiological processes such as cardiac contractility, blood pressure or body water homeostasis. It is becoming increasingly clear that cAMP acts in a spatially and temporally highly coordinated fashion to confer specificity to cellular signalling processes, which is a prerequisite for normal physiological function. Dysregulation of such compartmentalised cAMP signalling can cause or is associated with diseases such as heart failure, hypertension or various forms of diabetes insipidus.
Our aim is to elucidate molecular mechanisms underlying the coordination of cAMP signalling in the cardiovascular system and to reveal how its dysregulation causes disease. We aim to identify novel targets and small molecule modulators to provide starting points for new strategies towards the treatment of diseases for which a satisfactory treatment is not available, e.g. heart failure or water balance disorders such as diabetes insipidus.
A plethora of extracellular cues such as hormones or neurotransmitters stimulates synthesis or release of an intracellular ubiquitous “second” messenger. Different stimuli can act simultaneously and thereby can even cause an elevation of the same second messenger at the same time. This raises the pivotal question how each of the different stimuli elicits a specific cellular response. It is emerging that second messengers such as cyclic nucleotides, e.g. cyclic adenosine monophosphate (cAMP), or Ca2+ must be spatio-temporally orchestrated to ensure specificity.
Spatio-temporal coordination of cAMP signalling requires several players. Activation of G protein-coupled receptors (GPCRs) through an extracellular cue stimulates adenylyl cyclases (ACs) to synthesise cAMP, whereas strategically positioned phosphodiesterases (PDEs) hydrolyse it. This interplay of cAMP synthesis and degradation establishes gradients and local pools of cAMP. The main effector of cAMP is cAMP-dependent protein kinase (protein kinase A, PKA). The positioning of effectors, in particular of PKA, often involves direct protein-protein interactions of scaffolding proteins like the around 50 A-kinase anchoring proteins (AKAPs). They tether protein complexes to defined cellular compartments. AKAPs possess a conserved PKA binding domain, unique domains for direct interactions with other signalling proteins such as ACs, PDEs, phosphatases, further kinases, and kinase substrates, and anchoring domains that target the protein complex to specific cellular compartments. The direct protein-protein interactions and assembly of compartment-specific protein complexes establishes signalling hubs that provide the specificity that cAMP enables to elicit specific cellular responses.
Physiological processes that depend on such tightly controlled cAMP signalling include cardiac myocyte contractility, blood pressure control in vascular smooth muscle cells and vasopressin-mediated water reabsorption in renal collecting duct principal cells. Vasopressin fine-tunes body water homeostasis.
Our aim is to elucidate the role and the molecular mechanisms underlying the spatio-temporal coordination of cAMP signalling in these processes. Their dysregulation causes or is associated with widespread disease such as heart failure, hypertension or water balance disorders including diabetes insipidus. Since satisfactory treatment is not available for these conditions, elucidating the mechanisms underlying the control of local cAMP signalling may lead to new targets and concepts for their treatment.
Our projects focus on AKAPs and PDEs in the cardiovascular system.
The unifying property of AKAPs is their ability to compartmentalise cAMP signalling. However, they can also interact with proteins involved in various other signalling cascades, and thereby mediate crosstalk between second messengers, e.g. crosstalk between cAMP and Ca2+ signalling. An important goal of the group is the identification of AKAPs and their role in the control of cardiac myocyte contractility and vasopressin-mediated water reabsorption.
We have identified a new member of the AKAP18 family, AKAP18δ, and have shown that it interacts with PKA, SERCA2, phospholamban and a PDE, PDE3A, and that AKAP18δ is involved in the control of cardiac myocyte relaxation by the coordination of this protein complex. The complex mediates Ca2+ reuptake from the cytosol into the sarcoplasmic reticulum, which is an essential event for relaxation and thus diastole to occur.
We have shown that AKAP18δ also coordinates another signalling hub. It consists of AKAP18δ, PKA and another PDE, PDE4D. The complex plays a role in the control of vasopressin-mediated water reabsorption in renal collecting duct principal cells.
Recently, we have shown that the E3 ubiquitin ligase, STUB1 (also termed CHIP), functions as an AKAP and that it forms a complex with PKA, another kinase, CDK18, and the water channel aquaporin-2 (AQP2). This complex resides on intracellular vesicles in renal collecting duct principal cells and is also involved in the control of vasopressin-mediated water reabsorption.
Another AKAP we identified is GSKIP. Our analyses showed that it directly interacts with PKA and the kinase GSK3β and that it coordinates PKA and GSK3β in the control of the canonical Wnt signalling pathway. Wnt signalling controls numerous fundamental biological processes, e.g. in development, in the cell cycle or in immune responses.
We have contributed to characterising functioning of PDEs of the PDE4 family, and uncovered physiological roles. For example, we discovered a protein complex comprising PDE4D, PKA and AKAP18δ. PDE4D in the complex controls local cAMP levels, which is important for tuning water reabsorption in renal collecting duct principal cells.
More recently, we identified mutations in the gene encoding PDE3A in several families that are associated with hypertension and brachydactyly. The mutations cause hyperactivity of PDE3A and thus lower cAMP levels. We can fully reproduce the phenotype in model systems. Thus, an important goal of the group is to understand PDE3-directed local cAMP signalling that controls blood pressure. For this, we investigate vascular smooth muscle cells and cardiac myocytes.
A lack of pharmacological agents to specifically modulate local cAMP signalling limits options to delineate functions of defined processes. Protein-protein interactions are specific and diverse. Their modulation allows for selective interference with cellular functions and disease processes. Therefore, we aim to develop pharmacological agents for interference with defined protein-protein interactions as molecular tools in local cAMP systems, such as those directed by AKAP18δ.
Dysregulation of local cAMP signalling plays a role in various cardiovascular diseases. Our data argue that aberrant PDE3A signalling can cause hypertension with brachydactyly and that aberrant AKAP-PKA interactions are relevant in heart failure. Moreover, vasopressin-mediated water reabsorption depends on AKAP-PKA interactions and is dysregulated in various diseases such as heart failure and diabetes insipidus. There is a huge medical need for innovative strategies for the treatment of such diseases because there is no satisfactory therapy available. We are attempting to identify new targets in local cAMP signalling networks and develop novel small molecules modulating specific protein-protein interactions in disease-relevant networks. Novel small molecules may serve as starting points for the development of drug candidates. Indeed, so far we have identified peptidic and non-peptidic small molecules targeting AKAP-PKA interaction, the interaction between a particular AKAP, AKAP-Lbc and the small GTPase RhoA, and we contributed to the identification of small molecule activators of specific PDE4 isoforms. Moreover, we revealed that fluconazole, an approved antimycotic drug, promotes water reabsorption in renal collecting duct principal cells independent from vasopressin. It may thus have utility for the treatment of various forms of diabetes insipidus.
Our work is kindly supported by the Deutsche Forschungsgemeinschaft (DFG ) through individual grants and within the framework of the Collaborative Research Centre (CRC), The Bundesministerium für Bildung und Forschung (BMBF; Federal Ministry for Education and Science), the German-Israeli Foundation (GIF) and the MDC.