A new mode of cAMP signalling: the adenylyl cyclase-IP3 receptor junction

All cells separate themselves from their surroundings by wrapping themselves in an impermeable barrier, the plasma membrane, without which it would be impossible to maintain an intracellular composition distinct from that outside. But the plasma membrane is also a barrier to essential communication with the surroundings. Receptor proteins that span the plasma membrane overcome this problem by transmitting information across the barrier. These receptors selectively bind chemicals in the extracellular environment, change their shape, and the re-shaped intracellular part of the protein can then interact with intracellular proteins to initiate signalling cascades that lead to changes in cellular activity. Very commonly these protein-protein interactions at the plasma membrane lead to production of soluble intracellular chemicals, second messengers, that convey information to many different intracellular targets. The most remarkable feature of these signalling cascades is their economy: thousands of different extracellular stimuli, detected by a similar number of receptors converge to regulate the intracellular concentration of just a handful of second messengers. These then selectively regulate many hundreds of different cellular processes, including release of neurotransmitters, contraction, gene expression and cell growth and death. Defects within these signalling pathways are causes of common diseases and the proteins involved are among the most successful of drug targets. How can so few second messengers function to allow specific communication between so many extracellular stimuli and so many cellular responses? Three important features contribute to this versatility. First the second messengers, like Ca2+ and cAMP, are spatially organized within cells: an increase in Ca2+ concentration in one part of a cell can evoke very different effects to the same increase elsewhere. Second, interactions between signalling pathways allow complex information processing, such that a response may occur only when 2 pathways are simultaneously active. Third, signalling proteins, like the adenylyl cyclase that makes cAMP, come in different hues (subtypes) that differ in their regulation and interactions with additional proteins. Our understanding of the interplay between these features and their significance for reliable information processing is still in its infancy but there is real promise of new opportunities for therapeutic intervention. Our work is concerned with 2 ubiquitous signalling proteins: adenylyl cyclase (AC) and IP3 receptors (IP3R). The latter mediate most Ca2+ signals evoked by extracellular stimuli in animal cells. Our recent work suggests an entirely unexpected mode of communication between these 2 proteins, with cAMP passing directly from a specific subtype of AC (AC6) to a specific subtype of IP3R (IP3R2) to increase its sensitivity to IP3. We suggest that these signalling junctions, which may be a universal feature of second messenger pathways, allow robust signalling between the plasma membrane and intracellular proteins and autonomous processing of information within each junction. Our work seeks to identify the novel cAMP-binding site on the IP3R, to define the composition and structure of the AC6-IP3R junction, its regulation and its contribution to differential decoding of cAMP signals by the proteins that respond to it. Our work addresses a fundamental issue in cell biology, how information passes reliably from the plasma membrane to the cell interior, and it paves the way to identifying novel future drug targets.

Ca2+ and cAMP are ubiquitous intracellular messengers and interactions between them are common and important. We recently established that cAMP passes directly between type 6 adenylyl cyclase (AC6) to type 2 IP3R (IP3R2) directly sensitizing it to IP3 without involvement of any known mediator of cAMP action. Aside from identifying a new target for cAMP (IP3R), our results also revealed a new mode of cAMP signalling: the AC-IP3R junction, wherein cAMP is delivered at a super-saturating concentration directly from the enzyme that makes it (AC) to a tightly associated target (IP3R). We suggest that such junctions allow robust signalling, independent regulation of autonomous signalling junctions, and reciprocal interplay within individual junctions that might give rise to entirely local oscillations in cAMP and/or Ca2+. We speculate that such junctions may be more general and perhaps also mediate communication between phospholipase C and IP3R. This proposal aims to identify the novel cAMP-binding site that regulates IP3R, to establish the structural determinants of the IP3R-AC junction, its subcellular distribution and whether junctions are pre-assembled or assemble 'on demand' in response to extracellular stimuli. We aim to establish whether receptors coupled to AC can differentially regulate IP3R (via junctions) and protein kinase A. Finally, we aim to extend our analysis of AC-IP3R junctions to establish whether a similar mode of signal operates between phospholipase C and IP3R.

Training and skills Staff are encouraged to develop the skills and experience required to become independent. This involves engaging fully with every aspect of the project from developing proposals, managing budgets, reviewing and developing research programmes, to preparing publications and presenting work at meetings. All staff apply a range of state-of-the art techniques directly or via close collaboration. Staff are expected to gain experience of teaching by supervising project/PhD students, teaching practical classes and in a lecture on advanced techniques to PhD/final year students. All staff contribute fully to weekly lab meetings, where they present their work and critically evaluate the work of others. In my absence, lab meetings are chaired by postdocs. A major impact is the proven ability of my lab to train staff to be well-equipped to meet the future needs of industry and/or academia. International and interdisciplinary interactions BBSRC has identified international collaboration as a policy priority. Interactions with international partners is a strength of my lab. This allows mutual exchange of expertise. We have established links with Hasan in India (Bangalore, Drosophila neurogenetics applied to IP3R), Yaras in Turkey (Antalya, electrophysiological analysis of beta-cells), Falcke in Germany (Berlin, stochastic modelling), and Marchant in USA (Minnesota, optical single molecule analyses). Several short-term visitors from China, S America and Europe have also spent time in my lab. Within the UK, we have collaborated for > 20 years with Potter (Bath, medicinal chemistry). Many impacts arise from these diverse interactions. They encourage interactions at the boundaries between disciplines to the advantage of both parties and their research programmes. By fostering extensive international interactions, they help ensure that the UK remains fully engaged with a world-wide science community. Public understanding and schools Every year, under the auspices of the Nuffield Foundation, we accommodate 1-2 students for 6-8 weeks, during which they undertake a practical project. These placements allow bright students to experience science first-hand before they finalise their choice of university subject. Staff contribute to the Cambridge Science Festival, which attracts thousands of visitors. Typically, the contribution involves a hands-on demonstration of the actions of common drugs on waterfleas. We provide occasional ad hoc visits to local schools providing practical experience of, for example, insect biology and microscopy. Aside from publishing our research and presenting it at meetings, we work with press offices to maximize the impact of our more high profile publications. For our most recent paper in Nature, for example, we worked closely with staff at BBSRC Press Office to ensure wide coverage. We regard this as an important opportunity to bring the significance of fundamental research to the attention of a wide audience. The impacts of these activities are to encourage interest in science from pre-university students and to facilitate wide appreciation of the importance of addressing fundamental questions in biology. Health and wealth Our analyses of signalling junctions may eventually reveal opportunities selectively to disrupt the links between specific receptors and ubiquitous signalling pathways. A more immediate prospect is that we may succeed, through analyses of structure-activity relationships, in identifying/synthesizing a cAMP-related ligand that selectively interacts with IP3R. This would be a valuable tool for research and a starting point for drugs that might interfere with IP3R in vivo. The significance of the latter for treatment of specific diseases awaits our unravelling of the role of AC-IP3R junctions in native tissues. The impact of these findings, in the longer term, may be to provide pharmaceutical companies with a lead drug for selective disruption of IP3R activation.