Compartmentalization of GPCR-PKA signalling

Content of research project: Scaffolding proteins organize the cellular information flow from activated cell-surface receptors to intracellular effector cascades. A variety of G protein-coupled receptor (GPCR) bound scaffolding proteins recruit cytoplasmic downstream effector molecules to convert, propagate, and amplify the incoming signal leading to a physiological or pathological cell response. In a proof-of-concept study we determined the macromolecular organization of GPCR-controlled protein kinase A (PKA) complexes from osteosarcoma cells. Besides established kinase:scaffolding protein interactions (with A-kinase anchoring proteins, AKAP) we unexpectedly identified a binary protein:protein interaction between PKA and a specific GPCR. Hypotheses: We identified the first GPCR with intrinsic AKAP scaffolding function. GPR161 recruits the cAMP-sensing PKA to the primary cilium compartment (Bachmann et al., PNAS 2016). We hypothesize that complex formation between the receptor and PKA controls compartmentalized cAMP fluxes in the primary cilium where the cAMP-sensing kinase activity is critically involved in antagonistic Hedgehog (Hh) signaling. We assume that specific PKA regulatory and catalytic subunits interact with the carboxy-terminus of GPR161. We showed that GPR161 phosphorylation and PKA interaction affect kinase compartmentalization to the cilium. We propose that this complex acts as critical factor for Hh-mediated signal transmission in the primary cilium compartment. The detailed structural description of the GPR161:PKA interaction will unveil mechanistic details of complex formation. The characterization of further PKA associated components would describe general principles of GPCR/kinase signal integration through-cell specific PKA signalosomes. Methods: Cell-based reporter assays, kinase activity assays, structure determination, transcription factor activity measurements, biochemical interaction studies, protein chemistry, mass spectrometry, cell biology of the primary cilium, functional experiments using zebra fish embryos, systems biology based approaches, theoretical chemistry approaches. What is new and/or special about the project? So far a collection of interjacent scaffolding proteins have been described that physically link and compartmentalize PKA activity and GPCR functions. We published that one GPCR by itself contains intrinsic AKAP functions. This new receptor feature consolidates both, GPCR activities and cAMP-controlled PKA functions. PKA and GPR161 are critically involved in compartmentalized signal transduction in the primary cilium. We assume that compartmentalization GPR161:PKA signalosome act as signal integrator for additional cAMP-linked receptor pathways. Characterization and perturbation of the GPR161:PKA interaction will be relevant to understand and pharmaceutically target pathological signaling involving Hh signaling pathways and deregulated PKA activities. We assume that the cell-specific organization of PKA signalosomes regulate and organize interrelated GPCR and PKA functions. We combine affinity isolations with a subtractive phosphoproteomics approach to identify proliferation- relevant PKA interactors and/or substrates which are constituents of cell-specific PKA signalosomes and involved in colon cancer cell proliferation. Main researcher: Eduard STEFAN, PhD

Compartmentalization of signaling molecules participates in the precise coordination of cellular signal propagation. The primary cilium accomplishes this key function by acting as a spatiotemporally restricted signal-sensing platform. For the integrity of ciliary signaling, it is mandatory that the involved signaling pathways are precisely regulated through the adjustment of oscillating small molecules and the presence or absence of their effector molecules. Ciliary G protein-coupled receptor (GPCR) pathways participate in coordinating alterations of cAMP, a second messenger key molecule. In the cilium compartment cAMP fluctuations are sensed and propagated through protein kinase A (PKA) complexes. Locally restricted activities of PKA signalosomes are indispensable for the basal repression of Hedgehog (Hh) signaling. In this project we analyzed key aspects of the underlying signaling circuits, second messenger compartmentalization and the involvement of kinase interacting GPCRs. First, we showed that a macromolecular and ciliary-localized signaling complex, composed of the orphan GPCR Gpr161 and type I PKA holoenzymes, is involved in antagonizing Hh functions. The ciliary localized and cAMP-linked receptor pathway and the participating kinase signalosome are feasible targets to counteract Hh dysregulation, as found in diseases such as cancer. Second, using cell-based reporter and phospho-proteomics analyses, we identified new modes of kinase counter-regulation of signaling pathways related to the ubiquitin proteasome system. We observed that cAMP elevations affect cilia resorption. Thus, PKA substrates seem to contribute to cilium formation and its removal. In this case the respective signalosome is composed of a multimeric protein complex that includes two kinases and the ubiquitin proteasome system. We present and analyzed examples how compartmentalized enzyme activities contribute to ciliogenesis. Third, we analyzed kinase regulations on the molecular level. Using both, newly generated biosensors and established read-outs for GPCR activation, we have characterized Gpr161 signal transmission and general concepts of cell-based kinase regulation and perturbation. Overall, we discovered new examples how compartmentalized kinase signaling platforms are controlled and altered through post-translational modifications and defined bioactive small molecule binding. Insights into the molecular mechanism of binary kinase interactions on the cellular level could become relevant for identifying new means for perturbations of pathological receptor and effector pathways.