GPR161 structure uncovers the redundant role of sterol-regulated ciliary cAMP signaling in the Hedgehog pathway.

The orphan G protein-coupled receptor (GPCR) GPR161 plays a central role in development by suppressing Hedgehog signaling. The fundamental basis of how GPR161 is activated remains unclear. Here, we determined a cryogenic-electron microscopy structure of active human GPR161 bound to heterotrimeric Gs. This structure revealed an extracellular loop 2 that occupies the canonical GPCR orthosteric ligand pocket. Furthermore, a sterol that binds adjacent to transmembrane helices 6 and 7 stabilizes a GPR161 conformation required for Gs coupling. Mutations that prevent sterol binding to GPR161 suppress Gs-mediated signaling. These mutants retain the ability to suppress GLI2 transcription factor accumulation in primary cilia, a key function of ciliary GPR161. By contrast, a protein kinase A-binding site in the GPR161 C terminus is critical in suppressing GLI2 ciliary accumulation. Our work highlights how structural features of GPR161 interface with the Hedgehog pathway and sets a foundation to understand the role of GPR161 function in other signaling pathways.

GPR161 structure uncovers the redundant role of sterol-regulated ciliary cAMP signaling in the Hedgehog pathway.

The orphan G protein-coupled receptor (GPCR) GPR161 is enriched in primary cilia, where it plays a central role in suppressing Hedgehog signaling1. GPR161 mutations lead to developmental defects and cancers2,3,4. The fundamental basis of how GPR161 is activated, including potential endogenous activators and pathway-relevant signal transducers, remains unclear. To elucidate GPR161 function, we determined a cryogenic-electron microscopy structure of active GPR161 bound to the heterotrimeric G protein complex Gs. This structure revealed an extracellular loop 2 that occupies the canonical GPCR orthosteric ligand pocket. Furthermore, we identify a sterol that binds to a conserved extrahelical site adjacent to transmembrane helices 6 and 7 and stabilizes a GPR161 conformation required for Gs coupling. Mutations that prevent sterol binding to GPR161 suppress cAMP pathway activation. Surprisingly, these mutants retain the ability to suppress GLI2 transcription factor accumulation in cilia, a key function of ciliary GPR161 in Hedgehog pathway suppression. By contrast, a protein kinase A-binding site in the GPR161 C-terminus is critical in suppressing GLI2 ciliary accumulation. Our work highlights how unique structural features of GPR161 interface with the Hedgehog pathway and sets a foundation to understand the broader role of GPR161 function in other signaling pathways.

Tracking and blocking interdependencies of cellular BRAF-MEK oncokinase activities.

The selective targeting of mutated kinases in cancer therapies has the potential to improve therapeutic success and thereby the survival of patients. In the case of melanoma, the constitutively active MAPK pathway is targeted by a combinatorial inhibition of BRAF and MEK activities. These MAPK pathway players may display patient-specific differences in the onco-kinase mutation spectrum, which needs to be considered for the design of more efficient personalized therapies. Here, we extend a bioluminescence-based kinase conformation biosensor (KinCon) to allow for live-cell tracking of interconnected kinase activity states. First, we show that common MEK1 patient mutations promote a structural rearrangement of the kinase to an opened and active conformation. This effect was reversible by the binding of MEK inhibitors to mutated MEK1, as shown in biosensor assays and molecular dynamics simulations. Second, we implement a novel application of the KinCon technology for tracking the simultaneous, vertical targeting of the two functionally linked kinases BRAF and MEK1. Thus, we demonstrate that, in the presence of constitutively active BRAF-V600E, specific inhibitors of both kinases are efficient in driving MEK1 into a closed, inactive conformation state. We compare current melanoma treatments and show that combinations of BRAFi and MEKi display a more pronounced structural change of the drug sensor than the respective single agents, thereby identifying synergistic effects among these drug combinations. In summary, we depict the extension of the KinCon biosensor technology to systematically validate, anticipate, and personalize tailored drug arrangements using a multiplexed setup.

Feedback control of the Gpr161-Gαs-PKA axis contributes to basal Hedgehog repression in zebrafish.

Hedgehog (Hh) ligands act as morphogens to direct patterning and proliferation during embryonic development. Protein kinase A (PKA) is a central negative regulator of Hh signalling, and in the absence of Hh ligands, PKA activity prevents inappropriate expression of Hh target genes. The orphan G-protein-coupled receptor Gpr161 contributes to the basal Hh repression machinery by activating PKA. Gpr161 acts as an A-kinase-anchoring protein, and is itself phosphorylated by PKA, but the functional significance of PKA phosphorylation of Gpr161 in the context of Hh signalling remains unknown. Here, we show that loss of Gpr161 in zebrafish leads to constitutive activation of medium and low, but not maximal, levels of Hh target gene expression. Furthermore, we find that PKA phosphorylation-deficient forms of Gpr161, which we show directly couple to Gαs, display an increased sensitivity to Shh, resulting in excess high-level Hh signalling. Our results suggest that PKA feedback-mediated phosphorylation of Gpr161 may provide a mechanism for fine-tuning Gpr161 ciliary localisation and PKA activity.

Mutation-oriented profiling of autoinhibitory kinase conformations predicts RAF inhibitor efficacies.

Kinase-targeted therapies have the potential to improve the survival of patients with cancer. However, the cancer-specific spectrum of kinase alterations exhibits distinct functional properties and requires mutation-oriented drug treatments. Besides post-translational modifications and diverse intermolecular interactions of kinases, it is the distinct disease mutation which reshapes full-length kinase conformations, affecting their activity. Oncokinase mutation profiles differ between cancer types, as it was shown for BRAF in melanoma and non-small-cell lung cancers. Here, we present the target-oriented application of a kinase conformation (KinCon) reporter platform for live-cell measurements of autoinhibitory kinase activity states. The bioluminescence-based KinCon biosensor allows the tracking of conformation dynamics of full-length kinases in intact cells and real time. We show that the most frequent BRAF cancer mutations affect kinase conformations and thus the engagement and efficacy of V600E-specific BRAF inhibitors (BRAFi). We illustrate that the patient mutation harboring KinCon reporters display differences in the effectiveness of the three clinically approved BRAFi vemurafenib, encorafenib, and dabrafenib and the preclinical paradox breaker PLX8394. We confirmed KinCon-based drug efficacy predictions for BRAF mutations other than V600E in proliferation assays using patient-derived lung cancer cell lines and by analyzing downstream kinase signaling. The systematic implementation of such conformation reporters will allow to accelerate the decision process for the mutation-oriented RAF-kinase cancer therapy. Moreover, we illustrate that the presented kinase reporter concept can be extended to other kinases which harbor patient mutations. Overall, KinCon profiling provides additional mechanistic insights into full-length kinase functions by reporting protein-protein interaction (PPI)-dependent, mutation-specific, and drug-driven changes of kinase activity conformations.

KinCon: Cell-based recording of full-length kinase conformations.

The spectrum of kinase alterations displays distinct functional characteristics and requires kinase mutation-oriented strategies for therapeutic interference. Besides phosphotransferase activity, protein abundance, and intermolecular interactions, particular patient-mutations promote pathological kinase conformations. Despite major advances in identifying lead molecules targeting clinically relevant oncokinase functions, still many kinases are neglected and not part of drug discovery efforts. One explanation is attributed to challenges in tracking kinase activities. Chemical probes are needed to functionally annotate kinase functions, whose activities may not always depend on catalyzing phospho-transfer. Such non-catalytic kinase functions are related to transitions of full-length kinase conformations. Recent findings underline that cell-based reporter systems can be adapted to record conformation changes of kinases. Here, we discuss the possible applications of an extendable kinase conformation (KinCon) reporter toolbox for live-cell recording of kinase states. KinCon is a genetically encoded bioluminescence-based biosensor platform, which can be subjected for measurements of conformation dynamics of mutated kinases upon small molecule inhibitor exposure. We hypothesize that such biosensors can be utilized to delineate the molecular modus operandi for kinase and pseudokinase regulation. This should pave the path for full-length kinase-targeted drug discovery efforts aiming to identify single and combinatory kinase inhibitor therapies with increased specificity and efficacy.

Hedgehog and Gpr161: Regulating cAMP Signaling in the Primary Cilium.

Compartmentalization of diverse types of signaling molecules contributes to the precise coordination of signal propagation. The primary cilium fulfills this function by acting as a spatiotemporally confined sensory signaling platform. For the integrity of ciliary signaling, it is mandatory that the ciliary signaling pathways are constantly attuned by alterations in both oscillating small molecules and the presence or absence of their sensor/effector proteins. In this context, ciliary G protein-coupled receptor (GPCR) pathways participate in coordinating the mobilization of the diffusible second messenger molecule 3',5'-cyclic adenosine monophosphate (cAMP). cAMP fluxes in the cilium are primarily sensed by protein kinase A (PKA) complexes, which are essential for the basal repression of Hedgehog (Hh) signaling. Here, we describe the dynamic properties of underlying signaling circuits, as well as strategies for second messenger compartmentalization. As an example, we summarize how receptor-guided cAMP-effector pathways control the off state of Hh signaling. We discuss the evidence that a macromolecular, ciliary-localized signaling complex, composed of the orphan GPCR Gpr161 and type I PKA holoenzymes, is involved in antagonizing Hh functions. Finally, we outline how ciliary cAMP-linked receptor pathways and cAMP-sensing signalosomes may become targets for more efficient combinatory therapy approaches to counteract dysregulation of Hh signaling.

LAMTOR/Ragulator is a negative regulator of Arl8b- and BORC-dependent late endosomal positioning.

Signaling from lysosomes controls cellular clearance and energy metabolism. Lysosomal malfunction has been implicated in several pathologies, including neurodegeneration, cancer, infection, immunodeficiency, and obesity. Interestingly, many functions are dependent on the organelle position. Lysosomal motility requires the integration of extracellular and intracellular signals that converge on a competition between motor proteins that ultimately control lysosomal movement on microtubules. Here, we identify a novel upstream control mechanism of Arl8b-dependent lysosomal movement toward the periphery of the cell. We show that the C-terminal domain of lyspersin, a subunit of BLOC-1-related complex (BORC), is essential and sufficient for BORC-dependent recruitment of Arl8b to lysosomes. In addition, we establish lyspersin as the linker between BORC and late endosomal/lysosomal adaptor and mitogen activated protein kinase and mechanistic target of rapamycin activator (LAMTOR) complexes and show that epidermal growth factor stimulation decreases LAMTOR/BORC association, thereby promoting BORC- and Arl8b-dependent lysosomal centrifugal transport.

Gpr161 anchoring of PKA consolidates GPCR and cAMP signaling.

Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein-protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein-protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions.

In-vivo detection of binary PKA network interactions upon activation of endogenous GPCRs.

Membrane receptor-sensed input signals affect and modulate intracellular protein-protein interactions (PPIs). Consequent changes occur to the compositions of protein complexes, protein localization and intermolecular binding affinities. Alterations of compartmentalized PPIs emanating from certain deregulated kinases are implicated in the manifestation of diseases such as cancer. Here we describe the application of a genetically encoded Protein-fragment Complementation Assay (PCA) based on the Renilla Luciferase (Rluc) enzyme to compare binary PPIs of the spatially and temporally controlled protein kinase A (PKA) network in diverse eukaryotic model systems. The simplicity and sensitivity of this cell-based reporter allows for real-time recordings of mutually exclusive PPIs of PKA upon activation of selected endogenous G protein-coupled receptors (GPCRs) in cancer cells, xenografts of mice, budding yeast, and zebrafish embryos. This extends the application spectrum of Rluc PCA for the quantification of PPI-based receptor-effector relationships in physiological and pathological model systems.