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Blocking the interaction between cell-surface receptors and their ligands is a proven therapeutic strategy. Adhesion G protein-coupled receptors (aGPCRs) are key cell-surface receptors that regulate numerous pathophysiological processes, and their large extracellular regions (ECRs) mediate ligand binding and function. The aGPCR GPR56/ADGRG1 regulates central nervous system myelination and melanoma progression by interacting with its ligand, tissue transglutaminase 2 (TG2), but the molecular basis for this interaction is largely undefined. Here, we show that the C-terminal portion of TG2 directly interacted with the GPR56 ECR with high-nanomolar affinity, and used site-directed mutagenesis to identify a patch of conserved residues on the pentraxin/laminin-neurexin-sex-hormone-binding-globulin-like (PLL) domain of GPR56 as the TG2 binding site. Importantly, we also show that the GPR56-TG2 interaction was blocked by previously-reported synthetic proteins, termed monobodies, that bind the GPR56 ECR in a domain- and species-specific manner. This work provides unique tools to modulate aGPCR-ligand binding and establishes a foundation for the development of aGPCR-targeted therapeutics.

Summary: Adhesion G-protein-coupled receptors (aGPCRs) play critical roles in diverse cellular processes in neurobiology, development, immunity, and numerous diseases. The lack of molecular understanding of their activation mechanisms, especially with regard to the transmembrane domains, hampers further studies to facilitate aGPCR-targeted drug development. Latrophilin-1/ADGRL1 is a model aGPCR that regulates synapse formation and embryogenesis, and its mutations are associated with cancer and attention-deficit/hyperactivity disorder. Here, we established functional assays to monitor latrophilin-1 function and showed the activation of latrophilin-1 by its endogenous agonist peptide. Via a comprehensive mutagenesis screen, we identified transmembrane domain residues essential for latrophilin-1 basal activity and for agonist peptide response. Strikingly, a cancer-associated mutation exhibited increased basal activity and failed to rescue the embryonic developmental phenotype in transgenic worms. These results provide a mechanistic foundation for future aGPCR-targeted drug design. : Molecular Biology; Membrane Architecture; Protein Structure Aspects Subject Areas: Molecular Biology, Membrane Architecture, Protein Structure Aspects

Adhesion G protein-coupled receptors (aGPCRs) play critical roles in diverse biological processes, including neurodevelopment and cancer progression. aGPCRs are characterized by large and diverse extracellular regions (ECRs) that are autoproteolytically cleaved from their membrane-embedded signaling domains. Although ECRs regulate receptor function, it is not clear whether ECRs play a direct regulatory role in G-protein signaling or simply serve as a protective cap for the activating “Stachel” sequence. Here, we present a mechanistic analysis of ECR-mediated regulation of GPR56/ADGRG1, an aGPCR with two domains [pentraxin and laminin/neurexin/sex hormonebinding globulin-like (PLL) and G protein-coupled receptor autoproteolysis-inducing (GAIN)] in its ECR. We generated a panel of high-affinity monobodies directed to each of these domains, from which we identified activators and inhibitors of GPR56-mediated signaling. Surprisingly, these synthetic ligands modulated signaling of a GPR56 mutant defective in autoproteolysis and hence, in Stachel peptide exposure. These results provide compelling support for a ligand-induced and ECR-mediated mechanism that regulates aGPCR signaling in a transient and reversible manner, which occurs in addition to the Stachel-mediated activation.

Synthetic binding proteins are constructed using nonantibody molecular scaffolds. Over the last two decades, in-depth structural and functional analyses of synthetic binding proteins have improved combinatorial library designs and selection strategies, which have resulted in potent platforms that consistently generate binding proteins to diverse targets with affinity and specificity that rival those of antibodies. Favorable attributes of synthetic binding proteins, such as small size, freedom from disulfide bond formation and ease of making fusion proteins, have enabled their unique applications in protein science, cell biology and beyond. Here, we review recent studies that illustrate how synthetic binding proteins are powerful probes that can directly link structure and function, often leading to new mechanistic insights. We propose that synthetic proteins will become powerful standard tools in diverse areas of protein science, biotechnology and medicine.

A new algorithm automatically determines the structures of large macromolecules of unknown fold from low-resolution single-wavelength anomalous X-ray data and a partial model that failed with other methods., Determining macromolecular structures from X-ray data with resolution worse than 3 Å remains a challenge. Even if a related starting model is available, its incompleteness or its bias together with a low observation-to-parameter ratio can render the process unsuccessful or very time-consuming. Yet, many biologically important macromolecules, especially large macromolecular assemblies, membrane proteins and receptors, tend to provide crystals that diffract to low resolution. A new algorithm to tackle this problem is presented that uses a multivariate function to simultaneously exploit information from both an initial partial model and low-resolution single-wavelength anomalous diffraction data. The new approach has been used for six challenging structure determinations, including the crystal structures of membrane proteins and macromolecular complexes that have evaded experts using other methods, and large structures from a 3.0 Å resolution F1-ATPase data set and a 4.5 Å resolution SecYEG–SecA complex data set. All of the models were automatically built by the method to R free values of between 28.9 and 39.9% and were free from the initial model bias.

Adhesion G protein-coupled receptors (aGPCRs) play critical roles in diverse neurobiological processes including brain development, synaptogenesis, and myelination. aGPCRs have large alternatively spliced extracellular regions (ECRs) that likely mediate intercellular signaling; however, the precise roles of ECRs remain unclear. The aGPCR GPR56/ADGRG1 regulates both oligodendrocyte and cortical development. Accordingly, human GPR56 mutations cause myelination defects and brain malformations. Here, we determined the crystal structure of the GPR56 ECR, the first structure of any complete aGPCR ECR, in complex with an inverse-agonist monobody, revealing a GPCR-Autoproteolysis-Inducing domain and a previously unidentified domain that we termPentraxin/Laminin/neurexin/sex-hormone-binding-globulin-Like (PLL). Strikingly, PLL domain deletion caused increased signaling and characterizes a GPR56 splice variant. Finally, we show that an evolutionarily conserved residue in the PLL domain is critical for oligodendrocyte development invivo. Thus, our results suggest that the GPR56 ECR has unique and multifaceted regulatory functions, providing novel insights into aGPCR roles in neurobiology.

SummaryFibronectin leucine-rich repeat transmembrane proteins (FLRTs) are cell-adhesion molecules with emerging functions in cortical development and synapse formation. Their extracellular regions interact with latrophilins (LPHNs) to mediate synapse development, and with Uncoordinated-5 (UNC5)/netrin receptors to control the migration of neurons in the developing cortex. Here, we present the crystal structures of FLRT3 in isolation and in complex with LPHN3. The LPHN3/FLRT3 structure reveals that LPHN3 binds to FLRT3 at a site distinct from UNC5. Structure-based mutations specifically disrupt LPHN3/FLRT3 binding, but do not disturb their interactions with other proteins or their cell-membrane localization. Thus, they can be used as molecular tools to dissect the functions of FLRTs and LPHNs in vivo. Our results suggest that UNC5 and LPHN3 can simultaneously bind to FLRT3, forming a trimeric complex, and that FLRT3 may form transsynaptic complexes with both LPHN3 and UNC5. These findings provide molecular insights for understanding the role of cell-adhesion proteins in synapse function.

Optogenetic methods have contributed substantially to the experimental analysis of neural function and behavior. In the nematode Caenorhabditis elegans, a commonly used model organism for studying neural circuits, optogenetic stimulation of several neuromuscular networks has induced identical behavioral responses to those resulting from mechanical stimulation. This has lead to the acceptance of optogenetic methods as physiologically relevant stimuli. Despite the similarities in macroscopic phenomenology, the calcium signal in downstream neurons resulting from optogenetic stimulation has yet to be investigated and compared to the calcium signal in downstream neurons resulting from environmental stimulation. The optogenetic channel, channelrhodopsin-2, will be expressed under the control of the mec-4 sensory neuron promoter in C. elegans. Additionally, a synthetic calcium sensor, RCaMP1h, will be expressed separately under the control of a specific downstream interneuron promoter, dbl-1, in order to compare the calcium signaling in this interneuron, AVA, resulting from optogenetic and mechanical stimulation. Using software we developed, these freely swimming worms will be tracked and optogenetically stimulated using a light source localized to the sensory neurons of interest. Traditional mechanical stimulation with an eyelash will be carried out in tandem. The downstream calcium responses observed during each stimulation will be compared to determine if the optogenetic stimulation resulted in the same physiological response as mechanical stimulation. This study will provide insight into the relevance of optogenetics as a physiological probe of neural networks.