暂无摘要。

Intercellular signaling mediated by evolutionarily conserved planar cell polarity (PCP) proteins aligns cell polarity along the tissue plane and drives polarized cell behaviors during tissue morphogenesis. Accumulating evidence indicates that the vertebrate PCP pathway is regulated by noncanonical, β-catenin-independent Wnt signaling; however, the signaling components and mechanisms are incompletely understood. In the mouse hearing organ, both PCP and noncanonical Wnt (ncWnt) signaling are required in the developing auditory sensory epithelium to control cochlear duct elongation and planar polarity of resident sensory hair cells (HCs), including the shape and orientation of the stereociliary hair bundle essential for sound detection. We have recently discovered a Wnt/G-protein/PI3K pathway that coordinates HC planar polarity and intercellular PCP signaling. Here, we identify Wnt7b as a ncWnt ligand acting in concert with Wnt5a to promote tissue elongation in diverse developmental processes. In the cochlea, Wnt5a and Wnt7b are redundantly required for cochlear duct coiling and elongation, HC planar polarity, and asymmetric localization of core PCP proteins Fzd6 and Dvl2. Mechanistically, Wnt5a/Wnt7b-mediated ncWnt signaling promotes membrane recruitment of Daple, a nonreceptor guanine nucleotide exchange factor for Gαi, and activates PI3K/AKT and ERK signaling, which promote asymmetric Fzd6 localization. Thus, ncWnt and PCP signaling pathways have distinct mutant phenotypes and signaling components, suggesting that they act as separate, parallel pathways with nonoverlapping functions in cochlear morphogenesis. NcWnt signaling drives tissue elongation and reinforces intercellular PCP signaling by regulating the trafficking of PCP-specific Frizzled receptors.

G-protein-coupled receptors (GPCRs) are essential mediators of neuromodulation and prominent pharmacological targets. While activation of heterotrimeric G-proteins (Gαβɣ) by GPCRs is essential in this process, much less is known about the postreceptor mechanisms that influence G-protein activity. Neurons express G-protein regulators that shape the amplitude and kinetics of GPCR-mediated synaptic responses. Although many of these operate by directly altering how G-proteins handle guanine-nucleotides enzymatically, recent discoveries have revealed alternative mechanisms by which GPCR-stimulated G-protein responses are modulated at the synapse. In this review, we cover the molecular basis for, and consequences of, the action of two G-protein regulators that do not affect the enzymatic activity of G-proteins directly: Gα inhibitory interacting protein (GINIP), which binds active Gα subunits, and potassium channel tetramerization domain-containing 12 (KCTD12), which binds active Gβγ subunits.

The interactions of G-protein-coupled receptors (GPCRs) with other proteins are critical in several cellular processes but resolving their structural dynamics remains challenging. An increasing number of GPCR complexes have been experimentally resolved but others including receptor variants are yet to be characterized, necessitating computational predictions of their interactions. Although integrative approaches with multi-scale simulations would provide rigorous estimates of their conformational dynamics, protein-protein docking remains a first tool of choice of many researchers due to the availability of open-source programs and easy to use web servers with reasonable predictive power. Protein-protein docking algorithms have limited ability to consider protein flexibility, environment effects, and entropy contributions and are usually a first step towards more integrative approaches. The two critical steps of docking: the sampling and scoring algorithms have improved considerably and their performance has been validated against experimental data. In this chapter, we provide an overview and generalized protocol of a few docking protocols using GPCRs as test cases. In particular, we demonstrate the interactions of GPCRs with extracellular protein ligands and an intracellular protein effectors (G-protein) predicted from docking approaches and test their limitations. The current chapter will help researchers critically assess docking protocols and predict experimentally testable structures of GPCR complexes.

The heterotrimeric G-protein α subunit, Gαolf, acts to transduce extracellular signals through G-protein coupled receptors (GPCRs) and stimulates adenylyl cyclase mediated production of the second messenger cyclic adenosine monophosphate. Numerous mutations in the GNAL gene, which encodes Gαolf, have been identified as causative for an adult-onset dystonia. These mutations disrupt GPCR signaling cascades in in vitro assays through several mechanisms, and this disrupted signaling is hypothesized to lead to dystonic motor symptoms in patients. However, the cells and circuits that mutations in GNAL corrupt are not well understood. Published patterns of Gαolf expression outside the context of the striatum are sparse, conflicting, often lack cell type specificity, and may be confounded by expression of the close GNAL homolog of GNAS. Here, we use RNAScope in-situ hybridization to quantitatively characterize Gnal mRNA expression in brain tissue from wildtype C57BL/6J adult mice. We observed widespread expression of Gnal puncta throughout the brain, suggesting Gαolf is expressed in more brain structures and neuron types than previously accounted for. We quantify transcripts at a single cell level, and use neuron type specific markers to further classify and understand patterns of GNAL expression. Our data suggests that brain regions classically associated with motor control, initiation, and regulation show the highest expression of GNAL, with Purkinje Cells of the cerebellum showing the highest expression of any neuron type examined. Subsequent conditional Gnal knockout in Purkinje cells led to markedly decreased intracellular cAMP levels and downstream cAMP-dependent enzyme activation. Our work provides a detailed characterization of Gnal expression throughout the brain and the biochemical consequences of loss of Gαolf signaling in vivo in neurons that highly express Gnal.

GPCR-G protein signaling from endosomes plays a crucial role in various physiological and pathological processes. However, the mechanism by which endosomal G protein signaling is terminated remains largely unknown. In this study, we aimed to investigate the regulatory mechanisms involved in terminating the signaling of Gα subunits from endosomes. Through structural analysis and cell-based assays, we have discovered that SNX25, a protein that targets endosomes via its PXA or PXC domain, interacts with regulator of G protein signaling (RGS) proteins (including RGS2, RGS4, RGS8, and RGS17) in a redox-regulated manner. The interaction between SNX25 and these RGS proteins enhances their GTPase-accelerating activity towards Gαi/q and their ability to bind GDP-bound (inactive form) Gαi/q. As a result, SNX25 recruits these RGS proteins to endosomes, leading to the termination of endosomal Gαi/q signaling. Furthermore, we have found that the SNX25/RGS complex also exerts a negative regulatory effect on Gαi/q signaling from the plasma membrane. This is achieved by recruiting Gαi/q to endosomes and preventing its activation on the plasma membrane. Our findings shed light on the previously unknown role of redox-modulated SNX25 in inhibiting Gαi/q signaling, thereby uncovering a novel mechanism for terminating Gαi/q signaling from endosomes. Importantly, this study expands our understanding of the regulation of GPCR-Gαi/q signaling beyond the plasma membrane.

Transient receptor potential melastatin-8 (TRPM8) is a cation channel that is activated by cold and \"cooling agents\" such as menthol and icilin, which induce a cold sensation. The stimulation of TRPM8 activates an intracellular signaling cascade that ultimately leads to a change in the gene expression pattern of the cells. Here, we investigate the TRPM8-induced signaling pathway that links TRPM8 channel activation to gene transcription. Using a pharmacological approach, we show that the inhibition of phosphatidylinositol 4-phosphate 5 kinase α (PIP5K), an enzyme essential for the biosynthesis of phosphatidylinositol 4,5-bisphosphate, attenuates TRPM8-induced gene transcription. Analyzing the link between TRPM8 and Gq proteins, we show that the pharmacological inhibition of the βγ subunits impairs TRPM8 signaling. In addition, genetic studies show that TRPM8 requires an activated Gα subunit for signaling. In the nucleus, the TRPM8-induced signaling cascade triggers the activation of the transcription factor AP-1, a complex consisting of a dimer of basic region leucine zipper (bZIP) transcription factors. Here, we identify the bZIP protein c-Jun as an essential component of AP-1 within the TRPM8-induced signaling cascade. In summary, with PIP5K, Gq subunits, and c-Jun, we identified key molecules in TRPM8-induced signaling from the plasma membrane to the nucleus.

Ion channels are membrane proteins that may also have intracellular and extracellular domains that interact with other ligands. In many cases, these interaction sites are highly mobile and may undergo changes in the configuration upon binding with regulatory signaling molecules. Isothermal titration calorimetry (ITC) is a powerful technique to quantify protein-ligand interactions of purified samples in solution. This chapter describes a fragment-based analysis method using ITC to quantify the interactions between a domain of the voltage-gated Kv7 channel and the calcium-regulated protein calmodulin. This example can be used to quantify the interactions between specific domains of other ion channels and their regulatory signaling proteins.

TG2 is a unique member of the transglutaminase family as it undergoes a dramatic conformational change, allowing its mutually exclusive function as either a cross-linking enzyme or a G-protein. The enzyme\'s dysregulated activity has been implicated in a variety of pathologies (e.g., celiac disease, fibrosis, cancer), leading to the development of a wide range of inhibitors. Our group has primarily focused on the development of peptidomimetic targeted covalent inhibitors, the nature and size of which were thought to be important features to abolish TG2\'s conformational dynamism and ultimately inhibit both its activities. However, we recently demonstrated that the enzyme was unable to bind guanosine triphosphate (GTP) when catalytically inactivated by small molecule inhibitors. In this study, we designed a library of models targeting covalent inhibitors of progressively smaller sizes (15 to 4 atoms in length). We evaluated their ability to inactivate TG2 by measuring their respective kinetic parameters kinact and KI. Their impact on the enzyme\'s ability to bind GTP was then evaluated and subsequently correlated to the conformational state of the enzyme, as determined via native PAGE and capillary electrophoresis. All irreversible inhibitors evaluated herein locked TG2 in its open conformation and precluded GTP binding. Therefore, we conclude that steric bulk and structural complexity are not necessary factors to consider when designing TG2 inhibitors to abolish G-protein activity.

Prior evidence demonstrated that Regulator of G protein Signaling 6 (RGS6) translocates to the nucleolus in response to cytotoxic stress though the functional significance of this phenomenon remains unknown.
Utilizing in vivo gene manipulations in mice, primary murine cardiac cells, human cell lines and human patient samples we dissect the participation of a RGS6-nucleolin complex in chemotherapy-dependent cardiotoxicity.
Here we demonstrate that RGS6 binds to a key nucleolar protein, Nucleolin, and controls its expression and activity in cardiomyocytes. In the human myocyte AC-16 cell line, induced pluripotent stem cell derived cardiomyocytes, primary murine cardiomyocytes, and the intact murine myocardium tuning RGS6 levels via overexpression or knockdown resulted in diametrically opposed impacts on Nucleolin mRNA, protein, and phosphorylation.RGS6 depletion provided marked protection against nucleolar stress-mediated cell death in vitro, and, conversely, RGS6 overexpression suppressed ribosomal RNA production, a key output of the nucleolus, and triggered death of myocytes. Importantly, overexpression of either Nucleolin or Nucleolin effector miRNA-21 counteracted the pro-apoptotic effects of RGS6. In both human and murine heart tissue, exposure to the genotoxic stressor doxorubicin was associated with an increase in the ratio of RGS6/Nucleolin. Preventing RGS6 induction via introduction of RGS6-directed shRNA via intracardiac injection proved cardioprotective in mice and was accompanied by restored Nucleolin/miRNA-21 expression, decreased nucleolar stress, and decreased expression of pro-apoptotic, hypertrophy, and oxidative stress markers in heart.
Together, these data implicate RGS6 as a driver of nucleolar stress-dependent cell death in cardiomyocytes via its ability to modulate Nucleolin. This work represents the first demonstration of a functional role for an RGS protein in the nucleolus and identifies the RGS6/Nucleolin interaction as a possible new therapeutic target in the prevention of cardiotoxicity.