Project description:As a critical sphingolipid metabolite, sphingosine-1-phosphate (S1P) plays an essential role in immune and vascular systems. There are five S1P receptors, designated as S1PR1 to S1PR5, encoded in the human genome, and their activities are governed by endogenous S1P, lipid-like S1P mimics, or nonlipid-like therapeutic molecules. Among S1PRs, S1PR1 stands out due to its nonredundant functions, such as the egress of T and B cells from the thymus and secondary lymphoid tissues, making it a potential therapeutic target. However, the structural basis of S1PR1 activation and regulation by various agonists remains unclear. Here, we report four atomic resolution cryo-electron microscopy (cryo-EM) structures of Gi-coupled human S1PR1 complexes: bound to endogenous agonist d18:1 S1P, benchmark lipid-like S1P mimic phosphorylated Fingolimod [(S)-FTY720-P], or nonlipid-like therapeutic molecule CBP-307 in two binding modes. Our results revealed the similarities and differences of activation of S1PR1 through distinct ligands binding to the amphiphilic orthosteric pocket. We also proposed a two-step “shallow to deep” transition process of CBP-307 for S1PR1 activation. Both binding modes of CBP-307 could activate S1PR1, but from shallow to deep transition may trigger the rotation of the N-terminal helix of Gαi and further stabilize the complex by increasing the Gαi interaction with the cell membrane. We combine with extensive biochemical analysis and molecular dynamic simulations to suggest key steps of S1P binding and receptor activation. The above results decipher the common feature of the S1PR1 agonist recognition and activation mechanism and will firmly promote the development of therapeutics targeting S1PRs.
Project description:Activation of the first sphingosine-1-phosphate receptor (S1PR1 ) promotes permeability of the blood brain barrier, astrocyte and neuronal protection, and lymphocyte egress from secondary lymphoid tissues. Although an agonist often activates the S1PR1 , the receptor exhibits high levels of basal activity. In this study, we performed long-timescale molecular dynamics and accelerated molecular dynamics (aMD) simulations to investigate activation mechanisms of the ligand-free (apo) S1PR1 . In the aMD enhanced sampling simulations, we observed four independent events of activation, which is characterized by close interaction between Y3117.53 and Y2215.58 and increased distance between the intracellular ends of transmembrane (TM) helices 3 and 6. Although TM helices TM3, TM6, TM5 and, TM7 are associated with GPCR activation, we discovered that their movements are not necessarily correlated during activation. Instead, TM5 showed a decreased correlation with each of these regions during activation. During activation of the apo receptor, Y2215.58 and Y3117.53 became more solvated, because a water channel formed in the intracellular pocket. Additionally, a lipid molecule repeatedly entered the receptor between the extracellular ends of TM1 and TM7, providing important insights into the pathway of ligand entry into the S1PR1 .
Project description:Ezrin, radixin, and moesin (ERM) proteins link cortical actin to the plasma membrane and coordinate cellular events that require cytoskeletal rearrangement, including cell division, migration, and invasion. While ERM proteins are involved in many important cellular events, the mechanisms regulating their function are not completely understood. Our laboratory previously identified reciprocal roles for the sphingolipids ceramide and sphingosine-1-phosphate (S1P) in the regulation of ERM proteins. We recently showed that ceramide-induced activation of PP1α leads to dephosphorylation and inactivation of ERM proteins, while S1P results in phosphorylation and activation of ERM proteins. Following these findings, we aimed to examine known inducers of the SK/S1P pathway and evaluate their ability to regulate ERM proteins. We examined EGF, a known inducer of the SK/S1P pathway, for its ability to regulate the ERM family of proteins. We found that EGF induces ERM c-terminal threonine phosphorylation via activation of the SK/S1P pathway, as this was prevented by siRNA knockdown or pharmacological inhibition of SK. Using pharmacological, as well as genetic, knockdown approaches, we determined that EGF induces ERM phosphorylation via activation of S1PR2. In addition, EGF led to cell polarization in the form of lamellipodia, and this occurred through a mechanism involving S1PR2-mediated phosphorylation of ezrin T567. EGF-induced cellular invasion was also found to be dependent on S1PR2-induced T567 ezrin phosphorylation, such that S1PR2 antagonist, JTE-013, and expression of a dominant-negative ezrin mutant prevented cellular invasion toward EGF. In this work, a novel mechanism of EGF-stimulated invasion is unveiled, whereby S1P-mediated activation of S1PR2 and phosphorylation of ezrin T567 is required.
Project description:Activation of the GPCR sphingosine-1-phosphate receptor 1 (S1P1) by sphingosine-1-phosphate (S1P) regulates key physiological processes. S1P1 activation also has been implicated in pathologic processes, including autoimmunity and inflammation; however, the in vivo sites of S1P1 activation under normal and disease conditions are unclear. Here, we describe the development of a mouse model that allows in vivo evaluation of S1P1 activation. These mice, known as S1P1 GFP signaling mice, produce a S1P1 fusion protein containing a transcription factor linked by a protease cleavage site at the C terminus as well as a β-arrestin/protease fusion protein. Activated S1P1 recruits the β-arrestin/protease, resulting in the release of the transcription factor, which stimulates the expression of a GFP reporter gene. Under normal conditions, S1P1 was activated in endothelial cells of lymphoid tissues and in cells in the marginal zone of the spleen, while administration of an S1P1 agonist promoted S1P1 activation in endothelial cells and hepatocytes. In S1P1 GFP signaling mice, LPS-mediated systemic inflammation activated S1P1 in endothelial cells and hepatocytes via hematopoietically derived S1P. These data demonstrate that S1P1 GFP signaling mice can be used to evaluate S1P1 activation and S1P1-active compounds in vivo. Furthermore, this strategy could be potentially applied to any GPCR to identify sites of receptor activation during normal physiology and disease.
Project description:Neuropathic pain afflicts millions of individuals and represents a major health problem for which there is limited effective and safe therapy. Emerging literature links altered sphingolipid metabolism to nociceptive processing. However, the neuropharmacology of sphingolipid signaling in the central nervous system in the context of chronic pain remains largely unexplored and controversial. We now provide evidence that sphingosine-1-phosphate (S1P) generated in the dorsal horn of the spinal cord in response to nerve injury drives neuropathic pain by selectively activating the S1P receptor subtype 1 (S1PR1) in astrocytes. Accordingly, genetic and pharmacological inhibition of S1PR1 with multiple antagonists in distinct chemical classes, but not agonists, attenuated and even reversed neuropathic pain in rodents of both sexes and in two models of traumatic nerve injury. These S1PR1 antagonists retained their ability to inhibit neuropathic pain during sustained drug administration, and their effects were independent of endogenous opioid circuits. Moreover, mice with astrocyte-specific knockout of S1pr1 did not develop neuropathic pain following nerve injury, thereby identifying astrocytes as the primary cellular substrate of S1PR1 activity. On a molecular level, the beneficial reductions in neuropathic pain resulting from S1PR1 inhibition were driven by interleukin 10 (IL-10), a potent neuroprotective and anti-inflammatory cytokine. Collectively, our results provide fundamental neurobiological insights that identify the cellular and molecular mechanisms engaged by the S1PR1 axis in neuropathic pain and establish S1PR1 as a target for therapeutic intervention with S1PR1 antagonists as a class of nonnarcotic analgesics.
Project description:Sphingolipids represent an important class of bioactive signaling lipids which have key roles in numerous cellular processes. Over the last few decades, the levels of bioactive sphingolipids and/or their metabolizing enzymes have been realized to be important factors involved in disease development and progression, most notably in cancer. Targeting sphingolipid-metabolizing enzymes in disease states has been the focus of many studies and has resulted in a number of pharmacological inhibitors, with some making it into the clinic as therapeutics. In order to better understand the regulation of sphingolipid-metabolizing enzymes as well as to develop much more potent and specific inhibitors, the field of sphingolipids has recently taken a turn toward structural biology. The last decade has seen the structural determination of a number of sphingolipid enzymes and effector proteins. In these terms, one of the most complete arms of the sphingolipid pathway is the sphingosine-1-phosphate (S1P) arm. The structures of proteins involved in the function and regulation of S1P are being used to investigate further the regulation of said proteins as well as in the design and development of inhibitors as potential therapeutics.