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After the ligand binds to the cell-surface receptor, the activation of the receptor’s intracellular components sets off a chain of events that is called a signaling pathway or a signaling cascade. In a signaling pathway, second messengers, enzymes, and activated proteins interact with specific proteins, which are in turn activated in a chain reaction that eventually leads to a change in the cell’s environment (Figure 1). Interactions that occur before a certain point are defined as upstream events, and events after that point are called downstream events.
In certain cancers, the GTPase activity of the RAS G-protein is inhibited. This means that the RAS protein can no longer hydrolyze GTP into GDP. What effect would this have on downstream cellular events?
[reveal-answer q=”265793″]Show Answer[/reveal-answer]
[hidden-answer a=”265793″]ERK would become permanently activated, resulting in cell proliferation, migration, adhesion, and the growth of new blood vessels. Apoptosis would be inhibited.[/hidden-answer]
Signaling pathways can get very complicated very quickly because most cellular proteins can affect different downstream events, depending on the conditions within the cell. A single pathway can branch off toward different endpoints based on the interplay between two or more signaling pathways, and the same ligands are often used to initiate different signals in different cell types. This variation in response is due to differences in protein expression in different cell types. Another complicating element is signal integration of the pathways, in which signals from two or more different cell-surface receptors merge to activate the same response in the cell. This process can ensure that multiple external requirements are met before a cell commits to a specific response.
The effects of extracellular signals can also be amplified by enzymatic cascades. At the initiation of the signal, a single ligand binds to a single receptor. However, activation of a receptor-linked enzyme can activate many copies of a component of the signaling cascade, which amplifies the signal.
Toll-like Receptor Signaling Pathway
Toll-like receptors (TLRs) are a class of proteins that play a key role in innate immunity. They are single domain trans-membrane receptors belong to pattern recognition receptors (PRRs) which usually expressed in sentinel cells such as macrophages dendritic cells and many other non-immune cells such as fibroblasts and epithelial cells. They recognize structurally conserved molecules derived from microbes which are called pathogen-associated molecular patterns (PAMPs) or self-derived molecules derived from damaged cells, referred as damage associated molecules patterns (DAMPs). PAMPs include various bacterial cell wall components such as lipopolysaccharide (LPS), peptidoglycan (PGN) and lipopeptides, as well as flagellin, bacterial DNA and viral double-stranded RNA. DAMPs include intracellular proteins such as heat shock proteins as well as protein fragments from the extracellular matrix. PRRs activate downstream signaling pathways that lead to the induction of innate immune responses by producing inflammatory cytokines, type I interferon (IFN), and other mediators. These processes not only trigger immediate host defensive responses such as inflammation, but also prime and orchestrate antigen-specific adaptive immune responses. These responses are essential for the clearance of infecting microbes as well as crucial for the consequent instruction of antigen-specific adaptive immune responses.
Figure 2. The schematic diagram of the TLRs molecular structure.
Toll-like receptor family
The TLR family comprises 10 members (TLR1–TLR10) in human and 12 (TLR1–TLR9, TLR11–TLR13) in mouse. TLRs localize to the cell surface or to intracellular compartments such as the ER, endosome and lysosome. Cell surface TLRs include TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10, whereas intracellular TLRs are localized in the endosome and include TLR3, TLR7, TLR8, TLR9, TLR11, TLR12, and TLR13 (Figure 1). Cell surface TLRs mainly recognize microbial membrane components such as lipids, lipoproteins, and proteins. Intracellular TLRs recognize nucleic acids derived from bacteria and viruses, and also recognize self-nucleic acids in disease conditions such as autoimmunity.
The function of Toll-like receptor usually based on a dimerization process of two TLR molecules, but not always. For example, TLR-1 and TLR-2 will bind to each other to form a dimmer when recognize PAMPs molecules mainly including lipoproteins, peptidoglycans, lipotechoic acids (LTA, Gram-), zymosan, mannan, and tGPI-mucin. TLR-2 can also form a dimmer with TLR-6 when they recognize the same PAMPs listed above. TLR-4 can recognize lipopolysaccharide (LPS, Gram+) and form a homodimer with another TLR-4 molecule. TLR-5 can recognize bacterial flagellin, but they don’t form a dimmer. TLR-11 is functional in mice and mainly recognize uropathogenic bacterial. TLR-3, 7, 8, 9, 13 are expressed on the endosome surface in the cytoplasm. TLR3 recognizes viral double-stranded RNA (dsRNA), small interfering RNAs, and self-RNAs derived from damaged cells. TLR-7 is predominantly expressed in plasmacytoid DCs (pDCs) and recognizes single-stranded (ss) RNA from viruses. It also recognizes RNA from streptococcus B bacteria in conventional DCs (cDCs). TLR8 responds to viral and bacterial RNA. TLR-9 recognizes bacterial and viral DNA that is rich in unmethylated CpG-DNA motifs. TLR13 recognizes bacterial 23S rRNA and unknown components of vesicular stomatitis virus.
Although there are so many types of TLR molecule which recognize wide range of ligands, all these TLRs share a common structural framework in their extracellular, ligand-binding domains. These domains all adopt horseshoe-shaped structures built from leucine-rich repeat motifs. Typically, on ligand binding, two extracellular domains form an ‘‘m’’-shaped dimer sandwiching the ligand molecule bringing the transmembrane and cytoplasmic domains in close proximity and triggering a downstream signaling cascade (Figure 2).
Toll-like receptor signaling pathway
1. Toll-like receptor signaling cascade
Toll-like receptors allow sentinel cells such as macrophages to detect microbes through PAMPs such as LPS. LPS is a component of bacterial cell wall. The mechanism of lipopolysaccharide recognition by Toll-like receptors is complex and require several accessary proteins. A serum protein, LPS-binding protein binds LPS monomers and transfer it to a protein called CD14. CD14 can be soluble or bind to the cell surface through a glycosylphosphatidylinositol anchor. CD 14 delivers and loads LPS to the extracellular domain of Toll-like receptors. TLRs are able to detect LPS with the help of an accessary protein called MD-2. Then homodimerization of TLRs are induced when LPS bind to the complex of TLR-CD14-MD2. The conformational change of the extracellular domains initiate dimerization of cytoplasmic Toll IL-1 receptor (TIR) domain. The TIR conformational change provide a new scaffold that allows the recruitment of adaptor proteins to form a post receptor signaling complex. The TIR containing an adaptor protein myeloid differentiation primary-response protein 88 (MyD88).
MyD88 functions as an adaptor linking TLRs/IL-1Rs with downstream signaling molecules that have DDs. It recognize the conformational change in the TIR domain of the TLRs, binds to the new receptor complex, and transfer the signaling by amino (N)-terminal death domain (DD) interaction with IL-1R-associated kinases (IRAKs). These results a complex cascade with signaling invents that warns the cell of pathogen invasion. There are 4 IRAKs (IRAK 1, 2, 4, M). They contain an N-terminal DD and a central serine/threonine-kinase domain. IRAK1 and IRAK4 have intrinsic kinase activity, whereas IRAK2 and IRAK-M have no detectable kinase activity. IRAK4 activated by MyD88 and it continue to activate IRAK1. IRAK1 then activate the downstream TRAF6. TRAF6 is a member of the tumor necrosis factor receptor (TNFR)-associated factor (TRAF) family that mediates cytokine signaling pathways. Upon stimulation, TRAF6 is recruited to the receptor complex, and activated by IRAK-1 that binds to the TRAF domain of TRAF6. Then, the IRAK-1/TRAF6 complex dissociates from the receptor and associates with TGF-beta-activated kinase 1 (TAK1) and TAK1-binding proteins, TAB1 and TAB2. The complex of TRAF6, TAK1, TAB1, and TAB2 moves into the cytoplasm, where it forms a large complex with other proteins, such as the E2 ligases Ubc13 and Uev1A. The Ubc13 and Uev1A complex has been shown to catalyze the synthesis of a Lys 63-linked polyubiquitin chain of TRAF6 and thereby induce TRAF6-mediated activation of TAK1 and finally of NF-kB. These signal pathway described above are called MyD88-dependent pathway as the signal is starting from the MyD88 molecule. There is also another pathway called MyD88-independedt pathway, which signaling is not starting from MyD88. Instead, the signal starting from TRIF protein. TRIF interacts with TRAF6 and TRAF3.TRAF6 recruits the kinase RIP-1, which in turn interacts with and activates the TAK1 complex, leading to activation of NF-kB and MAPKs and induction of inflammatory cytokines. In contrast, TRAF3 recruits the IKK-related kinasesTBK1 and IKKi along with NEMO for IRF3 phosphorylation and activation. IRF3 forms a dimer and translocates into the nucleus from the cytoplasm, induces the expression of type I IFN.
TLRs signal actually mainly through the recruitment of specific adaptor molecules, leading to activation of the transcription factors NF-kB and IRFs, which dictate the outcome of innate immune responses. So this pathway downstream signaling is to activate IRFs transcription factor, the NF-kB signaling pathway and MAKP pathway. You can find more detail information about NF-kB and MAKP pathway from:
NF-kB signaling pathway, P38 signaling pathway and MAKP signaling pathway.
Of cause there some negative regulation by a number of molecules through various mechanisms to prevent or terminate the excessive immune responses that lead to detrimental consequences associated with autoimmunity and inflammatory disease. Activation of the MyD88-dependent pathway is suppressed by ST2825, SOCS1, and Cbl-b, and activation of the TRIF-dependent pathway is suppressed by SARM and TAG. These molecules associate with MyD88 or TRIF to prevent them from binding to TLRs or downstream molecules. TRAF3 activation is negatively regulated by SOCS3 and DUBA. TRAF6 is targeted by a number of inhibitory molecules such as A20, USP4, CYLD, TANK, TRIM38 and SHP. TAK1 activation is inhibited by TRIM30a and A20.
4. Relationship with diseases
As TLR is involved in LPS sensing and it could have a role in sepsis, targeting of TLRs is important for the treatment of several diseases. In addition to interfering with TLR responses to treat pathogen infections, an obvious clinical application of the knowledge gained from TLR studies was to use TLR ligands as vaccine adjuvants. Moreover, TLR inhibition has also been attempted in the clinic, the goal of which is to limit excessive inflammation that is presumably driven by the over activation of a particular TLR.
Results and Discussion
Ncad Ligation Activates p38 in a Cdo-, JLP-, and Bnip-2-Dependent Manner.
To assess whether Ncad ligation activates p38 signaling in myoblasts, C2C12 cells were plated at low density (without any cell–cell contact) on culture dishes or coverslips coated with Ncad ectodomain-Fc fusion protein (Ncad-Fc). Under these conditions, Ncad-expressing cells attach and spread as if adhering to neighboring cells and undergo Ncad-dependent signaling, but without other juxtacrine signals that may occur indirectly as a consequence of cell–cell adhesion (26). Dishes and coverslips coated with fibronectin (which like Ncad promotes myogenesis) (27) or poly-L-lysine (PLL) were used as controls. Cells were visualized by staining with phalloidin to reveal F-actin structures and harvested for analysis of the phosphorylated (activated) form of p38 (pp38) by Western blotting. Cells plated on Ncad-Fc for 2 h spread, formed stress fibers and filopodial extensions, and induced pp38 (Fig. 1 A and B). Furthermore, pp38 in these cells was localized in the nucleus and perinuclear region (Fig. 1C). Cells plated on fibronectin formed stress fibers and longer projections and achieved a similar overall surface area to those on Ncad-Fc, but did not produce pp38 (Fig. 1 A, B, and D). Cells plated on PLL attached but did not spread as well and did not produce pp38. Therefore, Ncad ligation specifically activated p38 in myoblasts. Furthermore, cells plated at low density on Ncad-Fc, but not PLL, for 24 h expressed myogenin and troponin T, early and later markers of differentiation, respectively (Fig. S1).
Ncad ligation activates p38 in myoblasts. (A) Photomicrographs of C2C12 cells cultured on Ncad-Fc (Ncad), fibronectin (FN), or poly-L-lysine (PLL) for 2 h and stained with rhodamine phalloidin to reveal F-actin structures. (B) Western blot analysis of pp38 and total p38 in cultures shown in A. (C) Photomicrograph of a C2C12 cell on Ncad-Fc substrate for 2 h stained with rhodamine phalloidin (red), anti-pp38 (green), and DAPI to reveal nuclei (blue). (D) Surface area of C2C12 cells cultured on the indicated substrates for 2 h. Values are means ± SD, n > 80 cells. *, P < 0.01 by Student's t test. (Scale bars, 10 μm.)
Because Ncad and Cdo associate in myoblasts and Cdo-containing complexes are involved in differentiation-dependent p38 activity (6, 7, 11), Cdo and Cdo-binding proteins were assessed for their importance in pp38 production by Ncad ligation. C2C12 cells were depleted of Cdo, JLP, and Bnip-2 by RNAi, and the cells were plated on Ncad-Fc cells that expressed a control RNAi sequence were also analyzed. Depletion of each protein diminished production of pp38 upon Ncad ligation to an extent that was roughly proportional to the extent of knockdown (Fig. 2 A and B). In contrast, phosphorylation of other MAP kinases, ERK and JNK, induced by Ncad ligation was not significantly affected by RNAi depletion of Cdo, JLP, or Bnip-2, indicating a specific requirement for Cdo and its associated proteins in Ncad-dependent p38 signaling (Fig. 2B and Fig. S2). Cdo also interacts with M-cadherin (11) and, consistent with potential cadherin redundancy in myoblasts, M-caderin ligation activated p38 in a Cdo-dependent manner (Fig. S3). Satellite cell-derived myoblasts from Cdo +/+ and Cdo −/− mice were also investigated. Similar to C2C12 cells, Cdo +/+ myoblasts activated pp38 when plated on Ncad-Fc but not on fibronectin or PLL. In contrast, Cdo −/− myoblasts displayed only trace production of pp38 upon Ncad ligation (Fig. 2C). Next, we asked whether disruption of Ncad-Cdo association interfered with p38 activation. A Cdo deletion mutant that lacks the first FnIII repeat (designated CdoΔFn1) is deficient in its ability to interact with Ncad but associates normally with other proteins known to interact with the Cdo ectodomain (11–13) expression of this protein in C2C12 cells inhibits differentiation (11). Expression of CdoΔFn1 in these cells also strongly diminished induction of pp38, but not pERK, by Ncad ligation (Fig. 2D).
Cdo, JLP, and Bnip-2 are involved in Ncad-induced p38 activation. (A) Western blots of C2C12 cells stably expressing siRNA against Cdo, JLP, or Bnip-2, or a control siRNA (Con), were probed with antibodies against the indicated protein or against β-tubulin as a control. (B) Western blot analysis of pp38, total p38, pERK, and total ERK2 in C2C12 cells expressing the indicated siRNA constructs and cultured for 2 h on Ncad or PLL substrates. (C) Western blot analysis of pp38 and total p38 in Cdo +/+ and Cdo −/− myoblasts cultured for 2 h on Ncad, FN, or PLL substrates. (D) Western blot analysis of endogenous Cdo and exogenous Cdo(ΔFn1), pp38, total p38, pERK, and total ERK2, in control or Cdo(ΔFn1)-expressing C2C12 cells cultured on Ncad or PLL substrates for 2 h. Note that although the Con and Cdo(ΔFn1) blots appear split, they are from the same autoradiograms.
Cdo-dependent p38 activation in differentiating myoblasts occurs through Cdo/Bnip-2-dependent Cdc42 activity (6). C2C12 cells plated on Ncad-Fc substrate induced Cdc42 activity significantly above that observed in cells on PLL, as assessed by levels of GTP-bound Cdc42 (Fig. 3A). Furthermore, RNAi-mediated depletion of Cdo strongly decreased levels of GTP-bound Cdc42 in cells plated on either substrate, suggesting that Cdo is not only required for Ncad-initiated Cdc42 activity but also involved in basal Cdc42 activity on PLL (Fig. 3A). We previously showed that stable overexpression of Cdc42GAP in C2C12 cells reduces steady-state levels of GTP-bound Cdc42, differentiation-associated p38 activation, and differentiation itself (6). Overexpression of Cdc42GAP also reduced induction of pp38 by Ncad ligation (Fig. 3B). Taking the results together, Ncad ligation induced pp38 in a short-term signaling assay in a manner very similar to the activation of pp38 seen in differentiating myoblasts (i.e., each is sensitive to disruption of Ncad-Cdo interaction, depletion of Cdo and its intracellular binding proteins JLP and Bnip-2, and reduction of Cdc42 activity) (6, 7, 10).
Cdc42, but not RhoA, is involved in Ncad-induced p38 activation. (A) Quantification by G-LISA of GTP-bound (activated) Cdc42 in C2C12 cells stably expressing siRNA against Cdo or a control siRNA plated on Ncad or PLL substrates. *, P < 0.01 **, P < 0.001 by Student's t test. See Methods for details. (B) Western blot analysis of pp38, total p38, and flag epitope in C2C12 cells stably transfected with control (Con) or Cdc42GAP(flag) expression vectors. (C) Photomicrographs of C2C12 cells cultured with or without cell-permeable C3 RhoA inhibitor and plated on Ncad-Fc substrate for 2 h and then stained with rhodamine phalloidin (red), anti-pp38 (green), and DAPI to reveal nuclei (blue). (Scale bar, 10 μm.) (D) Western blot analysis of pp38 and total p38 in C2C12 cells cultured with or without cell-permeable C3 RhoA inhibitor and plated on Ncad-Fc or PLL substrates for 2 h.
The small GTPase RhoA positively regulates myogenic differentiation (15), and stable expression of constitutively active or dominant-negative forms of RhoA enhances or reduces pp38 levels, respectively, in C2C12 cells cultured for 24–72 h in differentiation medium (20). Furthermore, RhoA is activated by cadherin ligation in several cell systems, including myoblasts (15, 26). To assess whether RhoA is directly involved in Ncad ligation-induced pp38, C2C12 cells were treated with the specific Rho inhibitor, C3 transferase. C3 decreased RhoA-GTP levels in C2C12 cells by >70% (Fig. S4), and C3-treated cells on Ncad-Fc substrate displayed a virtually complete loss of stress fibers, production of which is known to require RhoA activity (28) (Fig. 3C). In contrast, Ncad-mediated induction of pp38 was unaffected by C3, nor was the nuclear/perinuclear localization of pp38 (Fig. 3 C and D). Therefore, RhoA is not directly involved in Ncad-dependent p38 activation. The ability of RhoA to positively regulate p38 activity over a longer time course in differentiating myoblasts (19) may be related to its effects on MyoD expression and a consequent effect on MyoD’s linkage to p38 activity through feed-forward mechanisms (4, 29).
JLP, Bnip-2, and Active Cdc42 Cluster at Sites of Ncad-Ligation and Ncad-Cdo Interaction but Not at Sites of Shh-Cdo Interaction.
The results described above strongly suggest that the Cdo-dependent signaling complexes that activate p38 in differentiating myoblasts lie in a pathway initiated by Ncad ligation. High cell density promotes both Ncad ligation and p38 activity in C2C12 cells (15, 20). Ncad and Cdo associate in both high- and low-density cultures (i.e., Ncad ligation is not necessary for Ncad-Cdo interaction) (11), but Cdo and p38 coimmunoprecipitate only in high-density cultures (Fig. 4A), suggesting that Ncad ligation triggers this association. It would therefore be predicted that components of the Cdo complex would cluster at sites of active Ncad ligation. To test this notion, Ncad ectodomain-coated microspheres were allowed to settle onto adherent NIH 3T3 cells that transiently expressed fluorescently tagged forms of Ncad, Cdo, JLP, Bnip-2, or the Cdc42-binding domain of N-Wasp (wGBD), which interacts specifically with active, GTP-bound Cdc42 (30). Cadherin-coated beads attach to cells via cognate cellular cadherins, and additional cellular proteins that cluster at these sites of adhesion can be visualized as a fluorescent signal surrounding the bead (31–33). When Ncad-coated beads attached to cells that coexpressed DsRed-tagged Ncad and Cdo-GFP, each fluorescent protein was concentrated at such beads. In contrast, GFP itself clustered at Ncad beads much less efficiently (Fig. 4 B and C). The percentage of beads that clustered a given fluorescent protein in these assays was quantified. Eighty-five percent of Ncad beads clustered Ncad-DsRed, and 77% clustered Cdo, whether or not exogenous Ncad was expressed (exogenous Ncad is unnecessary as NIH 3T3 cells express abundant endogenous Ncad) (34), whereas only 20% of Ncad beads clustered GFP (Fig. 4C). Furthermore, PLL-coated beads also attached to NIH 3T3 cells but only 11% of these beads clustered any fluorescent protein, regardless of its identity (Fig. 4C).
Ncad ligation clusters Cdo, JLP, Bnip-2, and wGBD. (A) C2C12 cells were cultured at high or low cell density as indicated. Lysates were immunoprecipitated with antibodies to Cdo and immunoprecipitates and straight lysates blotted with antibodies to Cdo, p38, and Ncad. (B) (Upper) Photomicrograph of Ncad-coated beads attached to a cell that coexpresses Ncad-DsRed and Cdo-GFP. Arrow, a bead scored positive for clustering Ncad and Cdo arrowhead, a bead scored negative for clustering Ncad and Cdo. (Lower) Photomicrograph of an Ncad-coated bead attached to a cell that expresses GFP. (C) Percentage of Ncad-coated beads that clustered the indicated fluorescent protein. Values are means ± SD, n > 100 beads. *, P < 0.001 by Student's t test. (D) Photomicrographs of Ncad-coated beads attached to cells that coexpress nonfluorescent Cdo plus JLP-GFP, Bnip-2-GFP, or wGBD-GFP. Arrow, a bead scored positive for clustering Bnip-2-GFP arrowhead, a bead scored as negative. (E) Percentage of Ncad-coated beads that clustered the indicated fluorescent protein. Values are means ± SD, n > 100 beads. *, P < 0.01 **, P < 0.001 with differences referring to both the respective −Cdo control and the +Cdo/+GFP control. (Scale bars, 10 μm.)
NIH 3T3 cells express low levels of Cdo (35), and clustering of JLP-GFP, Bnip-2-GFP, and wGBD-GFP at Ncad beads was largely dependent on coexpression of nonfluorescent Cdo [Fig. 4 D and E note that the exogenous expression levels of Cdo-GFP and nonfluorescent Cdo were similar (Fig. S5)]. Seventy-six percent, 59%, and 43% of these beads were positive for JLP-GFP, Bnip-2-GFP, and wGBD-GFP clustering, respectively, all significantly above the 20% seen with GFP. The percentage of beads positive for clustering Ncad-DsRed, Cdo-GFP, JLP-GFP, Bnip-2-GFP, and wGBD-GFP progressively diminished in a manner consistent with the model in which cellular Ncad binds directly to Cdo, Cdo binds directly to JLP and Bnip2, and wGBD binds to Bnip-2 indirectly via Cdc42 (6, 7, 11). The diminishing percentages would be expected if the efficiency of each interaction were <100% and binding was specific (i.e., only beads that clustered Ncad were able to cluster Cdo, and only beads that clustered Cdo were able to cluster JLP and Bnip-2). When the percentage of “available” beads for a given interaction is considered, efficiency at each step ranged from 77 to 99%.
Cdo binds directly to Shh, and the ability of beads coated with an amino-terminal Shh signaling fragment (ShhN)-Fc fusion protein to cluster Cdo and Cdo-binding proteins was also assessed. Fifty-six percent of ShhN beads clustered Cdo, less than seen with Ncad beads but significantly above the GFP background of 20% (Fig. 5 A and B). In contrast, the percentage of ShhN beads that clustered JLP-GFP or Bnip-2-GFP (even in the presence of nonfluorescent Cdo) was no different from that seen with GFP alone. Therefore, Ncad beads clustered Cdo and its associated proteins that are involved in activation of p38, but Shh beads clustered only Cdo. Shh is reported to activate p38 signaling in primary astrocyte cultures (36). However, consistent with its inability to cluster Cdo-binding proteins, pp38 was not induced in C2C12 myoblasts by recombinant ShhN, whereas it was induced in response to a known pathway activator, hyperosmotic stress (0.7 M NaCl) (Fig. 5C). Despite the failure to induce pp38, the cells were responsive to ShhN, as demonstrated by activation of Gli1 expression (Fig. 5D).
Shh-coated beads cluster Cdo but not JLP or Bnip-2. (A) Photomicrographs of Shh-coated beads attached to cells that express Cdo or coexpress nonfluorescent Cdo plus JLP-GFP or Bnip-2-GFP. (Scale bar, 10 μm.) (B) Percentage of Shh-coated beads that clustered the indicated fluorescent protein. Values are means ± SD, n > 100 beads. *, P < 0.001. (C) Western blot analysis of pp38 and total p38 in C2C12 cultures treated with recombinant ShhN (0.5 μg/mL) for the indicated amounts of time. As a positive control cells were treated with NaCl (0.7 M) for 20 min. (D) qRT-PCR analysis of Gli1 expression in C2C12 cells treated ± ShhN (0.5 μg/mL) for 24 h. (E) Model for Ncad-stimulated p38 activation in myoblasts and for Cdo as a multifunctional coreceptor. Cdo bound in cis to ligated Ncad exists in a state that permits stable interaction with JLP/p38 and Bnip-2/Cdc42. In contrast, Cdo bound to Shh does not interact stably with these factors, although it permits the Shh signal to be transmitted to Ptch1, activating canonical Hedgehog pathway signaling. Note that activated MKK6 rescues the defective differentiation program caused by loss of Cdo or Bnip-2, but the role of MKK3/6 or other p38 activators has not been established, so a question mark accompanies their position. Note also that, Shh binds both Cdo and Ptch1, but the ternary complex shown is hypothetical.
Collectively, these results link cadherin-based cell–cell adhesion to a defined signaling pathway (i.e., Cdo → p38) that directly regulates the activity of a cell-type-specific differentiation program and suggest the following model: When Cdo is associated with ligated cadherins, its intracellular region undergoes a change in conformation and/or posttranslational modification that permits its stable association with Bnip-2 and JLP and, consequently, activation of p38. In contrast, such changes do not occur in Cdo when it binds Shh (Fig. 5E). ShhN-coated beads were somewhat less effective at clustering Cdo than Ncad-coated beads, even though Cdo was presumably bound directly to the ShhN bead and only indirectly to the Ncad bead (via cis interaction with endogenous Ncad, which bound the bead directly). The ability of cadherins to cluster into large adhesive complexes (37) may increase the avidity and/or stability of the association of Cdo and its binding partners at these sites of adhesion.
The results also allow a distinction to be drawn between Cdo’s actions as a putative coreceptor for cadherin and for Hedgehog signaling pathways. In the Hedgehog pathway, Cdo appears to sensitize cells to a given level of ligand, with signaling occurring via the canonical, Smoothened-dependent pathway, and no requirement for the Cdo intracellular region (13, 35). However, in Ncad-initiated signaling, Cdo confers to the cadherin a signaling capability (activation of the p38 pathway) that it does not possess intrinsically and that depends on the Cdo intracellular region for activity. Therefore, Cdo plays mechanistically distinct roles in modulating the signaling output of two different pathways: cytoplasmic signaling in the Ncad pathway and ligand binding in the Shh pathway. Furthermore, the notion of a shared coreceptor raises the possibility for cross-regulation between these pathways, as may occur in development of the cerebral cortex (38). This concept may also extend to other signaling pathways that share membrane-associated components.
The small GTPase Cdc42 initiates an apoptotic signaling pathway in Jurkat T lymphocytes.
Apoptosis plays an important role in regulating development and homeostasis of the immune system, yet the elements of the signaling pathways that control cell death have not been well defined. When expressed in Jurkat T cells, an activated form of the small GTPase Cdc42 induces cell death exhibiting the characteristics of apoptosis. The death response induced by Cdc42 is mediated by activation of a protein kinase cascade leading to stimulation of c-Jun amino terminal kinase (JNK). Apoptosis initiated by Cdc42 is inhibited by dominant negative components of the JNK cascade and by reagents that block activity of the ICE protease (caspase) family, suggesting that stimulation of the JNK kinase cascade can lead to caspase activation. The sequence of morphological events observed typically in apoptotic cells is modified in the presence of activated Cdc42, suggesting that this GTPase may account for some aspects of cytoskeletal regulation during the apoptotic program. These data suggest a means through which the biochemical and morphological events occurring during apoptosis may be coordinately regulated.
PI(3)P and RAB-7 Are Sequentially Enriched on Phagosomal Surfaces
We examined the maturation process of phagosomes containing apoptotic cells in C. elegans . Previously, we reported the transient recruitment of DYN-1 and the incorporation of early endosomes, which are labeled with HGRS-1, the worm homolog of mammalian endosomal protein Hrs, to the surface of phagosomes . Here, we examined two additional molecular events on phagosomal surfaces: the accumulation of PI(3)P [19,20] and the recruitment of small GTPase Rab7 . We monitored both events in developing embryos using modified versions of an established protocol (Materials and Methods) . In all time-lapse experiments described below, we chose to follow the engulfment and degradation of three particular apoptotic somatic cells, C1, C2, and C3, which are located near each other on the ventral side of an embryo and die almost simultaneously during embryogenesis, at approximately 330 min past the first cell division (the first cleavage) (Figure 1C(l) and 1D(a)). ABplaapppa, ABpraapppa, and ABplaapppp, three ventral hypodermal cells, engulf C1, C2, and C3, respectively (Figure 1C(f) and 1D(a)), during their extension to the ventral midline . In addition, all green fluorescent protein (GFP) or monomeric red fluorescence protein (mRFP1)  tagged reporters were expressed specifically in engulfing cells under the control of Pced-1, the ced-1 promoter .
The FYVE domain of C. elegans EEA-1, in a tandem repeat, specifically associates with PI(3)P . The FYVE-FYVE::mRFP1 reporter was detected primarily in cytoplasm as bright puncta, consistent with its endosomal localization (Figure 1C) . The 2xFYVE markers were also evenly distributed in nuclei (Figure 1D(d)). In engulfing cells, we observed bright mRFP1 circles surrounding cell corpses (Figure 1C and 1D). Consistent results have been obtained independently . In embryos that coexpressed Pced-1 ced-1::gfp and Pced-1 2xFYVE::mrfp1, CED-1::GFP, but not 2xFYVE::mRFP1, was enriched on extending pseudopods (Figure 1C). Strikingly, bright mRFP1 circles appeared on phagosomal surfaces within 4.1 ± 1.0 min (n = 16) after the closure of pseudopods (Figure 1C(d), 1C(j), and 1E), indicating that PI(3)P is specifically presented on the surface of nascent phagosomes. Whereas the CED-1::GFP signal rapidly disappeared from phagosomal surface after the enclosure of the phagocytic cup (within 8.4 ± 2.0 min, n = 17) (Figure 1C and 1E), PI(3)P remained on a phagosome until its content was completely degraded (Figure 1D and 1E). PI(3)P thus labels the surface of phagosomes for almost their entire duration.
C. elegans rab-7 encodes RAB-7, a likely ortholog of human Rab7 (Figure S1A and Text S1). In wild-type animals expressing Pced-1 gfp::rab-7, a functional GFP::RAB-7 was detected in the cytoplasm, and a portion of it is enriched on cytoplasmic puncta (Figures S1B and S3D). This punctate localization pattern is consistent with previous reports indicating that RAB-7 is localized to late endosomes and lysosomes [24,25]. In both embryos and adult hermaphrodite gonads, we observed robust GFP::RAB-7 signals around cell corpses (Figure S1C). Quantitative measurements of time-lapse images (Materials and Methods) indicated that the intensity of the GFP signal on phagosomal surfaces could reach as high as 2.5 times that in the cytosol of the same engulfing cell (Figure 1D(o)). In a time-lapse recording of embryos that coexpress GFP::RAB-7 and 2xFYVE::mRFP1, RAB-7 was recruited to the surface of a phagosome approximately 3 min after PI(3)P appeared on the same phagosome (Figure 1D and 1E). After the completion of engulfment, yet prior to the recruitment of RAB-7, a nascent phagosome appeared as a dark hole surrounded by evenly distributed GFP::RAB-7 in the engulfing cell cytoplasm (Figure 1D(h) and 1D(i)). Like 2xFYVE::mRFP1, GFP::RAB-7 persisted on the surface of a phagosome until the phagosome disappeared (Figure 1D and 1E, and Video S1).
To determine whether the enrichment of RAB-7 and PI(3)P was restricted to phagosomes containing C1, C2, and C3, we examined wild-type 1.5-fold to 2-fold stage embryos, which were at 420–460 min after first cleavage . We found that 91% and 73% of cell corpses distinguished under DIC optics are labeled with GFP::RAB-7 and 2xFYVE::GFP, respectively (Figures 2C). These results indicate that both PI(3)P and RAB-7 are enriched on the surface of all phagosomes, and suggest that they may play important roles during phagosome maturation.
Cell Signaling Ligands
Typically, cell signaling is either mechanical or biochemical and can occur locally. Additionally, categories of cell signaling are determined by the distance a ligand must travel. Likewise, hydrophobic ligands have fatty properties and include steroid hormones and vitamin D3. These molecules are able to diffuse across the target cell’s plasma membrane to bind intracellular receptors inside.
On the other hand, hydrophilic ligands are often amino-acid derived. Instead, these molecules will bind to receptors on the surface of the cell. Comparatively, these polar molecules allow the signal to travel through the aqueous environment of our bodies without assistance.
Obesity is a global epidemic that causes morbidity and impaired quality of life. The melanocortin receptor 4 (MC4R) is at the crux of appetite, energy homeostasis, and body-weight control in the central nervous system and is a prime target for anti-obesity drugs. Here, we present the cryo–electron microscopy (cryo-EM) structure of the human MC4R-Gs signaling complex bound to the agonist setmelanotide, a cyclic peptide recently approved for the treatment of obesity. The work reveals the mechanism of MC4R activation, highlighting a molecular switch that initiates satiation signaling. In addition, our findings indicate that calcium (Ca 2+ ) is required for agonist, but not antagonist, efficacy. These results fill a gap in the understanding of MC4R activation and could guide the design of future weight-management drugs.
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Cellular Communication in Yeasts
The first life on our planet consisted of single-celled prokaryotic organisms that had limited interaction with each other. While some external signaling occurs between different species of single-celled organisms, the majority of signaling within bacteria and yeasts concerns only other members of the same species. The evolution of cellular communication is an absolute necessity for the development of multicellular organisms, and this innovation is thought to have required approximately 2.5 billion years to appear in early life forms.
Yeasts are single-celled eukaryotes, and therefore have a nucleus and organelles characteristic of more complex life forms. Comparisons of the genomes of yeasts, nematode worms, fruit flies, and humans illustrate the evolution of increasingly complex signaling systems that allow for the efficient inner workings that keep humans and other complex life forms functioning correctly.
Kinases are a major component of cellular communication, and studies of these enzymes illustrate the evolutionary connectivity of different species. Yeasts have 130 types of kinases. More complex organisms such as nematode worms and fruit flies have 454 and 239 kinases, respectively. Of the 130 kinase types in yeast, 97 belong to the 55 subfamilies of kinases that are found in other eukaryotic organisms. The only obvious deficiency seen in yeasts is the complete absence of tyrosine kinases. It is hypothesized that phosphorylation of tyrosine residues is needed to control the more sophisticated functions of development, differentiation, and cellular communication used in multicellular organisms.
Because yeasts contain many of the same classes of signaling proteins as humans, these organisms are ideal for studying signaling cascades. Yeasts multiply quickly and are much simpler organisms than humans or other multicellular animals. Therefore, the signaling cascades are also simpler and easier to study, although they contain similar counterparts to human signaling. 
Watch this collection(http://openstaxcollege.org/l/bacteria_biofilm) of interview clips with biofilm researchers in “What Are Bacterial Biofilms?”
Structure, Function and Regulation of Tor Complexes from Yeasts to Mammals Part B
Malene Hansen , Pankaj Kapahi , in The Enzymes , 2010
A direct link between the cellular recycling process autophagy and organismal aging was first made in C. elegans (e.g., in long-lived daf-2 /insulin/IGF-1 receptor mutants)  . As in yeast, TOR regulates autophagy in C. elegans, since animals with reduced levels of TOR or daf-15/Raptor heterozygotes have increased levels of autophagy  . Consistent with this, rapamycin also induces autophagy in C. elegans (C. Kumsta and M. Hansen, unpublished observations). This induction in autophagy is critical for the life span extension observed in animals with reduced TOR activity. For example, inactivation during adulthood of genes with autophagy functions, for example, vps-34 and bec-1, are required for daf-15/Raptor heterozygotes to live long  , and tor RNAi fails to extend the life span of short-lived autophagy mutants, including atg-1/unc-51 and atg-18 mutants  . Consistent with an overlap in mechanisms between TOR and dietary restriction, dietary-restricted animals have increased autophagy [34, 41] and require autophagy genes during adulthood to live long [34, 41, 84] . Moreover, the induction of autophagy in dietary-restricted eat-2 mutants requires the transcription factor PHA-4/FOXA [ 34 ], suggesting that autophagy is transcriptionally regulated in response to nutrients. Taken together, these data strongly suggest that TOR, as well as dietary restriction, relies on the upregulation of the autophagy process to extend C. elegans life span.
Mammalian TLRs play an important role in recognizing and removing pathogenic microorganisms, mediating the production of downstream cytokines, and linking innate and acquired immunity.
With deep research, TLR signaling pathways are separated into two groups: a MyD88-dependent pathway that leads to the production of pro-inflammatory cytokines with the quick activation of NF-kappa B and MAPK, and a MyD88-independent pathway associated with the induction of IFN-&beta ＆ IFN-inducible genes and maturation of dendritic cells with slow activation of NF-kappa B ＆ MAPK.
TLRs are mostly located on the cell membrane and mainly sense extracellular pathogen components. TLRs mediated signaling pathways exert functions through their TIR domains. The TIR domains of these receptors (except the TLR3 receptor) can interact with the adapter MyD88 protein. The MyD88 protein further recruits IRAK family proteins. IRAK4 protein recruited into the receptor complex can phosphorylate and activate IRAK1 protein and IRAK2 protein, which promotes the oligomerization of TRAF6 protein, activating the ubiquitin ligase activity of TRAF6. TRAF6 protein, together with the E2 binding enzyme complex UBC13-UEV1A, can catalyze the polyubiquitination, activating TAK1, IKK, and MAPK.
TLR3 and TLR4 interact with TRIF by binding to their respective ligands, double-stranded RNA and lipopolysaccharide, respectively. TRIF subsequently links to TRAF6 and RIP1 proteins, activating the NF-&kappaB signaling pathway. TRIF proteins also bind to IKK-like kinases TBK1 and IKK-ɛ, phosphorylating their substrate IRF3 protein. The phosphorylated IRF3 protein forms a dimer, which enters the nucleus and assembles into an enhancer complex, together with the NF-&kappaB protein and activated transcription factor 2 (ATF2) form. The enhancer complex can induce the expression of IFN-&beta. The TLR4 signaling pathway has been well studied.
Some other TLRs such as TLR7, TLR8, and TLR9 can also induce IFN-I expression. TLR7 and TLR8 bind to viral single-stranded RNA, while TLR9 binds to unmethylated CpG DNA. After binding to the ligands, they can recruit cytoplasmic signal complexes composed of MyD88, IRAKs, and TRAF6 in the cytoplasm.