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Dept. of Natural SciencesUniversity of St. La Salle Bacolod City
RECEPTORSReceptor molecules are proteins with 3 domains: extracellular (cell surface molecule containing a binding site for the ligand, trans-membrane, and cytoplasmic domains. Receptors that bind to protein signal molecules usually have a large extracellular ligand-bindingdomain (light green). This domain, together with some of the trans- membrane segments, binds the protein ligand. Receptors that recognize small signal molecules e.g. adrenaline, have smallextracellular domains, and the ligand usually binds deep within theplane of the membrane to a site that is formed by amino acids from several trans-membrane segments.
G -PROTEINSG Protein-linked receptors havean extracellularN-terminus and a cytosolic C- terminus separated by 7transmembrane α helices connected by peptide loops. One of the extracellular segments has an unique messenger- binding site. The cytosolic loop between the 5th and 6th α helicesspecifically binds a particular G protein. Activated G Proteins bindto enzymes or other proteins and alter the target protein’s activity.
G-Proteins consist of large heterotrimeric, and small monomeric G-Proteins. The G-protein linked receptor changes conformation when a ligand binds to allow association of the heterotrimeric G- proteins (, , and subunits) with the receptor. Ligand-binding causes the Gα subunit to release its bound GDP, pick up a GTP and detach from the complex.
Either the GTP-Gα complex, the Gß- G complex or both bind target protein(s). The Gα will remain an activating messenger until the GTP is hydrolyzed by the Gα subunit. The "inactive" GDP-Gα will then reassociate with the Gß-G complex to rapidly turn down this pathway when the original stimulatory signal is removed. Some G proteins bind K+ or Ca+2 ion channels in neurotransmitters. Some activate kinases (enyzmes that phosphorylate). Some cause either the release or formation of major 2nd messengers such as cyclic AMP (cAMP) and Ca+2 ions.
http://highered.mcgraw- Enzymes hill.com/olc/dl/120107/anim0022.swf activated by G proteins catalyze the synthesis of intracellular 2nd messenger molecules. Because eachactivated enzyme generates many 2nd messenger molecules, the signal is greatly The signal is passed on by the messenger amplified at this molecules, which bind to target proteins step in the and other signaling proteins in the cell pathway. and influence their activity.
http://highered.mcgraw-hill.com/olc/dl/120069/bio07.swf How 2ndmessengers work. (a) The cyclic AMP (cAMP) pathway. An extracellular receptor binds to a signal molecule and, through a G protein, activates the membrane-bound enzyme, adenylyl cyclase. This enzyme catalyzes the synthesis of cAMP, which binds to the target protein to initiate the cellular change. (b) The Ca+2 pathway. An extracellular receptor binds to another signal molecule and, through another G protein, activates the enzyme phospholipase C. This enzyme stimulates the production of inositol trisphosphate (IP3), which binds to and opens calcium channels in the membrane of the ER. Ca+2 is released into the cytoplasm, effecting a change in the cell.
cyclic AMP is a 2nd messenger used by a major class of G proteins. cyclic AMP (cAMP) is generated by adenylylcyclase which is embedded in the plasma membranewith the enzymatic activity in the cytoplasm. Adenylyl cyclase is activated by binding an activated αsubunit of the Gs G-protein. Phosphodiesterasecontinually degrades cAMP so in the absence of theligand and active G-Protein, cAMP levels are reduced.
Protein kinase A (PKA), a cAMP-dependent kinase, is the main intracellular target of cAMP. In thetarget tissue, intracellular effects such as the activation of the cAMP pathway can control a number of cell functions. Localization of PKA to specific regions of the cell by anchoring proteins restricts the effects of cAMP to particular subcellularlocations. One example is activation of PKA to cause the stimulation of glycogen breakdown.http://bcs.whfreeman.com/thelifewire/content/chp15/15020.html
Regulation of glycogen metabolism by cAMP in liver and muscle cells. (a) An increase in cytosolic cAMP activates PKA, which inhibits glycogen synthesisdirectly and promotes glycogen degradation via a protein kinase cascade. At high cAMP, PKA also phosphorylates an inhibitor of phosphoprotein phosphatase (PP). Binding of the phosphorylated inhibitor to PP prevents this phosphatase from dephosphorylating the activated enzymes in the kinase cascade or the inactive glycogen synthase. (b) A decrease in cAMP inactivates PKA, leading to release of the active form of phosphoprotein phosphatase. The action of this enzyme promotes glycogen synthesis and inhibits glycogen degradation.
After activation by cAMP, PKA may move into the nucleus and phosphorylate specificgene regulatory proteins. Once phosphorylated,these proteins stimulate the transcription of a whole set of target genes. This type of signaling pathwaycontrols many processes in cells, ranging from hormone synthesis in endocrine cells to the production of proteins involved in long-term memory in the brain. Activated PKA can also phosphorylate and thereby regulate otherproteins and enzymes in the cytosol.
Activation of gene expression following ligand binding to Gs protein–coupled receptors. Receptor stimulation (1) leads to activation of PKA (2).Catalytic subunits of PKA translocate to the nucleus (3) and there phosphorylate andactivate the transcription factor CREB (4). Phosphorylated CREB associates with the co-activator CBP/P300 (5) to stimulate various target genes controlled by theCRE regulatory element.
G proteins couple receptor activation to the opening of K+channels in the plasma membrane of heartmuscle cells. (A) Binding of the neurotransmitter acetylcholine to its G-protein–linked receptor on heart muscle cells resultsin the dissociation of the Gprotein into an activated ßcomplex and an activated α subunit. (B) The activated ß complex binds to and opens a K+ channel in the heart cell plasmamembrane. (C) Inactivation of the α subunit by hydrolysis of bound GTP causes it to reassociatewith the ß complex to form an inactive G protein, allowing the K+ channel to close.
Hormone-induced activation and inhibition of adenylyl cyclase in adipose cells. Ligand binding to Gs-coupled receptors causes activation of adenylylcyclase, whereas ligand binding to Gi-coupled receptors causes inhibition of theenzyme. The G ß subunit in both stimulatory and inhibitory G proteins is identical; the Gα subunits and their corresponding receptors differ. Ligand-stimulatedformation of active Gα ·GTP complexes occurs by the same mechanism in both Gs and Gi proteins. However, Gsα ·GTP and Giα ·GTP interact differently with adenylyl cyclase, so that one stimulates and the other inhibits its catalytic activity.
Many G proteins use inositoltriphosphate (IP3) anddiacylglyceral (DAG) as 2nd messengers G Protein- linked receptors.
Phospholipase Cactivates 2 signalingpathways. PIP2 is hydrolyzed byactivated phospholipase C to yield IP3 and DAG.IP3 diffuses through the cytosoland triggers the release ofCa2+ from the ER by binding toand opening special Ca2+ channels in theER membrane. The large electrochemicalgradient for Ca2+ causes Ca2+ to rush out into the cytosol. Togetherwith Ca2+, the membrane-bound DAG helps to activate the enzymeprotein kinase C (PKC), which is recruited from the cytosol to thecytosolic face of the plasma membrane.
Integrated regulation of glycogenolysis mediated by several 2nd messengers. (a) Neuronal stimulation of striated muscle cells orepinephrine binding to - adrenergic receptors on their surfaces leads to increased cytosolic concentrations of the second messengers Ca+2 or cAMP, respectively. The key regulatory enzyme, glycogen (b) In liver cells, ß-adrenergic stimulation leads to phosphorylase kinase increased cytosolic concentrations of cAMP and two other (GPK), is activated by second messengers, diacylglycerol (DAG) and inositol Ca+2 ions and by a 1,4,5-trisphosphate (IP3). Enzymes are marked by white cAMP dependent boxes. (+) activation of enzyme activity, (-) inhibition.protein kinase A (PKA). http://highered.mcgraw-hill.com/olc/dl/120109/bio48.swf
Disruption of G Protein signaling causes several human diseases. Vibrio cholerae secretes the cholera toxin which alters salt and fluid in the intestine normally controlled by hormones that activate Gs G-Protein to increase cAMP. The cholera toxin enzymatically changes Gs so that it is unable to convert GTP to GDP. Gs can not then be inactivated and cAMP levels remain high causing intestinal cell to secrete salt and water. Eventually dehydration can lead to death (cholera).
TRANSFORMING GROWTH FACTOR-ß TGF-ß receptors activate gene regulatory proteins directly at the plasma membrane. These receptor serine/ threonine kinases autophosphorylate themselves and recruit and activate cytoplasmic gene regulatory proteins called SMADs. The SMADs then dissociate from the receptors and bind to other SMADs, and the complexes then migrate to the nucleus, where they stimulate transcription of specific target genes.
TGF-Smad signaling pathway. (1) In some cells, TGF binds to the type III TGF receptor (RIII), which presents it to the type II receptor (RII). (2) In other cells, TGF binds directly to RII, a constitutively phosphorylated and active kinase. (3) Ligand-bound RII recruits and phosphorylatesthe juxtamembrane segment of the type I receptor (RI), which does not directly bind TGF. This releases the inhibition of RI kinase activity that otherwise is imposed by the segment of RI between the membrane and kinase domain. (4) Activated RI then phosphorylates Smad3 or another R-Smad, causing a conformational change that unmasks its nuclear-localization signal (NLS). (5) Two phosphorylated moleculesof Smad3 interact with a co-Smad (Smad4), which is not phosphorylated, and with importin (Imp-), forming a large cytosolic complex.(5-6) After the entire complex translocates into the nucleus, RanGTP causes dissociation of Imp.(7) A nuclear transcription factor (e.g., TFE3) then associates with the Smad3/Smad4 complex, forming an activation complex that cooperatively binds in a precise geometry to regulatory sequences of a target gene. At the bottom is the activation complex for the gene encoding plasminogen activator inhibitor (PAI-1).
Oncoproteins (e.g., Ski and SnoN) and I-Smads (e.g., Smad7) act as negative regulators of TGF signaling. TGF signaling generally inhibits cell proliferation. Loss of various components of the pathway contributes toabnormal cell proliferation and malignancy. Schematic model of Ski-mediated down-regulation of the response to TGF stimulation. Ski binds to Smad4 in Smad3/Smad4 or Smad2/Smad4 signaling complexes and may partially disrupt interactions between the Smad proteins. Ski also recruits a protein termed N-CoR that binds directly to mSin3A, which in turn interacts with histone deacetylase (HDAC), an enzyme that promotes histone deacetylation. As a result, transcription activation induced by TGF and mediated by Smad complexes is shut down.
Blocking Growth Factors and Receptors Active growth receptors cause cell division by activating the MAP kinase pathway, and keep cells alive by activating PKB. Turning off growth factor receptors therefore tends both to stop cells dividing and to kill them. The drug trastuzumab is effective in slowing down the progression of breast cancer because it prevents a growth factor (RGF) from binding to its RTK. This slows cell division and promotes cell death in cancer cells.
CYTOKINE RECEPTORS Cytokine receptors are associated with cytoplasmic tyrosine kinases. Binding of a cytokine to its receptors causes the associated tyrosine kinases (called Janus kinases, or JAKs) to cross-phosphorylate and activate one another. The activated kinases then phosphorylate the receptor proteins on tyrosines. Gene regulatory proteins called STATs (signal transducers andactivators of transcription) present in the cytosol then attach to the phosphotyrosines on the receptor, and the kinases activate these proteins. The STATs then dissociate from the receptor proteins, dimerize and activate the transcription of specific target genes.
JAK-STAT signaling pathway. Following ligand binding to a cytokinereceptor and activation of an associatedJAK kinase, JAK phosphorylates several tyrosine residues on the receptor’s cytosolic domain. After an inactive monomeric STAT transcription factor binds to a phosphotyrosine in the receptor, it is phosphorylated by active JAK. Phosphorylated STATs spontaneously dissociate from the receptor and spontaneously dimerize. Because the STAT homodimer has 2 phosphotyrosine–SH2 domaininteractions, whereas the receptor-STAT complex is stabilized by only one such interaction, phosphorylated STATs tend not to rebind to the receptor. The STAT dimer, which has two exposed nuclear- localization signals (NLS), moves into the nucleus, where it can bind to promoter sequences and activate transcription of target genes.
Overview of signal-transduction pathways triggered by ligand binding to the erythropoietin receptor (EpoR), a typical cytokine receptor. Four major pathways can transduce a signal from the activated, phosphorylated EpoR-JAK complex. Each pathway ultimately regulates transcription of different sets of genes. (a) In the most direct pathway, the transcription factor STAT5 is phosphorylated and activated directly in the cytosol. (b) Binding of linker proteins (GRB2 or Shc) to an activated EpoR leads to activation of the Ras–MAP kinase pathway. (c, d) Twophosphoinositide pathways are triggered by recruitment of phospholipase C and PI-3 kinase to the membrane following activation of EpoR. Elevated levels of Ca2 and activated protein kinase B also modulate the activity of cytosolic proteins that are not involved in control of transcription.
Signaling from cytokine receptors is terminated by the phosphotyrosine phosphatase SHP1 and several SOCS proteins.Two mechanisms for terminating signal transduction from the erythropoietin receptor(EpoR). (a) SHP1, a protein tyrosine phosphatase, is present in an inactive form in unstimulatedcells. Binding of an SH2 domain in SHP1 to a particular phosphotyrosine in the activated receptorunmasks its phosphatase catalytic site and positions it near the phosphorylated tyrosine in the lipregion of JAK2. Removal of the phosphate from this tyrosine inactivates the JAK kinase. (b) SOCS proteins, whose expression is induced in erythropoietin-stimulated erythroid cells, inhibit or permanently terminate signaling over longer time periods. Binding of SOCS to phosphotyrosineresidues on the EpoR or JAK2 blocks binding of other signaling proteins (left). The SOCS box can also target proteins such as JAK2 for degradation by the ubiquitin proteasome pathway (right). Similar mechanisms regulate signaling from other cytokine receptors.
Studies with mutant mice reveal that both the erythropoietin receptor (EpoR) and JAK2 are essential for development of RBC. Mice in which both alleles of the EpoR or JAK2 gene are “knocked out” develop normally until embryonic day 13, at which time they begin to die of anemia due to the lack of RBC- mediated transport of oxygen to the fetal organs. The red organ in the wild- type embryos (/) is the fetal liver, the major site of RBC production at thisdevelopmental stage. The absence of color in the mutant embryos (/) indicates the absence of RBC containing hemoglobin. Otherwise the mutant embryosappear normal, indicating that the main function of the EpoR and JAK2 in early mouse development is to support RBC production.
RECEPTOR TYROSINE KINASES RTKs have a ligand-binding domain on the exterior, a single transmembrane domain, and the enzyme active site on the cytoplasmic domain.The RTK can be comprisedof either one protein or two proteins: a receptor and a tyrosine kinase. The separate nonreceptor tyrosine kinase has acytosolic tail that containstyrosine residue targets of the enzyme activity.
Cytokine receptors and receptor tyrosine kinases, transduce signals via their associated or intrinsic protein tyrosine kinases. The cytosolic domain of RTKs contains a protein tyrosine kinase catalytic site (1). In both types of receptor, ligand binding causes a conformational change that promotes formation of a functional dimeric receptor, bringing together 2 intrinsic or associatedkinases, which then phosphorylate each other on a tyrosine residue in the activation lip (2). Phosphorylation causes the lip to move out of the kinase catalytic site, thus allowing ATP or a protein substrate to bind. The activated kinase then phosphorylates other tyrosine residues in the receptor’s cytosolic domain (3). The resulting phosphotyrosines function as docking sites for various signal-transduction proteins.
The cytosolic domain of cytokine receptors associates with a separate JAK kinase (1). In both types of receptor, ligand binding causes a conformational change that promotes formation of a functional dimeric receptor, bringing together 2 intrinsic or associated kinases, which then phosphorylate eachother on a tyrosine residue in the activation lip (2). Phosphorylation causes the lip to move out of the kinase catalytic site, thus allowing ATP or a protein substrate to bind. The activated kinase then phosphorylates other tyrosineresidues in the receptor’s cytosolic domain (3). The resulting phosphotyrosines function as docking sites for various signal-transduction proteins.
Cytosolic proteins with SH2 (purple) or PTB (maroon) domains can bind to specific phosphotyrosine residues in activated RTKs or cytokinereceptors. In some cases, these signal-transduction proteinsthen are phosphorylated by the receptor’s intrinsic or associated protein tyrosinekinase, enhancing their activity. Certain RTKs and cytokine receptors utilize multidocking proteins such as IRS-1 to increase the number of signaling proteins that are recruited and activated.Subsequent phosphorylation of Recruitment of signal-transduction the IRS-1 by receptor kinase proteins to the cell membrane by activity creates additional binding to phosphotyrosine residues in docking sites for SH2- activated receptors. containing signaling proteins.
Activation of a receptor tyrosine kinase (RTK) stimulates the assembly ofan intracellular signaling complex that lead to cell growth, proliferation or differentiation. Binding of a signal molecule to the extracellular domain of RTK causes 2 receptor molecules to associate into a dimer. Dimer formationbrings the kinase domains of each intracellular receptor tail into contact withthe other; this activates the kinases and enables them to phosphorylate eachother on several tyrosine side chains. Each phosphorylated tyrosine serves as a specific binding site for a different intracellular signaling protein, which then helps relay the signal to the cell’s interior.
RTKs can activate several signal transduction pathways at once, including inositol-phospholipid-calcium pathway and the Ras pathway. Ras is an intracellular GTPase switch protein that acts downstream from most RTKs. Like G, Ras cycles between an inactive GDP-bound form and an active GTP-bound form. Ras cycling requires the assistance of two proteins, a guanine nucleotide–exchange factor (GEF) and a GTPase-activating protein (GAP). RTKs are linked indirectly to Ras via two proteins: GRB2, an adapter protein, and Sos, which has GEF activity The SH2 domain in GRB2 binds to a phosphotyrosine in activated RTKs, while its two SH3 domains bind Sos, thereby bringing Sos close to membrane-bound RasGDP and activating its nucleotide exchange activity. Binding of Sos to inactive Ras causes a large conformational change that permits release of GDP and binding of GTP, forming active Ras. GAP, which accelerates GTP hydrolysis, is localized near RasGTP by binding to activated RTKs.
Activation of Ras following ligand binding to receptor tyrosine kinases (RTKs).The receptors for epidermal growth factor (EGF) and many other growth factors are RTKs. The cytosolic adapter protein GRB2 binds to a specific phosphotyrosine on an activated, ligand-bound receptor and to the cytosolic Sos protein, bringing it near its substrate, theinactive Ras-GDP. The GEF activity of Sos then promotes formation of active RasGTP. Note that Ras is tethered to the membrane by a hydrophobic farnesyl anchor.
In unstimulated cells,Ras is in the inactiveform with bound GDP;binding of a ligand to itsRTK or cytokine receptorleads to formation of the active RasGTP complex.(1) Activated Ras triggers the downstream kinase cascade(2-6), culminating in activation of MAP kinase (MAPK). Inunstimulated cells, binding of the 14-3-3 protein to Rafstabilizes it in an inactive conformation. Interaction of theRaf N-terminal regulatory domain with RasGTP relievesthis inhibition, results in dephosphorylation of one of theserines that bind Raf to 14-3-3, and leads to activation ofRaf kinase activity (2-3). Note that in contrast to manyother protein kinases, activation of Raf does not depend on Kinase cascade thatphosphorylation of the activation lip. After inactive RasGDP transmits signalsdissociates from Raf, it presumably can be reactivated by downstream from activatedsignals from activated receptors, thereby recruiting RAS protein to MAP kinase.additional Raf molecules to the membrane.
Induction of gene transcription by activated MAP kinase. In the cytosol, MAPK phosphorylates and activates the kinase p90RSK,which then moves into the nucleus and phosphorylates the SRF transcription factor. After translocating into the nucleus,MAPK directly phosphorylates thetranscription factor TCF. Together, these phosphorylation events stimulate transcription of genes (e.g., c-fos) that contain an SRE sequence in their promoter. Byaltering the levels and activities of transcription factors, MAPK leads to altered transcription of genes that are important for the cell cycle. Genes on 22q11, 1q42, and 19p13 affect the MAPK/ERK pathway and are associated with schizophrenia, bipolar syndrome, and migraines.
RAS MUTATIONS, CANCER, AND DRUGS Mutations that stimulate cell proliferation by making Ras constantly active are a common feature of cancers. Like other GTPases, Ras turns itself off by hydrolyzing its bound GDP. Mutant Ras without GTPase activity is therefore always in the ON state and will be activating the pathway that terminates in MAPK and cell division at all times, even in the absence of a growth factor. Such mutant forms of Ras are found in about 20% of all human cancers. Many compounds can inhibit steps in the MAP/ERK pathway, and therefore are potential drugs for treating cancer (e.g. sorafenib - a Raf kinase inhibitor and R115777- prevents Ras from activating MAPK) Protein microarray analysis can be used to detect subtle changes in protein activity in signaling pathways.
Yeast and higher eukaryotes contain multiple MAP kinase pathways that are triggered by activation of various receptor classes including G protein–coupled receptors. Different extracellular signals induce activation of different MAP kinases, which regulate diverse cellular processes. The upstream components of MAP kinase cascades assemble into large pathway-specific complexes stabilized by scaffold proteins. This assures that activation of one pathway by a particular extracellular signal does not lead to activation of other pathways containing shared components.
Kinase cascade that transmits signals downstream frommating factor receptors in S. cerevisiae.The receptors for yeast αand mating factors arecoupled to the same trimeric G protein.Ligand binding leads to activation anddissociation of the G protein. In theyeast mating pathway, the dissociatedG activates a protein kinase cascadeanalogous to the cascade downstreamof Ras that leads to activation of MAPkinase. The final component, Fus3, isfunctionally equivalent to MAP kinase(MAPK) in higher eukaryotes.Association of several kinases with theSte5 scaffold contributes to specificityof the signaling pathway by preventingphosphorylation of other substrates.
Many RTKs and cytokine receptors can initiate the IP3/DAG signaling pathway by activating phospholipase C (PLC), a different PLC isoform than the one activated by G protein–coupled receptors. Activated RTKs and cytokine receptors can initiate another phosphoinositide pathway by binding PI-3 kinases, thereby allowing the catalytic subunit access to its membrane bound phosphoinositide (PI) substrates, which are phosphorylated at the 3rd position. The PH domain in various proteins binds to PI 3- phosphates, forming signaling complexes associated with the plasma membrane. Protein kinase B (PKB) becomes partially activated by binding to PI 3-phosphates. Its full activation requires phosphorylation by another kinase (PDK1), which also is recruited to the membrane by binding to PI 3- phosphates.
Activated PKB promotes survival of many cells by directly inactivating several pro-apoptotic proteins and down-regulating expression of others. Signaling via the PI-3 kinase pathway is terminated by the PTEN phosphatase, which hydrolyzes the 3-phosphate in PI 3-phosphates. Loss of PTEN, a common occurrence in human tumors, promotes cell survival and proliferation. A single RTK or cytokine receptor often initiates different signaling pathways in multiple cell types. Different pathways may be essential in certain cell signaling events but not in others.
Recruitment and activation of protein kinase B (PKB) in PI-3 kinase pathways. In unstimulated cells, PKB is in the cytosol with its PH domain bound to the catalytic domain, inhibiting its activity. Hormone stimulation leads to activation of PI-3 kinase and subsequent formation of phosphatidylinositol (PI) 3-phosphates.The 3-phosphate groups serve as docking sites on the plasma membrane for the PH domain of PKB and another kinase, PDK1. Full activation of PKB requiresphosphorylation both in the activation lip and at the C-terminus by PDK1.
The cytosolic domain of Eph receptors has tyrosine kinase activity. Within the Eph receptor family, the receptors exhibit some 30–70% homology in their extracellular domains and 65–90% homology in their kinase domains. Their ligands, the ephrins, either are linked to the membrane through a hydrophobic GPI anchor (class A) or are single-pass transmembrane proteins (class B). The core domains of various ephrin ligands show 30–70 % homology. Ephrin-B ligands and their receptors General structure of EPH can mediate reciprocalRECEPTORS and their ligands. signaling.
Like other RTKs, the INSULIN RECEPTOR has an extracellular domain that binds the transmitter, and a cytosolic domain with a tyrosine kinase activity. Unlike growth factor receptors, it exists as a dimer even in the absence of its ligand. After a large meal, the activatedreceptor translocates glucose across plasma membranes for conversion to glycogen.
RECEPTORS THAT ARE ION-CHANNELS Ligand-gated ion channelsLigands: neurotransmitters (acetylcholine,glutamate), cGNP, physical stimuli (touch,stretching), IP3 (receptor in ER membrane)Receptors: 4 or 5 subunits with a homologoussegment in each subunit lining the ion channelSignal transduction: (1) Localized changes inmebrane potential due to ion influx, (2)elevation of cytosolic Ca+2
LIGAND-GATED ION CHANNELSThese are mechanically gated channels which oftenhave cytoplasmic extensions that link the channel tothe cytoskeleton. They open on stimulation to allow passage of Na+, K+, Cl+ or Ca+2 ions.
INTRACELLULAR RECEPTOR PATHWAYS Nitric Oxide PathwayLigands: Nitric Oxide (NO)Receptor: Cytosolic guanylyl cyclaseSignal transduction: generation of cGMP Nuclear Receptor PathwaysLigands: lipophilic molecules including steroidhormones, thyroxine, retinoids, and fatty acids inmammals and ecdysone in DrosophilaReceptors: highly conserved DNA-binding domain,somewhat conserved hormone-binding domain, anda variable domain; located within nucleus or cytosolSignal Transduction: Activation of receptor’stranscription factor activity by ligand binding
NITRICOXIDE (NO) PATHWAYRegulation of contractility of arterial smooth muscle by nitric oxide (NO) and cGMP. Upon activation by acetylcholine, NO diffuses from the endothelium and activates an intracellular NO receptor with guanylyl cyclase activity in nearby smooth muscle cells.The resulting rise in cGMP leads to activation of protein kinase G (PKG), relaxation of themuscle, and thus vasodilation. The cell-surface receptor for atrial natriuretic factor (ANF)also has intrinsic guanylyl cyclase activity. Stimulation of this receptor on smooth muscle cells also leads to increased cGMP and subsequent muscle relaxation.
Operational model of the Wnt signaling pathway.(a) In the absence of Wnt, the kinase GSK3 constitutively phosphorylates -catenin. Phosphorylated ß-catenin isdegraded and hence does not accumulate in cells. Axin is ascaffolding protein that forms a complex with GSK3, ß- catenin, and APC, whichfacilitates phosphorylation of ß-catenin by GSK3 by an estimated factor of >20,000. The TCF transcription factor in the nucleus acts as a repressor of target genes unless altered by Wnt signal transduction. b) Binding of Wnt to its receptor Frizzled (Fz) recruits Dishevelled (Dsh) to the membrane. Activation of Dsh by Fz inhibits GSK3, permitting unphosphorylated - catenin to accumulate in the cytosol. After translocation to the nucleus, ß-catenin may act with TCF to activate target genes or, alternatively cause the export of TCF from the nucleus and perhaps its activation in cytosol.
(b) In the presence of Hh, inhibition of Smo by Ptc is relieved. Signaling from Smo causes hyperphos- phorylation of Fu and Cos2, and disassociation of the Fu/Cos2/Ci complex from microtubules. This leads to the stabilization of a full- length, alternately modified Ci, which functions as a transcriptional activator in conjunction with CREB binding protein (CBP). The exact membrane compartments in which Ptc and Smo respond to Hh and function are unknown; Hh signal causes Ptc to move from the surface to internal compartments while Smo does the opposite. Operational model of the Hedgehog (Hh) signaling pathway. (a) In the absence of Hh, Patched (Ptc) protein inhibits Smoothened (Smo) protein by anunknown mechanism. In the absence of Smo signaling, a complex containing the Fused (Fu),Costal-2 (Cos2), and Cubitis interuptus (Ci) proteins binds to microtubules. Ci is cleaved in a process requiring the ubiquitin/ proteasome-related F-box protein Slimb, generating the fragment Ci75, which functions as a transcriptional repressor.
Notch/Delta signaling pathway. The extracellular Binding of Notch to itssubunit of Notch on the responding cell is noncovalently ligand Delta on an associated with its transmembrane-cytosolic subunit. adjacent signaling cell (1) first triggers cleavage of Notch by the membrane-bound metalloprotease TACE (tumor necrosis factor alpha converting enzyme), releasing the extracellular segment (2). Presenilin 1, an integral membrane protein, then catalyzes an intramembrane cleavage that releases the cytosolic segment of Notch (3). Following translocation to the nucleus, this Notch segment interacts with several transcription factors that act to affect expression of genes that in turn influence the determination of cell fate during development (4).
Stimulation by TNFα or IL-1 induces activation of TAK1 NFKß signaling pathway kinase (1), leading to activation In resting cells, the dimeric of the trimeric IKB kinase (2a). transcription factor NFKß, composed of Ionizing radiation and other p50 and p65, is sequestered in the stresses can directly activate cytosol, bound to the inhibitor IKB. IKB kinase by an unknown mechanism (2b). Following phosphorylation of IKB by IKB kinase and binding of E3 ubiquitin ligase (3), polyubiquitination of IKB (4) targets it for degradation byproteasomes (5). The removal of IKB unmasks the nuclear- localization signals (NLS) inboth subunits of NFKB, allowing their translocation to the nucleus (6). Here NFKB activates transcription of numerous target genes (7),including the gene encoding the subunit of IKB, which acts to terminate signaling.
The NFKB transcription factor regulates many genes that permit cells to respond to infection and inflammation. In unstimulated cells, NFKB is localized to the cytosol, bound to an inhibitor protein, I-B. In response to extracellular signals, phosphorylation- dependent ubiquitination and degradation of IKB in proteasomes releases active NFKB, which translocates to the nucleus. Upon binding to its ligand Delta on the surface of an adjacent cell, the Notch receptor protein undergoes two proteolytic cleavages. The released Notch cytosolic segment then translocates into the nucleus and modulates gene transcription. Presenilin 1, which catalyzes the regulated intramembrane cleavage of Notch, also participates in the cleavage of amyloid precursor protein (APP) into a peptide that forms plaques characteristic of Alzheimer’s disease.
Proteolytic cleavage of APP, a neuronal plasma membrane protein. (Left ) Sequential proteolytic cleavage by α-secretase (1) and -secretase (2) produces an innocuous membrane-embedded peptide of 26 amino acids. -Secretase is a complex of several proteins, but the proteolytic site that catalyzes intramembrane cleavage probably resides within presenilin 1. (Right) Cleavage in the 1 2 extracellular domain by -secretase (1) followed by cleavage within the membrane by -secretase generates the 42-residue A42 peptide that has been implicated in formation of amyloid plaques in Alzheimer’s disease. In both pathways the cytosolic segment of APP is released into the cytosol, but its function is not known.
Osteoclasts initially bind to bone via integrin-mediated Bone resorption and itspodosomes. The subsequent activation of an osteoclast regulation by interaction withneighboring osteoblasts via the trimeric membrane proteins RANKL and RANK induces cytoskeletal reorganization, leading to formation of a specialized tight seal with bone. The activated osteoclast secretes into the extra-cellular space generated bythis seal a corrosive mixture of HCl and proteases that resorbs the bone . Osteoblasts can suppressbone resorption by secreting osteoprotegerin (OPG). Binding of this decoy receptor to RANKL blocks RANKL binding to RANK on osteoclasts and thus their activation.
Signaling pathways can be highly interconnected. Pathways from G-protein–linked receptors via adenylcyclase and via phospholipase C,and from enzyme-linked receptors viaphospholipase C and viaRas. The protein kinases in these pathways phosphorylate many proteins, including those belonging to the other pathways. Theresulting dense network of regulatory interconnections is symbolized by the red arrows radiating from each kinase shaded in yellow.
Networking of ECM, CAMs, junctional complexes, signaltransductionpathways and the genetic machinery:Facebook, Twitter, and other social networking sitespale in comparison!
CANCER and SIGNALTRANSDUCTION Oncogenes are calledproto-oncogenes (genes able to becomeoncogenes when altered bymutation to their cancer-causing condition). Shown are theprincipal classes of proto-oncogenes, with some typical representatives indicated.
Detailed knowledge of the signaling pathways involved and the structure of their constituent proteins will continue to provide important molecular clues for the design of specific therapies. Despite the close structural relationship between different signaling molecules (e.g., kinases), recent studies suggest that inhibitors selective for specific subclasses can be designed. In many tumors of epithelial origin, the EGF receptor exhibits constitutive (signal-independent) protein tyrosine kinase activity, and a specific inhibitor of this kinase (Iressa™) has proved useful in the treatment of several such cancers. The extracellular domains of many cell-surface receptors can now be produced by recombinant DNA techniques and have potential as therapeutic decoy receptors. Already in use is such a decoy receptor for TNF-α, which binds excess TNF-α associated with rheumatoid arthritis and other inflammatory diseases.
Drugs that target other signal-transducing proteins may be useful in controlling their abnormal activities. For example, inhibitors of farnesyl transferase (farnesyl groups anchor Ras to cell membranes) are being tested as therapeutic agents in cancers caused by expression of constitutively active Ras proteins. Detailed structural studies of the interaction between signal-transducing proteins offer exciting possibilities for designing new types of highly specific drugs. For instance, knowledge of the interface between the Sos and Ras proteins or between Ras and Raf could provide the basis of a drug that blocks activation of MAP kinase. As more signaling pathways become understood at a molecular level, additional targets for drug development will undoubtedly emerge.