4. CELL SIGNALLING
All cells receive and respond to specific chemical signals from their environment. This is accomplished by chain of events that mediates the transfer of signals from the cell's exterior to its interior thereby integrating and coordinating the functions of many cells such as changes in enzyme activity, gene expression and ion channel activity, etc.
Cell signaling generally involves:
- Synthesis and release of primary messenger by the signaling molecule.
- Transport and binding of the primary messenger to a specific receptor is expressed by the target cell.
- Transmission of the signal to the target cell’s interior resulting in various changes in cell’s activity.
In multicellular organisms, signaling mechanism can be categorized into three main types – endocrine, paracrine and autocrine signaling.
In endocrine signaling, cells transfer the signal molecules to distantly located target cells from their site of synthesis. It is a long range signaling in which the extracellular signal molecule is transported through the blood stream.
In paracrine signaling, the signaling molecules produced by a cell affect the target cells in neighboring places i.e., in close proximity, thereby altering the activity of those cells. The conduction of an electrical signal between the nerve cells at synapse via neurotransmitter (signal molecule) is an example of paracrine signaling.
In autocrine signaling, cells respond to the same cells that produces it. On stimulation with antigen, T-lymphocytes release some growth factor that in turn further increases the proliferation of T cells, resulting in the amplification of the immune system.
The transformation of extracellular signal to the cells interior is accomplished by the binding of the signal molecule to the surface receptor of the target cell. These receptors are intrinsic membrane proteins having the binding domain, which specifically recognizes the signal molecule (also known as ligand) and the intracellular domain, which is in contact with the cell cytoplasm and modulate activity of the cell. Signal transducing receptors can be divided into three main classes:
- Receptors that can span across the membrane and have intrinsic enzymatic activity. Examples of this class are tyrosine kinases (PDGF, EGF, FGF); serine-threonine kinases (TGFβ receptor); guanylate cyclase (ANP); tyrosine phosphatases (CD45); histidine kinase associated receptors.
- G-protein coupled receptors :- These receptors (7 transmembrane domains) are associated with heterotrimeric G-proteins. The G-proteins get activated when ligand binds with the receptor and initiate signaling cascades. These include pheromone receptors, hormone (glucagon, vasopressin, bradykinin, and angiotensin), odorant receptor, calcitonin receptor, somatostatin and adrenergic receptor.
- Intracellular receptors on binding with the ligand migrate towards the nucleus and modulate gene transcription. Such receptor consists of a ligand binding domain, a DNA binding domain and a transcriptional activator domain. Examples of this class include thyroid and steroid hormone receptors.
4.1. G-protein and G-protein coupled receptors
GPCR constitutes the largest family of cell surface receptors with seven membrane spanning helices. When ligand binds to the receptor, GPCR undergoes a conformational change that activates the heterotrimeric guanine nucleotide regulatory protein (G protein). This further transmits the signal to intracellular targets, eliciting a physiological response. GPCRs are found in all eukaryotes. The signal or primary stimulus can be pheromone, hormone, odorant, light, any neurotransmitter, neuropeptide or an antigen. Despite of extracellular physical and chemical diversity of the signal molecules, all GPCRs share the same basic heptahelical architecture.
Depending on the phylogenetic homology and functional similarity, the GPCR family can be divided into various classes;
In all, GPCRs can be grouped into 6 classes based on sequence homology and functional similarity:
- Class A (or 1) (Rhodopsin-like)
- Class B (or 2) (Secretin receptor family)
- Class C (or 3) (Metabotropic glutamate/pheromone)
- Class D (or 4) (Fungal mating pheromone receptors)
- Class E (or 5) (Cyclic AMP receptors)
- Class F (or 6) (Frizzled/Smoothened)
Glutamate (15 members), Rhodopsin (701 members), Adhesion (24 members), Frizzled receptor (24 members), Secretin receptor family (15 members) known as GRAFS. The largest class by far is class A, which accounts for nearly 85% of the GPCR genes.
GPCRs are integral membrane proteins with serpentine arrangement of α helices. The receptor consists of a single polypeptide with seven transmembrane helical segments (multipass in nature) by forming three extracellular loops (EL-1 to EL-3) and three intracellular loops (IL-1 to IL-3). The long IL-3 loop is involved in the interaction of the receptor with its coupled G-protein. As the ligand approaches the receptor, the seven transmembrane helices arranges themselves and forms a ligand binding pocket where the ligand interacts with the receptor. This further results in conformational changes in the receptor, causing activation of a G-protein.
GPCR are linked with heterotrimeric G-proteins. It is in active state when GTP is bounded to G-protein and in inactive state when bounded to GDP. All heterotrimeric G-proteins contains 3 subunits - α, β and γ. α and γ subunit remains anchored covalently to the plasma membrane while β subunit is conjugated with the γ subunit.
Thus, forming a heterotrimeric complex Gαβγ associated with GDP in inactive form. On binding of a ligand to the GPCR, it results in a conformational change of the receptor and activation of the G-protein complex. The activated Gα exchanges GTP from GDP and leads to the dissociation of Gα-GTP from Gβγ. This reaction is catalyzed by the association of guanine nucleotide exchange activity present in the IL3 of the GPCR itself. The resultant Gα-GTP and Gβγ specifically can bind to various downstream effector proteins of the signal transduction cascade and thereby switches it either on or off in different systems. After the signal propagation, the intrinsic GTPase activity of Gα leads to the hydrolysis of GTP into GDP thereby inactivating the G-protein cascade. The Gα-GDP being inactive reassociates with Gβγ. The total strength of signal amplification by a GPCR is determined by the duration of the ligand-receptor complex and receptor-effector protein complex and the deactivation time of the activated receptor and effectors through intrinsic enzymatic activity.
The rate of GTP hydrolysis can be altered due to the action of another protein Regulators of G-protein Signaling (RGS) which are a type of GTPase-activating protein, or GAP. The activation of a G-protein can trigger activation or inhibition of a particular effector.
The various type of effector proteins effect can be modulated by the G-protein are:
Calcium channels, potassium channels, adenylyl cyclase, phospholipase C (PLC), protein kinases, phosphoinositide 3-kinase (PI3-kinase), β-adrenergic receptor kinase, RhoGEFs (p115-RhoGEF, PDZ-RhoGEF, and LARG) and cGMP- specific phosphodiesterase.
Depending upon the function, G-protein can be of Gs family (stimulates adenylyl cyclase), Gi family (inhibits adenylyl cyclase, activates K+ channels) or Gt family (activates cyclic GMP phosphodiesterase) and others. For example, when the ligand binds the receptor, Gs stimulate the membrane bound adenylyl cyclase (effector molecule). Activated adenylate cyclase synthesizes cAMP from ATP.
4.1.2. cAMP and protein kinase A
Cyclic AMP (cAMP) is a secondary messenger which is formed from cytosolic ATP through adenylate cyclase enzyme. The adenylate cyclase becomes active when a G-protein linked receptor is coupled to Gs and the binding of ligand stimulates Gsα to attach to GTP. This as a result, activates the anchored adenylate cyclase to produce cAMP from ATP. On the other side, Gi inhibits the activity of adenylate cyclase, ceasing the formation of cAMP. So, depending on the type of ligand, the levels of cAMP in the cell can easily be regulated. Epinephrine and glucagon (ligand) stimulates the activation of adenylate cyclase while adenosine inhibits the enzyme and deactivate it.
cAMP is an allosteric activator of Protein kinase A (PKA). PKA regulates the activity of a wide variety of cellular proteins by catalyzing the transfer of terminal phosphate group from ATP to a serine or threonine found in the target protein. When inactive, PKA consists of a complex of two catalytic and two regulatory subunits. The binding of cyclic AMP to the regulatory subunits alters their conformation, causing them to detach from the two catalytic subunits. The released catalytic units thereby can phosphorylate and activate specific substrate protein molecules. The cAMP phosphodiesterase catalyzes the conversion and degradation of cAMP to 5' AMP further shutting down the signal transduction pathway. In some cases, the free catalytic subunits translocate in the nucleus and phosphorylate the transcription factor CREB (cAMP Response Element Binding Protein), leading to expression of specific target genes that contains a CRE (cAMP Response Element). Cyclic AMP inducible gene expression has a crucial role in regulating various physiological responses such as in regulating the cell proliferation and growth for the survival of cells.
4.1.3. Phosphatidylinositol signal pathway
Inositol-1,4,5-trisphosphate (IP3) and Diacylglycerol (DAG) are the secondary messengers, derived from the breakdown of membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2). PIP2 is a relatively uncommon phospholipid confined to the inner side of the plasma membrane. When activated, enzyme phospholipase C-β (PLC-β) catalyze the hydrolysis of PIP2. Four classes of phosphoinositide specific PLCs; PLC-β, PLC-γ, PLC-δ, PLC-ε are expressed by mammals.
The signaling pathway of PIP2 is as follows: the extracellular signal molecule binds with its membrane receptor, resulting in the activation of a specific G protein. The Gq in turn triggers the activation of phospholipase C (PLC-β) that hydrolyses PIP2 to form IP3 and DAG. DAG remains associated with the plasma membrane while the IP3 diffuses into the cytosol. IP3 interacts with the ligand gated Ca+2 channels present in the membrane of smooth endoplasmic reticulum and mitochondria thereby, increasing the Ca+2 level in the cell. The release of Ca+2 in the cytosol plays a critical role in regulating various cell functions.
- DAG along with intracellular Ca+2 activates protein kinase C (PKC), which phosphorylates the target protein at specific serine and threonine groups.
- This further activates CaM kinase pathway by association of Ca+2 with calcium modulated protein calmodulin (CaM) that in turn alters the conformation and activates CaM kinase II.
Calmodulin mediates the regulation of various physiological responses including cell motility, proliferation, differentiation, exocytosis, cytoskeletal assembly and intracellular alteration of both cAMP and calcium concentration.
The effectors of the Gα12/13 pathway are three RhoGEFs (p115-RhoGEF, PDZ-RhoGEF, and LARG), which, when bound to Gα12/13 allosterically, activate the cytosolic small GTPase, Rho. Once bound to GTP, Rho can then go on to activate various proteins responsible for cytoskeleton regulation such as Rho-kinase (ROCK). Most GPCRs that couple to Gα12/13 also couple to other sub-classes, often Gαq/11.
4.1.4. GPCR- Phosphodiesterase signaling pathway in rod cells
cGMP is a multifunctional secondary messenger that carries different cellular processes. It also facilitates the regulation of ion channel conductance, cellular apoptosis, and phosphodiesterase. It plays a pivotal role in photoreception by transforming the visual signals received in the form of light to nerve impulses. This is mediated by Rhodopsin, a G-protein coupled receptor in rod cells of retina of eye. Rhodopsin gets activated when a photon is absorbed, causing isomerization of its bounded chromophore 11- cis- retinal to all trans-retinal form. This results in conformational change in the rhodopsin protein followed by interaction with a G protein, transducin. Transducin (Gt) is a GTP binding protein, can bind to either GTP or GDP. The α subunit remains bounded with βγ in the dark. So, no signal is transmitted in that case. In the presence of light, rhodopsin gets activated and interacts with transducin (Gtαβγ). Gtα gets dissociated from Gtβγ and further Gtα –GTP stimulates the activity of cGMP-phosphodiesterase which converts cyclic GMP to 5'-GMP. Due to this, cGMP level decreases gradually in the cytosol and as a result of which cGMP dependent Na+ gated channels get closed. The Na+ channels that get closed in response to light are also permeable to Ca+2, so calcium level also decreases. This creates a hyperpolarized condition in the retinal cell.
Mechanism of action of cholera and pertussis toxin
Cholera toxin is an oligomeric complex secreted by the bacterium Vibrio cholerae. The protein complex is composed of six subunits: a single copy of the A-subunit and five copies of B-subunits. The A- subunit catalyzes ADP- ribosylation of the α-subunit of Gs protein using intracellular NAD+. Ribosylation causes alteration in α-subunit of Gs protein so that it is incapable of hydrolyzing its bound GTP. As a consequence of which it remains activated for a longer time and stimulates adenylyl cyclase continuously. This further result in an elevation in cGMP levels that leads to a large efflux of chloride ions and water into the intestinal lumen thereby causing severe dehydration associated with other symptoms of cholera.
Pertussis toxin, an enterotoxin is secreted by Bordetella pertussis. The toxin consists of two subunits. Mechanism is same as in cholera but ADP- ribosylation of the α-subunit of Gi protein (inhibitory protein) occurs. So that it prevents the dissociation of Giα from the βγ complex. As a result of this, the Giα remains bounded to GDP and are incapable to inhibit adenylate cyclase.
4.1.5. Gβγ signaling
The primary effectors of Gβγ are various ion channels, such as G-protein-regulated inwardly Rectifying K+ channels (GIRKs), P/Q type voltage gated Ca+2 channel and N-type voltage-gated Ca+2 Channels, as well as some isoforms of adenylate cyclase and phosphorylase-C, along with some Phosphoinositide-3-Kinase (PI3K) isoforms.
4.1.6. G-Protein-independent signaling
Some GPCRs are able to signal without G proteins and some times the heterotrimeric G-proteins may play functional roles independent of GPCRs that’s means they don’t required receptor for the activation of heterotrimer G protein .
- Mitogen-activated protein kinase (ERK-2) is activated by cAMP.
- β2-adrenoceptor also activates the ERK2 pathway after arrestin-mediated uncoupling of G-protein-mediated signaling. Therefore it seems likely that some mechanisms previously believed to be purely related to receptor desensitisation are actually examples of receptors switching their signaling pathway.
4.1.7. GPCR-independent signaling by heterotrimeric G-proteins
Heterotrimeric G-proteins may also take part in non-GPCR signaling. There is evidence for roles as signal transducers in nearly all other types of receptor-mediated signaling, including integrins, receptor tyrosine kinases (RTKs), cytokine receptors (JAK/STATs), as well as modulation of various other "accessory" proteins such as GEFs, Guanine-nucleotide Dissociation Inhibitors (GDIs) and protein phosphatases. There may even be specific proteins of these classes whose primary function is as part of GPCR-independent pathways, termed Activators of G-protein signalling (AGS).
4.1.8. Regulation of GPCR
GPCRs mediated signaling pathways are dynamically regulated in cells. Regulation plays an important role in maintaining the physiological balance in the target cells. This can be accomplished by inhibiting the coupling of GPCR protein (desensitization) and receptor mediated endocytosis (internalization).
After the prolonged period of stimulation, the ability of the receptor to activate adenylate cyclase through Gs is greatly reduced. This is due to the phosphorylation of many Serine and Threonine residues of the internal loop-3 and carboxyl terminal tail of the receptor. The phosphorylation reaction is catalyzed by the enzyme G-protein coupled receptor kinases (GRKs). The GRK'S phosphorylated receptor binds with the Arrestin protein which uncouples the G-protein and the receptor effectively switching it off for a short period of time.
This includes the removal and degradation of the cell surface receptor. This can be accomplished either by undergoing lysosomal degradation or remain internalized through receptor mediated endocytosis. Upon binding of β-arrestin to GPCR, it undergoes a conformational change that acts as a scaffolding protein for an adaptor complex AP-2, that in turn recruits clathrin protein. If several receptors recruit clathrin, they aggregate and invaginate inwardly and finally pinch off from the membrane. Such a process is known as down regulation of the receptors.
G-proteins may terminate their own activation due to their intrinsic GTP→GDP hydrolysis capability. However, this reaction proceeds at a slow rate (≈0.02 times/sec) and thus it would take around 50 seconds for any single G-protein to deactivate if other factors did not come into play
GPCRs are involved in a wide variety of physiological processes. Some examples of their physiological roles include:
- The visual sense: the opsins use a photoisomerization reaction to translate electromagnetic radiation(Light ) into cellular signals. Rhodopsin, for example, uses the conversion of 11-cis-retinal to all-trans-retinal for this purpose
- The sense of smell: receptors of the olfactory epithelium bind odorants (olfactory receptors) and pheromones (vomeronasal receptors)
- Behavioral and mood regulation: receptors in the mammalian brain bind several different neurotransmitters, including serotonin, dopamine, GABA, and glutamate
- Regulation of immune system activity and inflammation: Chemokine receptors bind ligands that mediates intercellular communication between cells of the immune system. Receptors such as histamine binds with inflammatory mediators and engage target cell types in the inflammatory response
- Autonomic nervous system transmission: Both the sympathetic and parasympathetic nervous systems are regulated by GPCR pathways, responsible for control of many automatic functions of the body such as blood pressure, heart rate, and digestive processes
- Cell density sensing: A novel GPCR role in regulating cell density sensing.
- Homeostasis modulation (e.g., water balance).
Retinitis pigmentosa (RP) is an inherited disease characterized by progressive degeneration of the retina and eventual blindness. Retinitis pigmentosa is because of mutations in rhodopsin gene of the rods. Mutation causes premature termination or improper folding of the rhodopsin protein.
Adenoma Mutation : Gain of function mutation causes a type of benign thyroid tumor, called an adenoma. Thyroid stimulating hormones (TSH) released from pituitary glands stimulate the thyroid gland to release thyroid hormone. However in Adenoma because of gain of function mutation thyroid adenomas secrete large quantities of thyroid hormone without having to be stimulated by TSH (the receptor is said to act constitutively). Therefore the TSH receptor constitutively activates a G protein on its inner surface and causes excessive thyroid hormone secretion and excessive cell proliferation that causes the tumor.
4.2. Receptor tyrosine kinase
These are a type of Enzyme- linked receptors with their ligand binding domain on the outer surface and cytosolic domain on the inner surface with intrinsic enzyme activity. Commonly known ligands for receptor tyrosine kinase are insulin, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factors (FGFs), hepatocyte growth factor (HGF), insulin, insulin like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), macrophage-colony-stimulating factor (M-CSF), and all the neurotrophins, including nerve growth factor (NGF).
Mostly RTKs are monomeric containing extracellular domain with ligand binding site, a single hydrophobic transmembrane spanning domain and a cytosolic domain with protein tyrosine kinase activity. When bounded with ligand to the extracellular domain, it can induce dimerization of the receptors. The protein kinase of each receptor monomer then phosphorylates a discrete set of tyrosine residues in the cytosolic domain of its dimer partner, a process termed autophosphorylation. On activation, the receptor can phosphorylate several protein substrates such as phospholipase C-γ (PLC-γ), Src-like type non receptor tyrosine kinase, GTPase activating protein. From the activated RTK, three major pathways through which signal can be transduced are as follows:
- Ras –MAP kinase pathway
- IP3/DAG pathway
- PI3 kinase pathway
4.2.1. Ras-MAP Kinase pathway
Ligand binding induces a conformational change in the receptor that activates the kinase activity and undergoes autophosphorylation. This further trigger multiple signal transduction pathway including the Ras-MAP kinase pathway. The Ras protein belongs to the family of GTPase. It is a small, monomeric GTP binding protein that stimulates a cascade of protein kinases responsible for cellular proliferation and differentiation. Ras acts as a GTPase switch that alters between an inactive GDP-bound form and active GTP-bound form and the reaction is mediated by GEF (Guanine nucleotide Exchange Factor) and GAP (GTPase-Activating Protein). The GEF protein interacts with the Ras-GDP complex resulting in the dissociation of the bound GDP and further activates Ras protein. Similarly on association of GTPase with Ras-GTP triggers the hydrolysis of bounded GTP and deactivates Ras.
The binding of a growth factor to an RTK causes the activation of Ras with the assistance of two cytosolic protein-GRB2 (adaptor protein) and Sos. The Src Homology domain, SH2 domain of GRB2 combines with the distinct phosphotyrosine residue of the receptor while SH3 domain binds with the Sos protein and in turn activates Ras. SH2 domains mediate a large number of phosphorylation-dependent protein–protein interaction. The SH2 domain binding specificity is determined by the amino acid sequence immediately adjacent to the phosphorylated tyrosine residues. For example, the SH2 domain of the Src protein-tyrosine kinase recognizes pTyr-Glu-Glu-Ile, whereas the SH2 domains of PI 3-kinase bind to pTyr-Met-X-Met (inwhich X can be any residue).
Adaptor proteins function as linkers that enable two or more signaling proteins to become joined together as part of a signaling complex. Adaptor proteins contain an SH2 domain and one or more additional protein–protein interaction domains. For instance, the adaptor protein Grb2 contains one SH2 and two SH3 (Src-homology 3) domains. SH3 domains bind to proline-rich sequence motifs. The SH3 domains of Grb2 bind constitutively to other proteins, including Sos and Gab.
Docking proteins, such as IRS, supply certain receptors with additional tyrosine phosphorylation sites. Docking proteins contain either a PTB domain or an SH2 domain and a number of tyrosine phosphorylation sites. Binding of an extracellular ligand to a receptor leads to autophosphorylation of the receptor, which provides a binding site for the PTB or SH2 domain of the docking protein. Once bound together, the receptor phosphorylates tyrosine residues present on the docking protein. These phosphorylation sites then act as binding sites for additional signaling molecules. Docking proteins provide versatility to the signaling process, because the ability of there receptor to turn on signaling molecules can vary with the docking proteins that are expressed in a particular cell.
Ras is anchored at the inner leaflet of the bilayer of the plasma membrane by a covalently attached lipid group. Ras is functionally similar to the heterotrimeric G proteins. However it is monomeric. Ras proteins are present in two different forms: an active GTP-bound form and an inactive GDP bound form. When Ras binds with GTP, it activates downstream signaling proteins. Ras is switched between OFF and ON by binding with GTP and GDP. The mutant version of Ras remains in the “on” position as the hydrolysis of GTP into GDP is prevented and keeping the cell in the proliferative mode. This lead to tumor formation.
4.2.2. The regulation of Ras
- GTPase-activating protein (GAPs) it stimulate hydrolysis of the bound GTP which inactivates the Ras.
- GAPs dramatically shorten the duration of a G protein mediated response. Mutation in GAPgenes cause neurofibromatosis. A disease in which patients develop large numbers of benign tumors (neurofibromas).
- Guanine nucleotide-exchange factors (GEFs)-GEFs replaced the boundGDP with a GTP and convert the Ras into active form.
- Guanine nucleotide-dissociation inhibitors (GDIs). GDIs are proteins that inhibit the release of a bound GDP from a monomeric G protein, thus maintaining the protein in the inactive, GDP-bound state.
Activation of receptor tyrosine kinase can be linked to several downstream signal transduction pathways such as the MAP kinase signaling cascade. MAP kinases (Mitogen Activated Protein kinases) are a family of serine/threonine kinases protein that further regulates cell growth and differentiation. After the activation of Ras, downstream serine/threonine phosphorylation proceeds through the following pattern:
- Ras activation stimulates the activation of Raf-protein. Raf is a serine-threonine kinase. Initiation of Raf leads to the activation of MEK (MAP kinase kinase), a second protein kinase.MEK is a dual-specificity protein kinase that activates and phosphorylates both threonine and tyrosine residues separated by an amino acid. The MEK phosphorylates and activates MAP kinase. This can further phosphorylate various nuclear and cytoplasmic proteins that mediate cellular responses by regulating expression of genes.
- 14different MAPKKKs, 7 different MAPKKs, and 13 different MAPKs have been identified in mammals.
For example, in case of insulin signaling pathway, ligand- bounded receptor gets activated and undergoes autophosphorylation at distinct tyrosine residues. It further phosphorylates the insulin receptor substrates (IRS) on tyrosine residues followed by the binding to the SH2 domain of several target proteins that are directly involved in assisting different effects of insulin.
The compound eye of Drosophila consists of around 750 units called ommatidia. Each ommatidium contains eight photosensitive retinula cells represented as R1-R8 cells. Sevenless (Sev), a receptor tyrosine kinase, controls the development of the R7 cell which is responsible for the vision in ultraviolet light. The mutants that lack functional R7 do not form ommatidia in eyes. A membrane bound protein, Bride of Sevenless (Boss) is expressed on the surface of R8 cells. Boss binds with the Sevenless RTK on the surface of nearby R7 cells. The mutant cells are unable to bind with the Boss and thus, R7 development does not occur.
4.2.3. Signaling by the Insulin Receptor
The blood glucose level in body is maintained by liver and monitored by pancreas. When blood glucose levels fall below a certain level, the alpha cells of the pancreas secrete glucagon. The glucagon acts on hepatocytes throughGPCRs and stimulates the breakdown of glycogen into glucose. This increase the blood glucose level.
Upon sensing the rise of blood glucose level normally after the meal pancreas secretes insulin. Insulin receptor works through RTK pathway.
Each insulin receptor is heterodimer composed of an α and a β chain, which are derived from a single precursor protein by proteolytic processing. The insulin-binding site is present on α chain and it is extracellular in nature. The α and β chains are linked together by disulfide bonds. Normally RTKs are monomeric but insulin receptors are dimeric. After binding of insulin as ligand to insulin receptor, the β chain of receptor is autophosphorylated due to its intrinsic kinase activity. Phosphorylated sites recruit SH2 domain-containing signaling proteins like IRS-1/2. IRSs are characterized by the presence of an N-terminal PH domain, a PTB domain and a long tail containing tyrosine phosphorylation sites.
The PTB domain may interact with phospholipids present at the inside leaflet of the plasma membrane. The PTB domain binds to receptor at phosphorylated sites, and the tyrosine phosphorylationsites provide docking sites for other SH2 domain-containing signaling proteins like PI 3-kinase, Grb2, and Shp2. PI 3-kinase convert PI 3,4-bisphosphate PI(3,4)P2 and PI 3,4,5-trisphosphate (PIP3). PIP3 remain in Inner leaflet of membrane and provide binding sites for PH domain-containing signalling proteins such as the serine-threonine kinases PKB andPDK1.
The glucose transport and glycogen synthesis is regulated by PKB. The glucose transporter GLUT4 carries out insulin-dependent glucose transport from the blood. PI 3-kinase and PKB stimulates the GLUT 4 containing vesicle to fuse with plasma membrane. PKB phosphorylates the GSK-3 a negative regulator of glycogen synthase. This result in an increase in glycogen synthase activity.
4.2.4. IP3/DAG pathway
Receptor tyrosine kinase (RTKs) can trigger various signaling cascades, such as in IP3/DAG pathway. It stimulates the activation of phospholipase C-γ (PLC-γ). On activation of the receptor, the SH2 domain of PLC-γ binds with distinct phosphotyrosine residues, thereby locating the enzyme near to the membrane bound PIP2. PLC-γ induces the breakdown of PIP2, resulting in the generation of two secondary messengers, DAG and IP3. Further more signaling promotes an increase in calcium concentration in the cytosol and to the activation of protein kinase C. This moreover phosphorylates other signal proteins and controls and regulates the physiological response of the cell.
4.2.5. PI-3/kinase pathway
The activated RTK induces phosphoinositide pathway or the PI-3 kinase pathway. PI-3 kinase is made up of two subunits, one comprising two SH2 domain and the other catalytic domain. On binding of SH2 domain of PI-3 kinase with specific phosphotyrosine residue, causes phosphorylation of phosphoinositide at the 3 position of the inositol ring. This results in the generation of PI 3,4-bisphosphate PI(3,4)P2 and PI 3,4,5-trisphosphate (PIP3) which acts as a docking site for several signal transduction pathway.
PIP3 primarily binds with the PH (Pleckstrin Homology) domain of Protein kinase B (PKB, also known as Akt), a serine-threonine kinase. When inactive PKB is located in the cytosol and on binding with the PIP3, PKB interacts with them and is anchored to the cell surface. Recruitment of PDK1 to the plasma membrane. Thus, positioning close to the PKB contributes to a frame in which PDK1 can phosphorylate and activate the serine-threonine kinase activity of PKB. This pathway can regulate the cell cycle including growth, proliferation, differentiation, motility, survival and intracellular trafficking of the cell.
The MAP kinase cascade is required for cells to respond to mating factors. In drosophila, the MAP kinase pathway differentiate the photoreceptors cell into the compound eye and in flowering plants, the pathway initiates a defense against pathogens.
4.2.6. GPCR NO signalling
- Acetylcholine binds to its receptor present on endothelial cell.
- Binding activates PLC which release IP3.
- IP3 increase the concentration of Ca+2 in cytosol by opening the IP3 gated Ca+2 channels present on cell surface.
- Ca+2 bins to calmodulin which activates the nitric oxide synthase.
- The NO formed in the endothelial cell diffuses across the plasma membrane and into the adjacent smooth muscle cells, where it binds and stimulates guanylylcyclase, the enzyme that synthesizes cyclic GMP(cGMP) is Guanylyl cyclase.
- Guanylyl cyclase activation relax the muscle.
- Smooth muscle relaxation causes vasodilation.
NO is involved in various biological processes including anticoagulation, neurotransmission, smooth muscle relaxation, and visual perception.
Nitroglycerine is prescribed by doctors to treat the pain of angina that results from a less blood flow to the heart. Nitroglycerine is metabolized to nitric oxide. NO stimulates the relaxation of the smooth muscles of blood vessels of the heart, increasing blood flow to the heart. During sexual arousal the nerve endings in the penis release NO. NO stimulates the relaxation of the smooth muscles of blood vessels of the penis, increasing blood flow to the penis and enlargement of the organ with blood. Viagra(sildenafil) is an inhibitor of cGMP phosphodiesterase, the enzyme that destroys cGMP. This leads to, increase in the levels of cGMP, which promotes the development and maintenance of an erection.
4.3. JAK-STAT Pathway
The JAK-STAT signaling mediates the transfer of message or signal from the cell exterior to the nucleus through a large number of cytokines, hormones and growth factors causing alteration in the transcription of specific genes. The pathway consists of cytokine receptors, a subtype of enzyme linked receptors that depends on cytoplasmic kinases to transfer signals into the cell. Intracellular activation and multimerization of the receptors occurs when ligand such as interferon, interleukins bind with the receptor. As a result, Jaks (a cytoplasmic tyrosine kinase) associated with the receptor gets activated.
In mammals, four types of Jaks are known- Jak1, Jak2, Jak3, and Tyk - and each is associated with specific cytokine receptors constituting two or more polypeptide chains. The dimerization (in some cases multimerization) brings the associated Jak (Janus kinase) of two receptor units in close proximity assisting both of them to cross-phosphorylate each other, thereby increasing the activity of their tyrosine kinase domains. Phosphorylated tyrosine acts as a docking site for STATs and other signaling pathways. STATs (Signal Transducer and Activator of Transcription) are latent transcription factor that are confined to the cytoplasm when inactive. There are many types of STATs each with a SH2 domain that plays a crucial role in signal transduction. The SH2 domain of the STAT binds with the phosphotyrosine residue of the activated cytokine receptor. Further, the Jak phosphorylate the STAT on tyrosine residue at the C-terminus, leading to its release from the receptor. The SH2 domain of the dissociated STAT facilitates its binding with a phosphotyrosine residue of the second STAT protein resulting in the formation of either a homodimer or a heterodimer. The STAT dimer translocate into the nucleus, where in it binds to the specific regulatory sequences and stimulates their transcription for the survival, proliferation and differentiation of cell.
Besides positive effectors, there are several negative regulators that often shut off the response. Some of them are as follows:
- Suppressors of Cytokine signaling (SOCs) : The activated STAT initiates the transcription of SOCs and ultimately the SOCs protein associates with the phosphorylated Jaks and by this process terminaes the pathway.
- Protein Inhibitors of Activated STAT (PIAS) : The PIAS protein binds with STAT dimers and inhibits the interaction of STAT with the DNA response element, thereby inhibiting the transcription of target proteins.
- PTPs (Protein Tyrosine Phosphatases): PTPs dephosphorylates the effector molecule, making them inactive, thus, negatively regulating the signaling.
4.4. TGF-β Pathway
The transforming growth factor β is a multifunctional enzyme that can either act as hormones, effector molecule, or local mediators to regulate many cellular responses. The ligand for the signaling can be TGFβ's themselves, Bone morphogenetic proteins (BMPs), Anti-müllerian hormone (AMH), Activin and nodal protein. These protein proceeds with the assistance of enzyme linked receptors containing a serine/threonine kinase domain on the cytoplasmic side of the membrane. These receptors mainly comprised of two classes- type I and type II which gets associated in a specific manner, needed for signaling. SARA (The SMAD Anchor for Receptor Activation) and HGS (Hepatocyte Growth factor-regulated tyrosine kinase Substrate) are the protein that further mediates the TGF β pathway. The signaling pathway proceeds as follows:
- TGF- β ligand binds to the type II homodimer causing to phosphorylate and activate type I receptor. Thus, forming a tetrameric complex.
- On activation, the receptor complex binds and phosphorylates regulatory protein, Smad 1, Smad 2, Smad 3. Phosphorylated Smad dissociates from the receptor and combines with the Smad 4.
- The Smad complex dissociates and enters into the nucleus and binds to the specific site in the DNA and regulates the expression of target genes.
The TGF β signaling is involved in various cellular processes including cell growth, cell differentiation, proliferation and apoptosis. The mechanism is regulated by feedback inhibition through several pathways such as clathrin mediated endocytosis, blocking the formation of Smad complex thus, shutting off the TGF- β pathway.
4.5. Intracellular Hormone Receptors
Steroid and thyroid hormone family of receptors works as transcription factors as after hormones binding they activates the gene expression. The steroid-thyroid hormone receptor superfamily [e.g. glucocorticoid (GR), vitamin D (VDR), retinoic acid (RAR) and thyroid hormone TR) receptors] Their receptor is located in cytoplasm and bind their lipophilic hormone ligands in this compartment as these hormones are capable of freely penetrating the hydrophobic plasma membrane. Upon binding ligand the hormone-receptor complex translocates to the nucleus and binds to specific DNA sequences termed hormone response elements (HREs). The binding of the complex to an HRE results in altered transcription rates of the associated gene. Analysis of the human genome has revealed 48 nuclear receptor genes.
Many of these genes are capable of yielding more than one receptor isoform. The nuclear receptors all contain a ligand-binding domain (LBD) and a DNA-binding domain (DBD). Steroid receptor III bind to DNA as homodimers eg estrogen receptor (ER), mineralocorticoid receptor (MR), progesterone receptor (PR), androgen receptor (AR), and the glucocorticoid receptor (GR). Steroid receptor I binds to DNA as heterodimers. The retinoid X receptors (RXRs), the liver X receptors (LXRs), the farnesoid X receptors (FXRs) and the peroxisome proliferator-activated receptors (PPARs) are the example of the receptor that bind with lipophilic ligands just like steroid hormone receptor and thyroid hormone receptors.
The steroid hormones are all derived from cholesterol. Moreover, with the exception of vitamin D, they all contain the same cyclopentanophenanthrene ring and atomic numbering system as cholesterol. Steroids with 21 carbon atoms are known as pregnanes, whereas those containing 19 and 18 carbon atoms are known as androstanes and estranes, respectively. Retinoic acid and vitamin D are not derived from pregnenolone, but from vitamin A and cholesterol respectively remaining all are steroid hormones are derived from pregneolone.
All the steroid hormones exert their action by passing through the plasma membrane and binding to intracellular receptors. The hormone – receptor complex work as transcription factor. The complex moves to nucleus binds to its DNA sequences known as hormone response elements and activates the genes.
4.6. Two component system :
In bacteria and plant, signal transduction mediates by two component system (TCS), involve in cell-cell communication and to respond extracellular signal. In bacteria two component systems is ubiquitous. TCS is not present in human and other mammals thus become target for drug.
Two component system contain a sensor, which is a homodimeric transmembrane protein called Histidine kinase placed, which is having autophosphorylating activity along with a conserved histidine residue and a response regulator located after histidine kinase, which contains a conserve aspartate residue. Histidine kinase (HK) has two domain, one histidine phospho transfer domain, which possesses specific histidine and second ATP binding domain. Response regulator (RR) also had two domain, one conserved receiver domain, which comprises conserved aspartate and second effector domain.
When a ligand comes and binds to the N terminal of histidine kinase, in turn causes the activation of histidine kinase autophosphorylating activity. As a result, it causes transfer of a phosphate residue from ATP to the conserved histidine present in kinase domain present at C terminal. This leads to the transfer of this phosphate from the histidine to conserve aspartate present in present in conserved receiver domain of response regulator. Phosphorylation of aspartate result in conformational change in RR, in turn causes the activation of effector domain of RR, as a result signal get generated to mediate cellular response specifically off or on gene expression.
Histidine kinase also present in hybrid form called hybrid histidine kinase, which histidine kinase also contain one internal receiver domain, as ligand bind to the hybrid histidine kinase, it autophosphorylates itself of histidine by same mechanism. Then transfer this phosphate to internal receiver domain’s aspartate residue ,after that this phosphate transfer to histidine phosphotransfer protein or histidine phosphotransferase, which transfer this phosphate to terminal response regulater containing conserved aspartate residue. This system is called as phosphorelay system.
4.7. Quorum sensing
Quorum sensing defines as a mechanism through which regulation of physiological process (motility, competence, conjugation, symbiosis, virulence, sporulation and antibiotic production) and cooperative activity takes place in bacteria because it control gene expression. Through this mechanism, communication between bacterial cells occurs by sensing and responding a secreted small low molecular weight signal molecule, which is diffusible in nature and known as autoinducer, which concentration define the bacterial cells density, because both had directly proportional correlation. This mechanism is help bacteria to carry out various function like, allow bacterial cells to identify their population density, in formation of biofilms, in colonization of bacteria, during protection against competitors and provide ability to adapt changing environment. Vibrio fischeri, a marine bioluminescent, is the first one into which quorum sensing gets described.
Quorum sensing responsible for initiation of coordinated activity governing gene’s expression, which is done when those gene expression governing transcriptional activator or sensor interact with its respective autoinducer, due to this signalling autoinducer also induce its own gene expression. Quorum sensing carried out in response to the bacterial population density and change according to the fluctuation takes place in bacterial population, in turn change the coordinated activity governing gene’s expression also takes place because in this situation interaction of gene expression governing transcriptional activator or sensor with its autoinducer also change with respect to situation. Alteration in gene expression takes place when autoinducer concentration is detected as minimal threshold stimulatory concentration level. Quorum sensing mechanism is used by both gram negative and gram positive bacteria.
In bacteria three quorum sensing classes present which are mentioned below:
First class is governed by LuxI/LuxR system which possesses acyl-homoserine lactone (AHL) as their signal molecule and this type of quorum sensing present in Gram-negative bacteria. LuxI like protein called ALH synthase responsible for the synthesis of acyl-homoserine lactone (AHL), AHL is formed by the coupling of homocystein moiety of S-adenosylmetionine (SAM) to a specific acyl-acyl carrier protein (acyl-ACP), in this coupling homocystein moiety joins with acyl side chain of acyl-ACP and lactonization of this intermediate result in the formation of acyl-HSL along with release of methylthioadenosine. Unique AHL is produced by each bacterial species as a result of a particular bacterial species member respond and recognise a specific signal molecule. After synthesis it get diffused and get recognised and binded by a cognate LuxR protein, in turn activation of LuxR occur then the complex of AHL-LuxR binds to the promoter of the target gene and transcription of that gene get starts.
This is the diagram of quorum sensing in Gram-negative bacteria, define transcriptional activation require the particular threshold concentration to activate the transcription of gene, below that concentration not any kind of transcription takes place.
Second class governs oligopeptide mediated two component system which possesses small peptide as their signal molecule and this type of quorum sensing present in Gram-positive bacteria. In Gram-positive bacteria autoinducer is not able to cross the plasma membrane and the sensor or receptor of this inducer called autoinducing peptide (AIP- 5to 25 amino acid) are transmembrane protein, here two-component signal transduction system are present which contain receptor of AIP is called histidine kinase protein along with a cytoplasmic response regulator which proceed the signal transduction by mediate the regulation of gene expression via peptide signalling. AIP get secreted into external environment form interior of the cell by ABC transpoter.
Third class governs by luxS encoded autoinducer 2 and this type of quorum sensing present in Gram-negative as well as Gram-positive bacteria.
Now LET'S TALK about the example of Vibrio fischeri, a marine bioluminescent. Vibrio fischeri reside in symbiotic relationship with a number of marine animal host. Vibrio fischeri produces light by the production of luciferase enzyme. Thus called bioluminescent and bacteria produce luminescence which is blue-green light, when bacteria is present in large concentration in response to AHLs quorum sensing. Light production takes place in specialized organ present in marine organism called light organ when bacteria get colonized in high concentration in this light organ but Vibrio fischeri does not produce luminescence when present in free state and this luminesence appears in dark.
Chemotaxis in bacteria
Chemotexis is a phenomenon which explains the movement of bacteria in response to chemical stimulus, in the specific direction. Chemotaxis plays an important role in bacteria’s flagella movement, searching of food and in case of protection like feel for poisons. If movement takes place towards higher concentration of chemical, it called as positive chemotaxis, as reverse, If movement takes place in opposite direction from the higher concentration of chemical, it called as negative chemotaxis. Chemotaxis inducer in motile cell called chemoattractant (chemokines and formyl peptides) and chemorepellent (amino acid, inorganic salts and some chemokines), if chemoattractant is presents cell moves in forward direction and if chemorepellent present then cell moves in opposite direction or away from the chemical. Both chemical perform their signalling by interact with its receptor, which is a transmembrane protein. Chemotaxis performs by two component system, which contains histidine kinase protein as transmembrane receptor along with a cytoplasmic response regulator which proceed the signal transduction by mediate the regulation of gene expression in response to particular chemical.
Flagellar rotation in E.coli governed by chemotaxis and movement of flagella correlated with the swimming behaviour of bacteria, during counter-clockwise flagellar rotation, bacteria move forward direction which is also called run along with this bacteria swim in straight line, this type of movement get achieved because counter-clockwise rotation causes alignment of flagella into a single rotating bundle. During clockwise flagellar rotation, bacteria movement in forward direction get cease along with this bacterium get tumble in place. This type of movement takes place because clockwise rotation breaks the flagella bundle separately, here each flagellum points in separate direction. If chemical gradient is not present, the movement of bacteria is random, in this case bacteria moves forward /run. Thus swims and after some time gets stop, thus gets tumble. If chemical gradient is present, in case of presence of chemoattractant tumble is less frequent and longer run occur or in case of presence of chemorepellent, longer run occur in opposite direction along with less tumble.
Flagellar movement is occured by two component system as mentioned above, here the receptor is known as Methyl-accepting Chemotaxis protein (MCP) and methylation of receptor done by a methyltransferase name CheR, CheW a adaptor protein binds to receptor in one side and bind to CheA to other side , thus linking the CheA with a sensor protein. CheA a sensor histidine kinase possess a conserve histidine residue. When a chemorepellent comes and binds to the MCP in turn activate the MCP, which activate the CheW and which activate the CheA in cascade manner, activated CheA cause autophosphorylation of its own conserve histidine residue and after that CheA transfer it phosphate to CheY, which is a response regulator and possess a conserve aspartate residue, as a result diffusion of ChsY takes place and it interacts with flagellar switch protein FliM or flagellar motor protein, this leads to the change of flagellum rotation from counter-clockwise to clockwise manner.
CheY is responsible for the control of flageller motor. As the change in rotation of single flagellum occurs, it causes disruption of entire flagella bundle, which result in tumble. Phosphorylation state of CheY persists for few sec, and CheY dephosphorylate by CheZ, which is responsible for signal termination and known as Asp specific phosphatise. Inactivation of CheY done by CheZ. Binding of attractant exert opposite effect, it cause inactivation of receptor, in turn phosphorylation of CheA and CheY get decreased, as a result counter clock wise rotation of flagella occurs thus bacteria runs and swims in forward direction. Bacteria get desensitized if higher concentration of ligand is present and which is more than the usual higher concentration.