Multicellular organism is a busy but well-organized cell community，in which complex cell communication and cellular signal transduction are carried out, playing an important role for multicellular organism to coordinate its function.

Signal transduction is the process by which a chemical or physical signal is transmitted through binding with receptors located on membrane or cytoplasm as a series of molecular events, most commonly protein phosphorylation catalysed by protein kinases, which ultimately results in a cellular response.

Extracellular signals received by cells include both physical and chemical signals. Of them, the most important is a large family of biological substances, which is called the first messenger and secreted by cells, regulating the function of organism and provide signals of communication among cells.

According to the characteristics and action pattern of extracellular signals, chemical signals can be classified into three groups:

A neurotransmitter is a chemical substance released from presynaptic terminals, which will bind to specific receptors in the postsynaptic cell membrane. For example, norepinephrine, acetylcholine and so on.

Function characteristics: operating with short distance within short time.

A hormone is a class of signaling molecules secreted by endocrine cells that are transported by the circulatory or lymphatic system to target distant organs to regulate physiology and behaviour. For example, arenaline, insulin, thyroid hormone and so on.

Function characteristics: operating with long distance, long lasting time and large scope.

Local chemical mediator is the biological substances produced and secreted by specific cells, released into extracellular fluid and act upon, proximal target cell (paracrine) or itself (autocrine) through local spreading effects, without entering blood. For example, growth factors, prostaglandins (PGs) and so on.

Functional characteristics: small scope with slow effect.

A receptor is a protein molecule located intracellular or on cell membrane, which can specifically recognize and bind with chemical signals from outside a cell. These chemical signals are refferred to as ligands. When such chemical signals bind to a receptor, they cause some form of cellular/tissue response, e.g. a change in the electrical activity of a cell.

Receptor proteins can be classified by their location. Transmembrane receptors include ion channel-linked (ionotropic)receptors, G protein-linked (metabotropic)hormone receptors, and tyrosine-specific protein kinase receptor. Intracellular receptors are those found inside the cell, and include cytoplasmic receptors and nuclear receptors.

Transmembrane receptor is glycoprotein laid on cell membrane. These proteins are typically composed of at least two different domains: a transmembrane domain which includes the ion pore, and an extracellular domain which includes the ligand binding location.

Ligand-gated ion channels, also commonly referred as ionotropic receptors, are a group of transmembrane ion channel proteins which open to allow ions such as Na+, K+,Ca2+, and/or Cl? to pass through the membrane in response to the binding of a chemical messenger (i.e. a ligand), such as a neurotransmitter.

One of the classic ion channels is nicotinic acetylcholine receptor (nAchR):

Acetylcholine is released from presynaptic membrane, binding with nicotinic acetylcholine receptors located on postsynaptic membrane - ligand-gated cation channels open - Na+ enter into cytoplasm, with K+ flow out of cytoplasm - membrane potential depolarization - voltage-gated ion channels open - enormous Na+ come out into cytoplasm of muscle cells - action potential of muscle cells is induced - Ca2+ release - skeletal muscle contraction.

① G protein-coupled receptors (GPCRs) comprise the largest protein superfamily in mammalian genomes. They share a common seven-transmembrane topology and mediate cellular response to a variety of extracellular signals ranging from photons and small molecules to peptides and proteins.

G proteins, also known as guanine nucleotide-binding proteins, are a family of proteins that act as molecular switches inside cells, and are involved in transmitting signals from a variety of stimuli outside a cell to its interior. Their activity is regulated by factors that control their ability to bind to and hydrolyze guanosine triphosphate (GTP) to guanosine diphosphate (GDP). When they are bound to GTP, they are 'on', and, when they are bound to GDP, they are 'off'. G proteins belong to the larger group of enzymes called GTPases. G protein is made up of alpha (α), beta(β) and gamma (γ) subunits. In addition, the beta and gamma subunits can form a stable dimeric complex referred to as the beta-gamma complex.

G proteins located within the cell are activated by G protein-coupled receptors (GPCRs) that span the cell membrane. Signaling molecules bind to a domain of the GPCR located outside the cell, and an intracellular GPCR domain then in turn activates a particular G protein. The G protein activates a cascade of further signaling events that finally results in a change in cell function. G protein-coupled receptor and G proteins working together transmit signals from many hormones, neurotransmitters, and other signaling factors.

② Functional mechanism:

In the inactive state, the GPCR is bound to a heterotrimeric G protein complex. Binding of an agonist to the GPCR results in a conformational change in the receptor that is transmitted to the bound Gα subunit of the heterotrimeric G protein via protein domain dynamics. The activated Gα subunit exchanges GTP in place of GDP which in turn triggers the dissociation of Gα subunit from the Gβγ dimer and from the receptor. The dissociated Gα interact with other intracellular proteins to continue the signal transduction cascade depending on the α subunit type (Gαs, Gi, Gp).

③ Biological property of G protein:

Gαs

Gαs activates the cAMP-dependent pathway by stimulating the production of cyclic AMP (cAMP) from ATP. This is accomplished by direct stimulation of the membrane-associated enzyme adenylate cyclase. cAMP can then act as a second messenger that goes on to interact with and activate protein kinase A (PKA). PKA can phosphorylate a myriad downstream targets.

Coupled receptor: β-adrenaline, ACTH, LH, Histone H2, calcitonin, glucagon, purine 2 receptor.

Gαi

Gαi inhibits the production of cAMP from ATP.

Coupled receptor: α2-adrenaline, vasodilatin, somatostatin receptor, DA2, purine 1 receptor.

Gp

phospholipase C pathway coupled receptor: α1-adrenaline, α-thrombin, vasopressin, bradykinin, metabotropic glutamate receptor.

A tyrosine protein kinase (TPK) is a transmembrane enzyme that can transfer a phosphate group from ATP to a protein in a cell. It functions as an “on” or “off” switch in many cellular functions. Tyrosine kinases are a subclass of protein kinase.

The phosphate group is attached to the amino acid tyrosine on the protein. Tyrosine kinases are a subgroup of the larger class of protein kinases that attach phosphate groups to other amino acids (serine and threonine). Phosphorylation of proteins by kinases is an important mechanism in communicating signals within a cell (signal transduction) and regulating cellular activity, such as cell division.

The ligands of TPK receptor mainly include some growth and differentiation factors, such as epidermal growth factor (EGF), platelet derived growth factor (PDGF) and insulin.

One of the classic TPKR is epidermal growth factor receptor (EGFR):

Growth factor ligand activation - EGFR undergoes a transition from an inactive monomeric form to an active homodimer - EGFR dimerization stimulates its intrinsic intracellular protein-tyrosine kinase activity - autophosphorylation of several tyrosine (Y) residues in the C-terminal domain of EGFR - this autophosphorylation elicits downstream activation and signaling by several other proteins that associate with the phosphorylated tyrosines through their own phosphotyrosine-binding SH2 domains - these downstream signaling proteins initiate several signal transduction cascades, principally the MAPK, Akt and JNK pathways, leading to DNA synthesis and cell proliferation

Intracellular receptors are receptors located inside the cell rather than on its cell membrane. Classic hormones that use intracellular receptors include thyroid and steroid hormones. Examples are the class of nuclear receptors located in the cell nucleus and cytoplasm and the IP3 receptorlocated on the endoplasmic reticulum. The ligands that bind to them are usually intracellular second messengers like inositol trisphosphate (IP3) and extracellular lipophilic hormones like steroid hormones. Some intracrine peptide hormones also have intracellular receptors.

Second messengers are intracellular signaling molecules released by the cell after receptor activation to trigger physiological changes such as proliferation, differentiation, migration, survival, and apoptosis. Secondary messengers are therefore one of the initiating components of intracellular signal transduction cascades. Examples of second messenger molecules include cyclic AMP, cyclic GMP, inositol trisphosphate, diacylglycerol and calcium.

The diverse effects of cAMP are mediated through the action of cAMP-dependent protein kinases (cAPKs). These enzymes, also referred to as protein kinases A (PKAs), modify the activities of target enzymes in various cell types by phosphorylating specific serine and threonine residues. Phosphorylation of many enzymes increases their catalytic activity, whereas phosphorylation of others decreases their activity.

As discussed later, other second messengers also activate protein kinases via a similar mechanism: in the absence of the second messenger, the kinase shows low activity; binding of the second messenger increases the kinase activity. Each type of second messenger - dependent kinase is inhibited by the binding of a peptide sequence, called apseudosubstrate, to the active site. The pseudosubstrate sequence can be part of a distinct regulatory subunit, as in cAPKs, or it can be part of a regulatory domain within the same polypeptide chain that contains the active site. Binding of a second messenger to the inactive kinase induces a conformational change that leads to release of the part of the regulatory domain bound to the kinase active site or to dissociation of the regulatory subunit; in both cases, the active sites of the kinase are unmasked and their enzymatic activities activated.

Synthesis:

Cyclic AMP is synthesized from ATP by adenylate cyclase located on the inner side of the plasma membrane and anchored at various locations in the interior of the cell. Adenylate cyclase is activated by a range of signaling molecules through the activation of adenylate cyclase stimulatory G (Gs)-protein-coupled receptors. On the contrary, adenylate cyclase is inhibited by agonists of adenylate cyclase inhibitory G (Gi)-protein-coupled receptors. cAMP can be hydrolysed to 5'-AMP by phosphodiesterase, losing its biological function.

Functions:

cyclic AMP works by activating protein kinase A (PKA). PKA is normally inactive as a tetramericholoenzyme, consisting of two catalytic and two regulatory units (C2R2), with the regulatory units blocking the catalytic centers of the catalytic units. Cyclic AMP binds to specific locations on the regulatory units of the PKA, and causes dissociation between the regulatory and catalytic subunits, thus enabling those catalytic units to phosphorylate substrate proteins. Protein kinase A can phosphorylate specific proteins that bind to promoter regions of DNA, causing increases in transcription.

Cyclic guanosine monophosphate (cGMP) is a cyclic nucleotide derived from guanosine triphosphate(GTP). cGMP acts as a second messenger much like cyclic AMP.

Synthesis:

Guanylate cyclase (GC) catalyzes cGMP synthesis. This enzyme, which includes membrane-bound GC and soluble GC (sGC), converts GTP to cGMP. Peptide hormones such as the atrial natriuretic factor activate membrane-bound GC, while sGC is typically activated bynitric oxide to stimulate cGMP synthesis.

Functions:

cGMP is a common regulator of ion channel conductance, glycogenolysis, and cellular apoptosis. It also relaxes smooth muscle tissues. In blood vessels, relaxation of vascular smooth muscles lead to vasodilationand increased blood flow.

cGMP is involved in the regulation of some protein-dependent kinases. For example, PKG (protein kinase G) is a dimer consisting of one catalytic and one regulatory unit, with the regulatory units blocking the active sites of the catalytic units. cGMP binds to sites on the regulatory units of PKG and activates the catalytic units, enabling them to phosphorylate their substrates such as histon, phosphorylase kinase, glycogen synthase and pyruvate kinase.

Inositol trisphosphate (IP3), together with diacylglycerol (DAG), is a secondary messenger molecule used in signal transduction and lipid signaling in biological cells. While DAG stays inside the membrane, IP3 is soluble and diffuses through the cell. It is made by hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), a phospholipid that is located in the plasma membrane, by phospholipase C (PLC).

Functions:

Increases in the intracellular Ca2+ concentrations are often a result of IP3 activation. When a ligand binds to a G protein-coupled receptor (GPCR) that is coupled to a Gq heterotrimeric G protein, the α-subunit of Gq can bind to and induce activity in the PLC isozyme PLC-β, which results in the cleavage of PIP2 into IP3 and DAG.

IP3 (also abbreviated Ins3P) is a soluble molecule and is capable of diffusing through the cytoplasm to the ER, or the sarcoplasmic reticulum (SR) in the case of muscle cells, once it has been produced by the action of PLC. Once at the ER, IP3 is able to bind to the Ins3P receptor (Ins3PR) on a ligand-gated Ca2+ channel that is found on the surface of the ER. The binding of IP3 (the ligand in this case) to InsP3R triggers the opening of the Ca2+ channel, and thus release of Ca2+ into the cytoplasm. In heart muscle cells this increase in Ca2+ activates the ryanodine receptor-operated channel on the SR, results in further increases in Ca2+ through a process known as calcium-induced calcium release. IP3 may also activate Ca2+channels on the cell membrane indirectly, by increasing the intracellular Ca2+ concentration.

Most intracellular Ca2+ ions are sequestered in the mitochondria and endoplasmic reticulum (ER) or other vesicles. Cells employ various mechanisms for regulating the concentration of Ca2+ ions free in the cytosol, which usually is kept below 0.2 μM. Ca2+ ATPases pump cytosolic Ca2+ ions across the plasma membrane to the cell exterior or into the lumens of the endoplasmic reticulum or other intracellular vesicles that store Ca2+ ions. As we discuss below, a small rise in cytosolic Ca2+ induces a variety of cellular responses.

Binding of many hormones to their cell-surface receptors on liver, fat, and other cells induces an elevation in cytosolic Ca2+ even when Ca2+ ions are absent from the surrounding medium. In this situation, Ca2+ is released into the cytosol from the endoplasmic reticulum (ER) and other intracellular vesicles. The mechanism by which a hormone-receptor signal on the cell surface is transduced to the ER became clear in the early 1980s, when it was shown that a rise in the level of cytosolic Ca2+ often is preceded by an increase in IP3.

Since it is water soluble, IP3 can diffuse within the cytosol carrying a hormone signal from the cell surface to the ER surface. Here, IP3 binds to a Ca2+-channel protein composed of four identical subunits, each containing an IP3-binding site in the large N-terminal cytosolic domain. IP3 binding induces opening of the channel allowing Ca2+ ions to exit from the ER into the cytosol. The resulting rise in the cytosolic Ca2+ level is only transient, however, because Ca2+ ATPases located in the plasma membrane and ER membrane actively pump Ca2+ from the cytosol to the cell exterior and ER lumen, respectively. Furthermore, within a second of its generation, IP3 is hydrolyzed to inositol 1,4-bisphosphate, which does not stimulate Ca2+ release from the ER.

When various phosphorylated inositols found in cells are added to preparations of ER vesicles, only IP3 causes release of Ca2+ ions from the vesicles. This simple experiment demonstrates the specificity of the IP3 effect. In addition, not all cells respond identically to IP3. This differential sensitivity may reflect expression of different isoforms of the IP3-sensitive Ca2+ channel in the ER membrane and/or variation in the Ca2+ content of the ER itself. Because of this variability, different types of cells may exhibit very different responses to the same extracellular signal.

Opening of the IP3-sensitive Ca2+ channels is potentiated by cytosolic Ca2+ ions, which increases the affinity of the receptor for IP3, resulting in greater release of stored Ca2+. High concentrations of cytosolic Ca2+, however, inhibit IP3-induced release of Ca2+ from intracellular stores by decreasing the affinity of the receptor for IP3. The complex regulation of the IP3 receptor in ER membranes can lead to rapid oscillations in the cytosolic Ca2+ level when the IP3pathway in cells is stimulated. For example, stimulation of hormone-secreting cells in the pituitary by luteinizing hormone - releasing factor causes rapid, repeated spikes in the cytosolic Ca2+ level; each spike is associated with a burst in secretion of luteinizing hormone (LH). The purpose of the fluctuations of Ca2+, rather than a sustained rise in cytosolic Ca2+, is not understood. One possibility is that a sustained rise in Ca2+ might be toxic to cells.

Opening of Ryanodine Receptors Releases Ca2+ Stores in Muscle and Nerve Cells

In addition to IP3-sensitive Ca2+ channels, muscle cells and neurons possess other Ca2+ channels called ryanodine receptors (RYRs), because of their sensitivity to the plant alkaloid ryanodine. In skeletal muscle cells, these receptors are located in the membrane of the sarcoplasmic reticulum (SR) and associate with the cytoplasmic domain of the dihydropyridine receptor, a voltage-sensing protein in the plasma membrane. A change in potential across the plasma membrane induces a conformational change in the RYR, so that Ca2+ ions are released from the sarcoplasmic reticulum into the cytosol .

While the IP3-induced increase in Ca2+ requires activation of SOCs in the plasma membrane to sustain Ca2+-mediated cellular responses, release of Ca2+ from internal stores in muscle cells is sufficient to induce muscle contraction. Like the IP3 receptor, the ryanodine receptor is stimulated by a small rise in cytosolic Ca2+, although it is not inhibited at high Ca2+ concentrations. Thus, Ca2+ feeds forward to activate RYRs. Repolarization of the muscle membrane closes the RYR channels and renders them insensitive to cytosolic Ca2+.

In smooth muscle cells, RYRs are found within the SR membrane; in neurons, these receptors are located in the ERmembrane. In both cases, the RYRs are closely associated with Ca2+ channels in the plasma membrane. It is unclear how the opening of these Ca2+ channels in response to a voltage change or neurotransmitter leads to opening of RYRs. Recent studies indicate that a newly identified intracellular signaling molecule, cyclic ADP ribose, promotes release of Ca2+ via RYRs from internal stores in neurons.

The precise spatial control of Ca2+ release from intracellular stores also plays an important role in cell physiology. For instance, in arterial smooth muscle cells, the local release of Ca2+ from the regions of the sarcoplasmic reticulum in close apposition to the plasma membrane leads to muscle relaxation. This local release is mediated by RYRs in the SR membrane and leads to opening of K+ channels in the plasma membrane; as a result K+ flows out of the cell, hyperpolarizing it. This hyperpolarization serves to inhibit Ca2+-dependent contraction. In contrast, global release of intracellular Ca2+ throughout the muscle leads to contraction. Spatial control of Ca2+ release is also important for the control of IP3-mediated Ca2+ channels.

Ca2+-Calmodulin Complex Mediates Many Cellular Responses

Localized increases in the cytosolic level of free Ca2+ are critical to its function as a second messenger. In secretory cells, such as the insulin-producing β cells in the pancreatic islets, a rise in Ca2+ triggers the exocytosis of secretory vesicles and the release of insulin. In smooth or striated muscle cells, a rise in Ca2+ triggers contraction; in both liver and muscle cells, an increase in Ca2+ activates the degradation of glycogen to glucose 1-phosphate. As discussed already, small increases in cytosolic Ca2+ and the associated cellular responses often are preceded by a hormone-induced rise in IP3.

A small cytosolic protein called calmodulin, which is ubiquitous in eukaryotic cells, mediates many cellular effects of Ca2+ ions. Each calmodulin molecule binds four Ca2+ ions. Binding of Ca2+ causes calmodulin to undergo a conformational change that enables the Ca2+-calmodulin complex to bind to and activate many enzymes, such as myosin light-chain kinase, which regulates the activity of myosin. Because Ca2+ binds to calmodulin in a cooperative fashion, a small change in the level of cytosolic Ca2+ leads to a large change in the level of active calmodulin.

One well-studied enzyme activated by the Ca2+-calmodulin complex is cAMP phosphodiesterase; as noted earlier, this enzyme degrades cAMP and terminates its effects. This reaction thus links Ca2+ and cAMP, one of many examples in which these two second messengers interact to finetune certain aspects of cell regulation. As with other second messengers, the Ca2+-calmodulin complex also activates several protein kinases that, in turn, phosphorylate transcription factors, thereby modifying their activity and regulating gene expression.

(1) Familial hypercholesterolemia(FH)

Patient's LDL receptor protein gene defects cause patient's the liver cell membrane's LDL receptor lacks or reduces, and reduces the liver cell's ability of uptaking in LDL from the blood, and causes the cholesterol content is very high in patient's blood, expressing the lasting hypercholesteremia. Cholesterol sedimentates at the vascular wall, then the vessel becomes small and appears the atherosclerotic plaques, and causes the myocardial obstruction and cerebral obstruction; patients usually died of coronary heart disease.

(2) Myasthenia gravis(MG):

Because the patient's body produces the antibody of anti- N type acetylcholine receptor (N-AchR-Ab), the antibody combines with the N-type acetylcholine receptor (AchR), and closes the effects of N-type acetylcholine and promotes the decompositon of acetylcholine receptor(AchR), causing the sum of receptor to decrease apparently in the patient's body. The anti-N type receptor (N-AchR) antibody by interfering the combination of itself with receptor, causing the joint site of the neuro and mascle(NMJ) and the N-Ach released from neuron's presynaptic membrane cannot combine with the myocyte's postsynaptic membrane, thus blocks the transduction of nervous impulse, leads to the signal transduction disorder, and causes the muscle contraction failure.

(3)Diabetes

The obesity is a factor to induce the diabetes. The obesities come into being because of the reduction of insulin receptors at the adipose cell membrane and muscular membrane decrease, decrease of affinity to insulin , and reduction of the somatic cell's sensitivity. It is caused by reduction of the insulin antibody, causing the malabsorption of the glucose, and makes the blood glucose increased.

Insulin can increase the cell membrane's permeability to glucose, and promote the transport of glucose from outer cell into the inner cell, creating the conditions for cell to absorb the glucose.

Cholera is a kind of gastrointestinal infectious disease, which is caused by Vibrio cholera. Vibrio cholera is adhered to the small intestinal mucosa, reproducing and forming the cholera toxin and then leads to the acute diarrhea.

Mechanism: The Gs protein's α subunit has the modification site of the cholera toxin, this kind of modification can make Gs protein ribosylation (ADP-ribosylation). The result is that α subunit combines with GTP while segregating with β、γ subunits, but it inhibits the activity of theα subunit 's GTP enzyme(GTPase ). GTP enzyme cannot hydrolyze GTP to GDP. Gs protein is lasting at the state of activation, which causes the adenylate cyclase (AC) is activated persistently. The intracellular cAMP increases so large that the anion channel(pathway) open persistently, and so a lot of Cl- and HCO3- enter the enteric cavity from the inner cells, and then the osmotic pressure between the extracellular and the intracellular loses the balance. A large amount of water enters the enteric cavity, leading to intense diarrhea and severe dehydration.

Pertussis is the acute respiratory infectious disease, caused by the bordetella pertussis. Its characteristics is the paroxysmal spasmodic cough. After the bordetella pertussis invades into the susceptible person's respiratory tract, it adheres to the cilium at the respiratory tract's epithelial cells. The bordetella pertussis reproduces and forms the pertussis toxin, which makes the cilium on the respiratory tract's epithelial cells paralysis and the cell necrosis, and the respiratory produces thick secretion and defects receptors, retenting secretion constantly stimulates respiratory nerve ending, causing the spasmodic cough through the coughing centre.

Mechanism: The pertussis toxin makes the Gi protein's α subunit ribosylation (ADP-ribosylation). Its result is that it stops the α subunit's GDP from replacing by the GTP, making it lose the inhabited effect on the adenylate cyclase(AC) so that the intracellular cAMP increases largely and leads to a lot of body fluids to secret into the lung and cause the intense cough.

Phorbol ester is the T lymphocytic activator and the potential carcinogen for the skin mucosa. Because the phorbol ester's molecular structure is similar to the second messenger (diacylglycerol, DAG), it can take the place of DAG and combines with protein kinase C (PKC) in the cells, but it is unlike the DAG decomposed quickly. It can excite PKC permanently and irreversibly, which causes the cells reproduces and grows uncontrollably and finally forms the tumor.

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