Cell Signaling


Cell signaling is part of a complex system of communication that governs basic cellular activities and coordinates cell actions. The ability of cells to perceive and correctly respond to their microenvironment is the basis of development, tissue repair, and immunity as well as normal tissue homeostasis. Errors in cellular information processing are responsible for diseases such as cancer, autoimmunity, and diabetes. By understanding cell signaling, diseases may be treated effectively and, theoretically, artificial tissues may be yielded.


Different types of signaling:


A cell can communicate signals to other cells in various ways.


Direct signaling is a transfer of ions or small molecules from one cell to its neighbor through pores in the membrane. Those pores are built out of membrane proteins and are called gap junctions. This is the fastest mode of cell-cell communication and is found in places where extremely fast and well-coordinated activity of cells in needed. An example of this process can be found in the heart. The muscle cells in the heart communicate with each other via gap junctions which allow all heart cells to contract almost simultaneously.


Endocrine signaling utilizes hormones. A cell secretes chemicals into the bloodstream. Those chemicals affect the behavior of distant target cells.


Paracrine signaling is a way for a cell to affect the behavior of neighboring cells by secreting chemicals into the common intercellular space. This is an important process during embryonic development.


Autocrine signaling is a way for a cell to alter its own extracellular environment, which in turn affects the way the cell functions. The cell secretes chemicals outside of its membrane and the presence of those chemicals on the outside modifies the behavior of that same cell. This process is important for growth.


The juxtacrine signaling also known as contact dependent signaling in which two adjacent cells must make physical contact in order to communicate. This requirement for direct contact allows for very precise control of cell differentiation during embryonic development.  Notch signaling is an example for juxtacrine signaling.


Synaptic signaling is found in the nervous system. It is a highly specific and localized type of paracrine signalling between two nerve cells or between a nerve cell and a muscle cell. We will go into details of synaptic signaling when we cover the human nervous system.


Except autocrine signaling molecules others actively participate in the intercellular signaling process.   They are also otherwise called as extracellular signaling because signaling molecule araised from extracellular region.



Receptors for cell signaling mainly are of two types namely cell surface receptors and intracellular or internal receptors.  Those signaling molecules which are capable of diffusing into cytosol of the cell can interact with internal receptors and execute signaling process.  Steroid molecules and Nitric oxide are examples of signaling molecules which can bind to internal receptors.  They participate in intracellular signaling process.

Signaling molecules like proteins which are unable to enter into cells can interact with the cell surface receptors and execute its signaling process.  Cell surface receptors are transmembrane proteins whose extracellular portion has the binding site for the signaling molecule and intracellular portion activates proteins in the cytosol that in different ways eventually regulate gene transcription in the nucleus.


Signaling Pathways:


In some cases, receptor activation caused by ligand binding to a receptor is directly coupled to the cell's response to the ligand. For example, the neurotransmitter GABA can activate a cell surface receptor that is part of an ion channel. GABA binding to a GABA A receptor on a neuron opens a chloride-selective ion channel that is part of the receptor. GABA A receptor activation allows negatively charged chloride ions to move into the neuron which inhibits the ability of the neuron to produce action potentials. However, for many cell surface receptors, ligand-receptor interactions are not directly linked to the cell's response. The activated receptor must first interact with other proteins inside the cell before the ultimate physiological effect of the ligand on the cell's behavior is produced. Often, the behavior of a chain of several interacting cell proteins is altered following receptor activation. The entire set of cell changes induced by receptor activation is called a signal transduction mechanism or pathway.


In the case of Notch-mediated signaling, the signal transduction mechanism can be relatively simple. Activation of Notch can cause the Notch protein to be altered by a protease. Part of the Notch protein is released from the cell surface membrane and can act to change the pattern of gene transcription in the cell nucleus. This causes the responding cell to make different proteins, resulting in an altered pattern of cell behavior. Cell signaling research involves studying the spatial and temporal dynamics of both receptors and the components of signaling pathways that are activated by receptors in various cell types.



Wnt Pathway:


The name Wnt was coined as a combination of Wg (wingless) and Int  and can be pronounced as 'wint'. The wingless gene had originally been identified as a segment polarity gene in Drosophila melanogaster that functions during embryogenesis and also during adult limb formation during metamorphosis. The INT genes were originally identified as vertebrate genes near several integration sites of mouse mammary tumor virus (MMTV). The Int-1 gene and the wingless gene were found to be homologous, with a common evolutionary origin evidenced by similar amino acid sequences of their encoded proteins.


The canonical Wnt pathway describes a series of events that occur when Wnt proteins bind to cell-surface receptors of the Frizzled family, causing the receptors to activate Dishevelled family proteins and ultimately resulting in a change in the amount of β-catenin that reaches the nucleus. Dishevelled (DSH) is a key component of a membrane-associated Wnt receptor complex which, when activated by Wnt binding, inhibits a second complex of proteins that includes axin, GSK-3, and the protein APC. The axin/GSK-3/APC complex normally promotes the proteolytic degradation of the β-catenin intracellular signaling molecule. After this "β-catenin destruction complex" is inhibited, a pool of cytoplasmic β-catenin stabilizes, and some β-catenin is able to enter the nucleus and interact with TCF/LEF family transcription factors to promote specific gene expression


Hedgehog pathway:


The hedgehog signaling pathway is one of the key regulators of animal development conserved from flies to humans. The pathway takes its name from its polypeptide ligand, an intercellular signaling molecule called Hedgehog (Hh) found in fruit flies of the genus Drosophila. Hh is one of Drosophila's segment polarity gene products, involved in establishing the basis of the fly body plan.  The appearance of the stubby and "hairy" larvae inspired the name 'hedgehog' when the gene mutated.


In mammals, when there is no hedgehog protein present, the patched (PTC) receptors bind a second transmembrane protein called smoothened (Smo).  However, when Hh protein binds to patched, the Smo protein separates from Ptc enabling Smo to activate a zinc-finger transcription factor designated GLI. GLI migrates into the nucleus when it activates a variety of target genes. Hedgehog signaling plays many important developmental roles in the animal kingdom. For example, wing development in Drosophila and development of the brain, GI tract, fingers and toes in mammals.


Mutations or other sorts of regulatory errors in the hedgehog pathway are associated with a number of birth defects as well as some cancers. Basal-cell carcinoma, the most common skin cancer (and, in fact, the most common of all cancers in much of the world), usually reveals mutations causing extra-high hedgehog or suppressed patched activity (both leading to elevated GLI activity).


Cell surface receptors:


Cell surface receptors are integral membrane proteins and, as such, have regions that contribute to three basic domains:


Extracellular domains: Some of the residues exposed to the outside of the cell interact with and bind the hormone - another term for these regions is the ligand-binding domain.


Transmembrane domains: Hydrophobic stretches of amino acids are "comfortable" in the lipid bilayer and serve to anchor the receptor in the membrane.


Cytoplasmic or intracellular domains: Tails or loops of the receptor that are within the cytoplasm react to hormone binding by interacting in some way with other molecules, leading to generation of second messengers. Cytoplasmic residues of the receptor are thus the effector region of the molecule.


Several distinctive variations in receptor structure have been identified. As depicted below, some receptors are simple, single-pass proteins; many growth factor receptors take this form. Others, such as the receptor for insulin, have more than one subunit. Another class, which includes the beta-adrenergic receptor, is threaded through the membrane seven times. Receptor molecules are neither isolated by themselves nor fixed in one location of the plasma membrane. In some cases, other integral membrane proteins interact with the receptor to modulate its activity. Some types of receptors cluster together in the membrane after binding hormone.


Types of Cell surface receptors:


  1. G-protein coupled receptors
  2. Receptor tyrosine kinase receptors
  3. Cytokine receptors and Non-tyrosine kinase receptors
  4. Integrin receptors
  5. Toll-like receptors
  6. Ligand gated ion-channels receptors
  7. Receptors with other enzymatic activities


G-protein coupled receptors:


G protein-linked receptors are seven-pass transmembrane proteins. This means that the polypeptide chain traverses the membrane seven times. When a chemical - a hormone or a pharmaceutical agent - binds to the receptor on the outside of the cell, this triggers a series of chemical reactions, including the movement and binding of the G-protein, transformation of GTP into GDP and activation of second messengers. Second messengers (e.g., cyclic AMP) start a cascade of enzymatic reactions leading to the cellular response. This signaling method is quite fast and, more importantly, it amplifies the signal. Binding of a single hormone molecule quickly results in thousands of molecules of second messengers acting on even more molecules of enzymes and so on. Thus, the response to a small stimulus can be very large.



Receptor Tyrosine Kinase:




In contrast to the G protein-coupled receptors, other cell surface receptors are directly linked to intracellular enzymes. The largest family of such enzyme-linked receptors are the receptor protein-tyrosine kinases, which phosphorylate their substrate proteins on tyrosine residues. This family includes the receptors for most polypeptide growth factors, so protein-tyrosine phosphorylation has been particularly well studied as a signaling mechanism involved in the control of animal cell growth and differentiation. Indeed, the first protein-tyrosine kinase was discovered in 1980 during studies of the oncogenic proteins of animal tumor viruses, in particular Rous sarcoma virus, by Tony Hunter and Bartholomew Sefton. The EGF receptor was then found to function as a protein-tyrosine kinase by Stanley Cohen and his colleagues, clearly establishing protein-tyrosine phosphorylation as a key signaling mechanism in the response of cells to growth factor stimulation.


By now more than 50 receptor protein-tyrosine kinases have been identified, including the receptors for EGF, NGF, PDGF, insulin, and many other growth factors. All these receptors share a common structural organization: an N-terminal extracellular ligand-binding domain, a single transmembrane α helix, and a cytosolic C-terminal domain with protein-tyrosine kinase activity. Most of the receptor protein-tyrosine kinases consist of single polypeptides, although the insulin receptor and some related receptors are dimers consisting of two pairs of polypeptide chains. The binding of ligands (e.g., growth factors) to the extracellular domains of these receptors activates their cytosolic kinase domains, resulting in phosphorylation of both the receptors themselves and intracellular target proteins that propagate the signal initiated by growth factor binding.


The first step in signaling from most receptor protein-tyrosine kinases is ligand-induced receptor dimerization. Some growth factors, such as PDGF and NGF, are themselves dimers consisting of two identical polypeptide chains; these growth factors directly induce dimerization by simultaneously binding to two different receptor molecules. Other growth factors (such as EGF) are monomers but have two distinct receptor binding sites that serve to crosslink receptors.


Ligand-induced dimerization then leads to autophosphorylation of the receptor as the dimerized polypeptide chains cross-phosphorylate one another. Such autophosphorylation plays two key roles in signaling from these receptors. First, phosphorylation of tyrosine residues within the catalytic domain may play a regulatory role by increasing receptor protein kinase activity. Second, phosphorylation of tyrosine residues outside of the catalytic domain creates specific binding sites for additional proteins that transmit intracellular signals downstream of the activated receptors.


The association of these downstream signaling molecules with receptor protein-tyrosine kinases is mediated by protein domains that bind to specific phosphotyrosine-containing peptides. The best-characterized of these domains are called SH2 domains (for Src homology 2) because they were first recognized in protein-tyrosine kinases related to Src, the oncogenic protein of Rous sarcoma virus. SH2 domains consist of approximately a hundred amino acids and bind to specific short peptide sequences containing phosphotyrosine residues. The resulting association of SH2-containing proteins with activated receptor protein-tyrosine kinases can have several effects: It localizes the SH2-containing proteins to the plasma membrane, leads to their association with other proteins, promotes their phosphorylation, and stimulates their enzymatic activities. The association of these proteins with autophosphorylated receptors thus represents the first step in the intracellular transmission of signals initiated by the binding of growth factors to the cell surface.


Cytokine receptors and Non-tyrosine kinase receptors:


Rather than possessing intrinsic enzymatic activity, many receptors act by stimulating intracellular protein-tyrosine kinases with which they are noncovalently associated. This family of receptors (called the cytokine receptor superfamily) includes the receptors for most cytokines (e.g., interleukin-2 and erythropoietin) and for some polypeptide hormones (e.g., growth hormone). Like receptor protein-tyrosine kinases, the cytokine receptors contain N-terminal extracellular ligand-binding domains, single transmembrane α helices, and C-terminal cytosolic domains. However, the cytosolic domains of the cytokine receptors are devoid of any known catalytic activity. Instead, the cytokine receptors function in association with nonreceptor protein-tyrosine kinases, which are activated as a result of ligand binding.


The first step in signaling from cytokine receptors is thought to be ligand-induced receptor dimerization and cross-phosphorylation of the associated nonreceptor protein-tyrosine kinases. These activated kinases then phosphorylate the receptor, providing phosphotyrosine-binding sites for the recruitment of downstream signaling molecules that contain SH2 domains. Combinations of cytokine receptors plus associated nonreceptor protein-tyrosine kinases thus function analogously to the receptor protein-tyrosine kinases discussed in the previous section.


The nonreceptor protein-tyrosine kinases associated with the cytokine receptors fall into two major families. Many of these kinases are members of the Src family, which consists of Src and eight closely related proteins. As already noted, Src was initially identified as the oncogenic protein of Rous sarcoma virus and was the first protein shown to possess protein-tyrosine kinase activity, so it has played a pivotal role in experiments leading to our current understanding of cell signaling. In addition to Src family members, the cytokine receptors are associated with nonreceptor protein-tyrosine kinases belonging to the Janus kinase, or JAK, family. Members of the JAK family appear to be universally required for signaling from cytokine receptors, indicating that JAK family kinases play a critical role in coupling these receptors to the tyrosine phosphorylation of intracellular targets. In contrast, members of the Src family play key roles in signaling from antigen receptors on B and T lymphocytes but do not appear to be required for signaling from most cytokine receptors.


Integrin Receptors:


Integrins are produced by a wide variety of cell types, and play a role in the attachment of a cell to the extracellular matrix (ECM) and to other cells, and in the signal transduction of signals received from extracellular matrix components such as fibronectin, collagen, and laminin. Ligand-binding to the extracellular domain of integrins induces a conformational change within the protein and a clustering of the protein at the cell surface, in order to initiate signal transduction. Integrins lack kinase activity, and integrin-mediated signal transduction is achieved through a variety of intracellular protein kinases and adaptor molecules such as integrin-linked kinase (ILK), focal-adhesion kinase (FAK), talin, paxillin, parvins, p130Cas, Src-family kinases, and GTPases of the Rho family, the main protein coordinating signal transduction being ILK. As shown in the figure, cooperative integrin and receptor tyrosine kinase signaling determine cellular survival, apoptosis, proliferation, and differentiation.


Important differences exist between integrin-signaling in circulating blood cells and that in non-circulating blood cells such as epithelial cells. Integrins at the cell-surface of circulating cells are inactive under normal physiological conditions. For example, cell-surface integrins on circulating leukocytes are maintained in an inactive state in order to avoid epithelial cell attachment. Only in response to appropriate stimuli are leukocyte integrins converted into an active form, such as those received at the site of an inflammatory response. In a similar manner, it is important that integrins at the cell surface of circulating platelets are kept in an inactive state under normal conditions, in order to avoid thrombosis. Epithelial cells, in contrast, have active integrins at their cell surface under normal conditions, which help to maintain their stable adhesion to underlying stromal cells, which provide appropriate signals in order to maintain their survival and differentiation.


Toll-like Receptors:


Toll-like receptors (TLRs) are a class of single membrane-spanning non-catalytic receptors that recognize structurally conserved molecules derived from microbes once they have breached physical barriers such as the skin or intestinal tract mucosa, and activate immune cell responses. They play a key role in the innate immune system.  They receive their name from their similarity to the protein coded by the Toll gene identified in Drosophila in 1985 by Christiane Nüsslein-Volhard.



When activated, Toll-like receptors (TLRs) recruit adapter molecules within the cytoplasm of cells in order to propagate a signal. Four adapter molecules are known to be involved in signaling. These proteins are known as MyD88, Tirap (also called Mal), Trif, and Tram. The adapters activate other molecules within the cell, including certain protein kinases (IRAK1, IRAK4, TBK1, and IKKi) that amplify the signal, and ultimately lead to the induction or suppression of genes that orchestrate the inflammatory response. In all, thousands of genes are activated by TLR signaling, and, together, the TLRs constitute one of the most powerful and important gateways for gene modulation.


Following activation by ligands of microbial origin, several reactions are possible. Immune cells can produce signalling factors called cytokines which trigger inflammation. In the case of a bacterial factor, the pathogen might be phagocytosed and digested, and its antigens presented to CD4+ T cells. In the case of a viral factor, the infected cell may shut off its protein synthesis and may undergo programmed cell death (apoptosis). Immune cells that have detected a virus may also release anti-viral factors such as interferons.


The discovery of the Toll-like receptors finally identified the innate immune receptors that were responsible for many of the innate immune functions that had been studied for many years. Interestingly, TLRs seem only to be involved in the cytokine production and cellular activation in response to microbes, and do not play a significant role in the adhesion and phagocytosis of microorganisms.


Ligand gated Ion channels:


When a signaling molecule binds to an ion channel on the outside of the cell, this triggers the change of the 3D conformation of the protein and the channel opens, allowing the ions to move in or out of the cell following their electrical gradients and thus altering the polarization of the cell membrane. Some ion channels respond to non-chemical stimuli in the same way, including changes in electrical charge or mechanical disturbance of the membrane.

Receptors with other enzymatic activity:


Although the vast majority of enzyme-linked receptors stimulate protein-tyrosine phosphorylation, some receptors are associated with other enzymatic activities. These receptors include protein-tyrosine phosphatases, protein-serine/threonine kinases, and guanylyl cyclases. The functions of most of these receptors are less well understood than those of either the G protein-coupled receptors or the receptors associated with protein-tyrosine kinase activity.


Protein-tyrosine phosphatases remove phosphate groups from phosphotyrosine residues, thus acting to counterbalance the effects of protein-tyrosine kinases. In many cases, protein-tyrosine phosphatases play negative regulatory roles in cell signaling pathways by terminating the signals initiated by protein-tyrosine phosphorylation. However, some protein-tyrosine phosphatases are cell surface receptors whose enzymatic activities play a positive role in cell signaling. A good example is provided by a receptor called CD45, which is expressed on the surface of T and B lymphocytes. Following antigen stimulation, CD45 is thought to dephosphorylate a specific phosphotyrosine that inhibits the enzymatic activity of Src family members. Thus, the CD45 protein-tyrosine phosphatase acts (somewhat paradoxically) to stimulate nonreceptor protein-tyrosine kinases.


The receptors for transforming growth factor β (TGF-β) and related polypeptides are protein kinases that phosphorylate serine or threonine, rather than tyrosine, residues on their substrate proteins. TGF-β is the prototype of a family of polypeptide growth factors that control proliferation and differentiation of a variety of cell types, generally inhibiting proliferation of their target cells. The cloning of the first receptor for a member of the TGF-β family in 1991 revealed that it is the prototype of a unique receptor family with a cytosolic protein-serine/threonine kinase domain. Since then, receptors for additional TGF-β family members have similarly been found to be protein-serine/threonine kinases. The binding of ligand to these receptors results in the association of two distinct polypeptide chains, which are encoded by different members of the TGF-β receptor family, to form heterodimers in which the receptor kinases cross-phosphorylate one another. The activated TGF-β receptors then phosphorylate members of a family of transcription factors called SMADs, which translocate to the nucleus and stimulate expression of target genes.


Some peptide ligands bind to receptors whose cytosolic domains are guanylyl cyclases, which catalyze formation of cyclic GMP. As discussed earlier, nitric oxide also acts by stimulating guanylyl cyclase, but the target of nitric oxide is an intracellular enzyme rather than a transmembrane receptor. The receptor guanylyl cyclases have an extracellular ligand-binding domain, a single transmembrane α helix, and a cytosolic domain with catalytic activity. Ligand binding stimulates cyclase activity, leading to the formation of cyclic GMP—a second messenger.


Other receptors bind to cytoplasmic proteins with additional biochemical activities. For example, the cytokine tumor necrosis factor (TNF) induces cell death, perhaps as a way of eliminating damaged or unwanted cells from tissues. The receptors for TNF and related death-signaling molecules are associated with specific proteases, which are activated in response to ligand binding. Activation of these receptor-associated proteases triggers the activation of additional downstream proteases, ultimately leading to degradation of a variety of intracellular proteins and death of the cell.



Secondary messenger


In cell physiology, a secondary messenger system (also known as a second messenger system) is a method of cellular signaling, whereby a diffusable signaling molecule is rapidly generated/released which can then go on to activate effector proteins within the cell to exert a cellular response. Secondary messengers are a component of signal transduction cascades.


Secondary messenger systems can be activated by diverse means, either by activation of enzymes that synthesise them, as is the case with the activation of cyclases which synthesise cyclic nucleotides, or by opening of ion channels to allow influx of metal ions, such as in Ca2+ signalling. These small molecules may then go on to exert their effect by binding to and activating effector molecules such as protein kinases, ion channels and a variety of other proteins, thus continuing the signalling cascade.


Types of secondary molecules


There are three basic types of secondary messenger molecules:


!    Hydrophobic molecules: water-insoluble molecules, like diacylglycerol, and phosphatidylinositols, which are membrane-associated and diffuse from the plasma membrane into the juxtamembrane space where they can reach and regulate membrane-associated effector proteins

!    Hydrophilic molecules: water-soluble molecules, like cAMP, cGMP, IP3, and Ca2+, that are located within the cytosol

!    Gases: nitric oxide (NO) and carbon monoxide (CO), which can diffuse both through cytosol and across cellular membranes.


These intracellular messengers have some properties in common:


·        They can be synthesized/released and broken down again in specific reactions by enzymes or ion channels.

·        Some (like Ca2+) can be stored in special organelles and quickly released when needed.

·        Their production/release and destruction can be localized, enabling the cell to limit space and time of signal activity.


Different terminologies used to differentiate intracellular messengers or molecules namely Primary effector, Secondary messenger and Secondary effector.  Primary effectors include Adenylate cyclase, Guanylate cyclase, Phospholipase-C, Phospholipase-A and Receptor tyrosine kinase.  Secondary messenger include cAMP, cGMP, IP3 and DAG.  Secondary effector include Protein kinase-A, Protein kinase-G, Protein kinase-C and Calcium ions. 



Second Messenger




Cyclic AMP

Epinephrine and nor-epinephrine, glucagon, luteinizing hormone, follicle stimulating hormone, thyroid-stimulating hormone, calcitonin, parathyroid hormone, anti-diuretic hormone

Activates Protein kinase-A


Cyclic GMP

Atrial naturetic hormone, nitric oxide

Activates Protein kinase-G and opens cation channels in rod cells



Epinephrine and norepinephrine, angiotensin II, antidiuretic hormone, gonadotropin-releasing hormone, thyroid-releasing hormone.

Activates Protein kinase-C




Epinephrine and norepinephrine, angiotensin II, antidiuretic hormone, gonadotropin-releasing hormone, thyroid-releasing hormone.

Opens calcium channels in ER


Nitric oxide (NO) as second messenger - The gas nitric oxide is a free radical that diffuses through the plasma membrane and affects nearby cells. NO is made from arginine and oxygen by the enzyme NO synthase, with citrulline as a by-product. NO works mainly through activation of its target receptor, the enzyme soluble guanylate cyclase, which, when activated, produces the second messenger cyclic-guanosine monophosphate (cGMP). NO can also act through covalent modification of proteins or their metal cofactors. Some of these modifications are reversible and work through a redox mechanism. In high concentrations, NO is toxic, and is thought to be responsible for some damage after a stroke. NO serves multiple functions. These include:

  1. Relaxation of blood vessels
  2. Regulation of exocytosis of neurotransmitters
  3. Cellular immune response
  4. Modulation of the Hair Cycle
  5. Production and maintenance of penile erections
  6. Activation of apoptosis by initiating signals that lead to H2AX phosphorylation.