How cells respond to signals: intracellular signalling pathways
A chemical signal that reaches a cell is carried inward by a receptor that changes shape when it is activated. It then passes along a chain of messengers and effectors, at which it can be amplified or suppressed. Each family of receptor drives its own pathways and signals on its own time scale, and many pathways end by changing which genes the cell expresses. Disturbances in the pathways can lead to cancer and inflammatory disease, and the pathways are targets for drugs that act inside the cell.
Receptor activation
Each cell is a living organism that interacts with its environment, and it alters its behaviour and even its size and shape in response to information it receives. For the cells of the human body, some of that information comes as chemical signals produced by other cells. A receptor that passes through the cell membrane, once activated, triggers a sequence of chemical signals within the cell. Proteins play crucial roles in these intracellular signalling pathways, both as signalling chemicals and as receptors.
The bond between a ligand and its receptor can cause a temporary change in the receptor protein's conformation, which can then trigger other events. For example, a ligand outside the cell can bind a receptor that crosses the membrane, and the resulting change in the shape of the receptor's intracellular domains can transmit a signal into the cell even though the ligand stays outside. Some receptor proteins have no chemical ligand at all and change conformation in response to other stimuli, such as light, temperature, mechanical stress or osmotic pressure.
The signal transduction pathway
Some signalling chemicals directly trigger a simple response, such as the opening of a ligand-gated ion channel, and others trigger a long and complicated cascade of signals within the cell. The basic elements of such a cascade are these:
- A ligand is a molecule that binds reversibly to a receptor, and first messengers are signalling compounds that come from outside the cell.
- Transduction typically begins when a receptor, the signal transducer, becomes occupied by a ligand or responds to some other stimulus. The receptor then undergoes a conformational change, which activates a primary effector.
- The primary effector may change the cell's behaviour directly, for instance by opening an ion channel through the membrane, or may trigger production of another signalling chemical inside the cell, the second messenger. A second messenger may activate a secondary effector, which may produce another second messenger, and so on.
- Each component within the cascade is a node. An efficient node amplifies a signal, which is called signal gain, so that one signalling molecule can generate a response involving hundreds to millions of molecules.
- Conversely, a signal can be suppressed by blocking an intracellular receptor or degrading a second messenger, so the nodes and the enzymes that regulate them were becoming important therapeutic targets.
- Each cell has many pathways, and they interact in complicated ways that cell biologists describe in borrowed radio terms: networks, noise and crosstalk.
A kinase is an enzyme that catalyses phosphorylation, the transfer of a phosphate group from a high-energy phosphate donor such as adenosine triphosphate to a specific substrate. Protein phosphorylation, the addition of a covalently bound phosphate group to an amino acid residue, often changes the protein's shape and function.
Receptor types and the duration of their signals
Each type of receptor signals on its own time scale: a ligand-gated ion channel typically provides a signal lasting only a few milliseconds; the signal from an activated G-protein-coupled receptor is typically measured in seconds; kinase-linked receptors produce a signal that can last for hours; and intracellular, or nuclear, receptors produce signals that typically last for hours.
Ligand-gated ion channels
Ligand-gated ion channels open or close a channel through which an ion, such as a calcium ion, can travel into or out of the cell. Many postsynaptic receptors are of this kind: binding the neurotransmitter that serves as their ligand changes their conformation and opens the channel, and the resulting flow of ions produces depolarisation in excitatory receptors or hyperpolarisation in inhibitory ones.
G-protein-coupled receptors
G-protein-coupled receptors are named for their association with guanine nucleotide-binding proteins. In the unactivated state the receptor's intracellular portion is linked to a heterotrimeric G protein, made of three different subunits, Gα, Gβ and Gγ, and the Gα subunit may be bound to guanosine diphosphate (GDP). When the receptor is activated, it undergoes a conformational change, and the complex of Gα with guanosine triphosphate (GTP) and the remaining Gβγ dimer then break free from the receptor and can interact with other receptors downstream in the cascade. Meanwhile the receptor can bind another G protein and start a new round of signalling. The dissociated G protein's signalling ends when the GTP is degraded to GDP, often by the Gα subunit's own GTPase activity, and Gα recombines with a Gβγ dimer into a new heterotrimer that can bind the receptor again.
G-protein classes and their second messengers
The classes of G protein are defined by their Gα subunits. Some receptors can activate more than one subtype, but they usually prefer one. The Gαs subtype activates the enzyme adenylyl cyclase, which catalyses formation of the second messenger cyclic adenosine monophosphate (cAMP). Cyclic AMP is an important second messenger in intracellular signalling cascades. An activated adenylyl cyclase can generate many molecules of cAMP, which amplifies the signal. Cyclic AMP often exerts its effect by binding and activating protein kinase A, also called cAMP-dependent kinase, which phosphorylates target proteins.
A Gαq/11 subtype activates phospholipase C, which splits the membrane phospholipid PIP2 into two second messengers, inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 is water soluble: it diffuses through the cytosol and binds a ligand-gated calcium channel on the endoplasmic reticulum, or on the sarcoplasmic reticulum in muscle cells, and the calcium ions that then enter the cytosol bind calcium-binding proteins such as calmodulin. DAG, in contrast, is lipid soluble, stays in the membrane and activates protein kinase C, which phosphorylates particular target proteins. A Gα12/13 subtype binds RhoGEF proteins, which activate the small GTPase Rho, a protein involved in regulating the cell's cytoskeleton.
Phosphodiesterase inhibitors
Phosphodiesterases break the phosphodiester bond in second messengers such as cyclic AMP and cyclic guanosine monophosphate. Cyclic AMP is broken down by cyclic nucleotide phosphodiesterases, which so suppress and shorten its signal. Several drugs work by selectively inhibiting one of these enzymes. For example, theophylline exerts its anti-inflammatory effect in the respiratory tract by inhibiting phosphodiesterase 4, which is found in a wide range of inflammatory cells, whereas inhibitors of phosphodiesterase 5 are used for erectile dysfunction. Some older drugs, such as theophylline and caffeine, have been found to work by inhibiting an enzyme in an intracellular signalling pathway.
Kinases
Kinases play an important role in intracellular cascades, and dysregulation of many of them has been linked to disease. By 2017 many kinase inhibitors were being investigated in clinical trials, and more than two dozen had been approved for human use, mainly in oncology but some for rheumatoid arthritis or macular degeneration. Some protein kinases serve as receptors, such as the insulin receptor and other growth hormone receptors, which are receptor tyrosine kinases, and others serve as secondary effectors further down the cascade.
Receptor tyrosine kinases
A receptor tyrosine kinase has an extracellular domain that binds a ligand and an intracellular domain that works as a kinase. It consists of two subunits that form a dimer anchored in the membrane. Ligand binding stabilises the dimer, which allows the tyrosine residues of the intracellular domains to be activated by phosphorylation. Once activated, the receptor starts phosphorylation cascades that affect cell metabolism and differentiation. Activated receptors of this kind activate small G proteins, such as those of the Ras and Rho families. Guanine nucleotide exchange factors such as SOS1, once activated, can activate more small G proteins and so amplify the signal.
Raf proteins are central components of the mitogen-activated protein kinase (MAPK) pathway that regulates cell proliferation. Mutations that increase their catalytic activity have been identified in many human tumours, so the Raf kinase family has emerged as a promising target in the treatment of many cancers. For example, sorafenib is used in unresectable hepatocellular carcinoma, advanced renal cell carcinoma and refractory thyroid carcinoma.
The JAK–STAT pathway
Members of the type 1 and type 2 cytokine receptor families have no catalytic kinase activity of their own. They rely instead on Janus kinases (JAKs) to phosphorylate and activate the downstream proteins in their pathways. Activated JAKs phosphorylate and activate transcription factors called STATs, signal transducers and activators of transcription. In 2017 JAK inhibitors, also known as jakinibs, were being investigated in autoimmune disease, and tofacitinib, approved for rheumatoid arthritis, is an example.
Integrins
Integrins are transmembrane receptors that facilitate the adhesion of cells to an extracellular matrix. They are heterodimers of an α and a β subunit. They have no kinase activity and transmit their signals through various intracellular protein kinases and adaptor molecules, especially integrin-linked kinase.
Epithelial cells normally carry active integrins, which help keep them attached to stromal cells. Circulating leukocytes, in contrast, normally carry inactive integrins, which the inflammatory process can activate. For example, a T lymphocyte's weak binding to its specific antigen on an antigen-presenting cell triggers signalling pathways in the T cell that activate its β2 integrins. The activated integrins let the T cell adhere strongly to the antigen-presenting cell for long enough to be fully stimulated, and they may then return to an inactive state so that the T cell can disengage.
Toll-like receptors
Toll-like receptors are single, membrane-spanning, noncatalytic receptors, usually expressed on the immune system's sentinel cells such as macrophages and dendritic cells, and they recognise molecules broadly shared by pathogens such as bacteria and viruses. They are named for their similarity to the toll protein identified in Drosophila. When activated, they recruit adaptor proteins, which drive the formation of protein complexes. These complexes activate other downstream proteins, kinases among them, in a pathway that ultimately alters gene expression.
Intracellular receptors and gene expression
Many signalling pathways end by activating an intracellular receptor that goes on to alter the expression of a particular gene. Some intracellular receptors are activated by lipid-soluble first messengers that have diffused through the cell membrane, such as steroid hormones and vitamins A and D, and others by second messengers produced by a cascade inside the cell. The receptors can be found in the cytosol or in the nucleus, and a cytosolic receptor–ligand complex has to pass into the nucleus before it can interact with a gene. There the complex's DNA-binding domain binds DNA. An activated steroid receptor generally binds a receptor-specific hormone-responsive element in the promoter region of a gene that the hormone–receptor complex activates. The complex can so alter the gene's expression, for example by increasing or decreasing its transcription and thus the production of the protein it encodes.
The pathways in disease and as drug targets
The pathways play important roles in health and disease. Some oncogenes code for a receptor protein, such as the oestrogen receptor, that is constitutively activated, or in the "on" state all the time, so that it activates its pathway even when its ligand is absent. Disruptions in the pathways that regulate immune cells can result in autoimmune or inflammatory disease. Disruptions in the pathways that would ordinarily suppress a cell's division or command it to commit suicide, by apoptosis or programmed cell death, can lead to cancer.
Signal transduction pathways were therefore becoming increasingly important therapeutic targets, particularly in oncology but also in immunology. In oncology the goal is to suppress the proliferation of cancer cells without undue harm to the body as a whole, and in immunology it is typically to suppress an inappropriate inflammatory response without dangerous immune suppression or other undesirable effects. Agents were being designed to act selectively on a particular target within a particular pathway in a particular set of cells, and understanding how they work requires understanding the pathways. Research into intracellular signalling pathways has been yielding new medications for inflammatory and neoplastic disease.