Good communication is as stimulating as black coffee and just as hard to sleep after.
—Anne Morrow Lindbergh (American author and aviator, 1906–2001)
In: Gift from the Sea (1955)
Soluble chemical signals sent from one cell to another are a basic means by which cell communication takes place. Hormones, growth factors, and neurotransmitters are chemical communication signals. The cellular recipient of the signal is known as the target cell and binds the signaling molecule via a protein receptor that may be on the cell surface or within its cytoplasm or nucleus. The binding to the receptor initiates the signaling process and results in a cascade of reactions that amplify the signal and produce the desired effect within the cell. The types of receptors that are engaged by signaling molecules are grouped into distinct cell signaling mechanisms for purposes of study and description, although a great deal of overlap exists in these classifications.
Biochemical processes within target cells are regulated in response to signaling molecules. The first cell signaling chapter focuses on G protein signaling. Receptors coupled to G proteins amplify the message sent by the signaling molecule by regulating the production of intracellular signaling molecules. The second chapter in this unit concerns signaling by catalytic receptors that possess their own enzymatic activity which is stimulated by ligand binding to the receptor. The third chapter in the unit examines steroid hormone signaling, which is readily distinguished from the other two forms by the location of steroid hormone receptors in the interior of the cell as opposed to on the membrane’s surface.
G protein and catalytic receptor signaling, and steroid hormone signaling to some extent, involves phosphorylation of amino acid residues within cellular proteins by protein kinases. While cellular programs are turned on for hours or more when serine or threonine residues are phosphorylated, the stimulatory effects of tyrosine phosphorylation are more fleeting, and a rapid cellular response ensues. Overstimulation of critical signaling pathways can cause heightened activation within a cell, with malignant consequences and no rest for the cell if the inappropriate stimulation cannot be halted.
G proteins are intracellular signaling proteins that are named for their ability to bind to guanosine triphosphate (GTP). They also possess GTPase activity, the ability to hydrolyze GTP to GDP. Two categories of G proteins are described: heterotrimeric G proteins and the Ras superfamily of G proteins (Figure 17.1).
FIGURE 17.1.Heterotrimeric and Ras superfamily G proteins.
Ras superfamily members are often called “small G proteins” since they are monomers that resemble one subunit of the heterotrimeric G proteins. Ras proteins receive their signals from catalytic receptors that have been activated by their ligand (see Chapter 18). The overall effects of Ras signaling often involve induction of cell proliferation, cell differentiation, or vesicle transport.
Heterotrimeric G proteins consist of three subunits, ?, ?, and ?. The signaling process is initiated by ligand binding to receptors linked to G proteins tethered to the inner membrane leaflet. Activation of the G protein then enables it to regulate a specific membrane-bound enzyme. Products of reactions catalyzed by activated enzymes include second messengers that amplify the signal sent to the cell by the hormone or neurotransmitter that bound to its receptor and acted as the first message (Figure 17.2).
Many second messengers activate serine/threonine protein kinases, enzymes that phosphorylate their substrates on serine and threonine amino acid residues. Changes in phosphorylation status of target proteins, many of which are enzymes, can alter their activity. The overall result is the biological response of the cell to the hormone or neurotransmitter. The biological response is often the regulation of a biochemical pathway or the expression of a gene.
FIGURE 17.2.Overview of G protein signaling.
ECEPTORS AND HETEROTRIMERIC G PROTEIN SIGNALING
Many hormones and neurotransmitters have receptors on their target cells that are linked to G proteins. G protein–linked receptors are the most common form of cell surface receptor. These receptors have extracellular hormone-binding regions as well as intracellular portions that interact with the G protein to send the message from the hormone into the cell to evoke a response.
G protein–linked receptors
G protein–linked receptors are transmembrane proteins with seven membrane-spanning regions (Figure 17.3). Close to 400 distinct G protein–coupled receptors have been identified in humans (367 were reported through 2009). Most are expressed in multiple tissues. Over 90% of them are expressed in the brain. All use the same basic process to stimulate G proteins to regulate the production of second messengers.
FIGURE 17.3.Structure of G protein–coupled receptors.
All heterotrimeric G proteins use the same basic scheme shown for the Gs type of G protein (Figure 17.4). An unoccupied G protein–linked receptor does not interact with the G protein in close proximity to its intracellular domain, such as the Gs shown here. Ligand binding to the receptor creates an occupied receptor that undergoes a conformational change and is then able to interact with the G protein. (A ligand is a molecule that binds specifically to a particular receptor.
Hormones and neurotransmitters are ligands of G protein–linked receptors.) In response to the receptor binding to the G protein complex, the G? subunit of the G protein releases GDP and binds GTP. The G protein is now active and the ? subunit dissociates from the ? and ? subunits. The active ? subunit then interacts with an enzyme whose function is regulated by the G protein. Adenylyl cyclase is the enzyme activated by Gs protein signaling to have the ability to convert ATP to cyclic AMP (cAMP) and inorganic phosphate (PPi). cAMP is the second messenger in Gs signaling.
The type of G protein that is activated and the second messenger it regulates depend on the ligand, the type of receptor, and the type of target cell. When hormone is no longer present, the receptor will revert to its resting state. GTP is hydrolyzed to GDP (by the GTPase of the G protein), the enzyme, such as adenylyl cyclase, is inactivated, and the ? subunit will reassociate with ? and ? subunits to stop the signaling process.
FIGURE 17.4.Activation of G proteins.
HETEROTRIMERIC G PROTEINS AND THE SECOND MESSENGERS THEY REGULATE
Distinct members of the heterotrimeric G protein family exist through the association of various forms of the three subunits, ?, ?, and ? (Figure 17.5). At least 15 different ? subunits are known. Combinations of different ?, ?, and ? subunits form the heterotrimeric subunits. GDP is bound to the ?, subunit of the G protein when all three subunits are joined together in the inactive form. Certain G? subunits interact with certain enzymes.
For example, Gs interacts with adenylyl cyclase as described above. G? subunits are distinguished from each other by subscripts including s, i, and q (G?s, G?i, and G?q). The identity of the enzyme determines which second messengers will be produced (or inhibited). Adenylyl cyclase and phospholipase C are two enzymes regulated by G proteins that are responsible for regulating messengers with important signaling roles.
FIGURE 17.5.Heterotrimeric G proteins.
Two different G? proteins regulate the activity of adenylyl cyclase; the G?s system stimulates its activity while the G?i inhibits it. Epinephrine (adrenaline) is a hormone that signals with cAMP as the second messenger. In liver, muscle, and adipose cells, the biological response that results is the breakdown of stored carbohydrates (glycogen) and fat for use as energy. Glucagon is a hormone that also stimulates glycogen breakdown in liver (see also LIR Biochemistry, pp. 131–134). In the heart, the number of beats per minute (heart rate) is increased by this signaling process.
1. G?s: The active G? stimulates adenylyl cyclase (see Figure 17.4). This enzyme uses ATP as a substrate to produce the second messenger cAMP. The enzyme phosphodiesterase converts cAMP to 5?-AMP, ensuring that the amount of cAMP in the cell is low. cAMP activates cAMP-dependent protein kinase A, known as protein kinase A (PKA) (Figure 17.6). The activation process involves cAMP binding to the regulatory or R subunits of PKA, enabling the release of catalytic or C subunits. Freed C subunits of PKA are active. PKA phosphorylates its protein substrates, many of which are enzymes, on serine and threonine residues. Phosphorylation regulates the activity of proteins and enzymes and can lead to intracellular effects. Protein phosphatases can dephosphorylate the phosphorylated proteins to regulate their activity. Over time, the G?s will hydrolyze GTP to GDP to terminate the activation of adenylyl cyclase and the production of cAMP.
FIGURE 17.6.Activation of PKA by cAMP.
2. G?i: When G?i is activated, it interacts with the active adenylyl cyclase to inhibit its ability to produce cAMP. In response, PKA will not be activated and its substrates will not be phosphorylated.
A variety of neurotransmitters, hormones, and growth factors initiate signaling through G?q (Figure 17.7). After a hormone binds to its Gq-linked receptor, the intracellular domain of the occupied receptor interacts with Gq. The ? subunit of Gq releases GDP and binds GTP. The ? subunit dissociates from the ? and ? subunits and then the ? subunit activates phospholipase C to cleave the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2).
The products of this cleavage are inositol 1,4,5-trisphosphate (IP3), which is released into the cytosol, and diacylglycerol (DAG), which remains within the plasma membrane. IP3 binds to a specific receptor on the endoplasmic reticulum, causing release of sequestered calcium. Calcium and DAG together activate the calcium-dependent protein kinase named protein kinase C (PKC). IP3, DAG, and calcium are second messengers in this system. PKC catalyzes phosphorylation of cellular proteins that mediate cellular responses. Effects of intracellular calcium are mediated by the calcium-binding protein calmodulin (Figure 17.8).
After calcium is released from the endoplasmic reticulum in response to the signaling of hormones or neurotransmitters, the transient increase in intracellular calcium concentration favors formation of the calmodulin-calcium complex. The calmodulin-calcium complex is an essential component of many calcium-dependent enzymes. Binding of the complex to inactive enzymes results in their conversion to active enzymes.
FIGURE 17.7. Generation of second messengers in response to G?q activation of phospholipase C.
FIGURE 17.8. Calmodulin mediates many effects of intracellular calcium.
Toxins and G? proteins that regulate adenylyl cyclase
Both cholera and pertussis toxins alter G? subunits and higher than normal concentrations of cAMP in infected cells. Cholera toxin is produced by Vibrio cholera bacteria that produce cholera toxin when they infect intestinal epithelial cells. This toxin modifies the G?sbunit so that it cannot hydrolyze GTP and adenylyl cyclase remains active indefinitely. Diarrhea and dehydration result from excessive outflow of water into the gut in response to excess cAMP.
Cholera can be fatal without appropriate hydration therapy. Bordetella pertussis is a bacterium that infects the respiratory tract and causes pertussis or whooping cough. Vaccination now prevents many young children from dying from the effects of pertussis. However, it remains a major health threat. The World Health Organization reports that there were 39 million cases and 297,000 deaths attributed to pertussis in 2000.
Ninety percent of all cases are reported in developing countries but the numbers of cases have been rising in the United States each year. This devastating disease is caused by pertussis toxin produced by the infecting bacteria. It inhibits G?i so that G?i cannot inhibit adenylyl cyclase. Adenylyl cyclase remains active indefinitely, producing excess cAMP. Coughing can lead to vomiting and dehydration. Antibiotics and hydration therapy are used in treatment.
RAS G PROTEINS
Ras G proteins are homologous to the ? subunits of heterotrimeric G proteins. They do not regulate membrane-bound enzymes or induce the production of second messengers. Instead, their activation by GTP allows them to initiate a cytoplasmic phosphorylation cascade that terminates with activation of gene transcription. In this signaling scheme, Ras proteins are viewed as relay switches between cell surface receptors and a cascade of serine/threonine kinases that regulate nuclear transcription factors. Such signaling is important in the regulation of cell proliferation. The aberrant function of Ras proteins may contribute to the malignant growth properties of cancer cells.
Ras proteins are involved in signaling by certain hormones and growth factors that are ligands of catalytic receptors (see also Chapter 18). A linear pathway from the cell surface to the nucleus has been described, with Ras acting as an intermediary (Figure 17.9). Ligand binding to catalytic receptors can cause phosphorylation of tyrosine residues within the receptors. The receptor’s phosphotyrosines provide “docking” or binding sites for intracellular adaptor proteins such as SHC and Grb2 that contain regions known as SH2 domains. Ras-specific guanine exchange factor (GEF) SOS joins the complex, followed by Ras.
The SHC-SOS-Ras complex exchanges GTP for GDP on Ras, activating Ras. Ras-GTP promotes binding and phosphorylation of Raf, a serine protein kinase (also known as MAPKKK for mitogen-activated protein kinase kinase kinase). A phosphorylation cascade then includes mitogen-activated protein kinases kinases (such as MEK) that phosphorylate and activate mitogen-activated protein kinase (MAPK, also known as extracellular signal-regulated kinases or ERK), enabling it to translocate to the nucleus where it phosphorylates a transcription factor (such as ELK). The cascade terminates with transcription of genes for immediate early genes involved in cell division. Hydrolysis of GTP to GDP by Ras terminates the signaling process.
FIGURE 17.9. Ras signaling via activation of a cytoplasmic serine/threonine cascade.
This linear pathway is now recognized to be only a part of a very complex signaling circuit in which Ras proteins are involved. Ras signaling involves a complex array of pathways, where cross talk, feedback loops, branch points, and multicomponent signaling complexes are seen.
Ras mutations and cell proliferation
Mutations in Ras genes result in Ras proteins that cannot hydrolyze GTP to GDP to inactivate the signaling process. The Ras protein then remains in the active state without stimulation of the receptor and continues to send signals to induce progression through the cell cycle. The result is excessive cell proliferation that can lead to malignancy.
- G proteins are intracellular signaling proteins named for the ability to bind to and hydrolyze GTP.
- Two categories of G proteins are described: heterotrimeric G proteins that regulate second messenger production and Ras superfamily small G proteins.
- Heterotrimeric G proteins are composed of ?, ?, and ? subunits and are activated by ligand binding to G protein–linked receptors.
- Active G protein–linked receptors interact with membrane-bound enzymes and regulate their function.
- Products of reactions catalyzed by G protein–linked enzymes are second messengers that amplify the signal sent to the cell by the ligand. Second messengers often regulate the activity of certain serine/threonine protein kinases.
- Adenylyl cyclase and phospholipase C are enzymes regulated by G proteins.
- Adenylyl cyclase is regulated by Gs proteins that stimulate its activity and Gi proteins that inhibit its activity.
- cAMP is the second messenger whose production is regulated by adenylyl cyclase.
- cAMP activates PKA.
- Phospholipase C is activated by Gq proteins that stimulate its activity to cleave the membrane lipid PIP2.
- IP3 and DAG are products of this cleavage and are the second messengers.
- IP3 induces the release of calcium from the endoplasmic reticulum.
- Calcium and DAG activate PKC.
- Calcium binds to calmodulin which regulates the activity of other proteins.
- The GTP-binding protein Ras is an intermediary in signaling via some catalytic receptors.
- Activated Ras can stimulate the MAP kinase cascade of serine/threonine phosphorylations that can result in stimulation of gene transcription.
- Ras signaling is involved in the stimulation of cell proliferation. Mutations in Ras can cause unregulated cell division and malignancy.