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15.1Receptor-Mediated Intracellular Signaling Pathways

Intracellular signal transduction is the result of a combination of the various mechanisms described in Chapter 14. Signal transduction is involved in all steps of cellular processes, from the transduction of extracellular stimuli into the cell via signal molecules, the appropriate processing of the received information within the cell, and to the subsequent response by the cell. Cell responses are diverse, ranging from short-term ones such as morphological changes and the release of substances stored in cells, to long-term changes such as cellular differentiation and proliferation, which are caused by changes in the genetic expression of cells.

This chapter introduces the four main receptor types that react to signal molecules from the extracellular environment: enzyme-linked, G protein-coupled, ion-channel-linked, and transcription factor receptors. Specific examples will be given for each of these receptor types to understand the signal transduction pathways through which stimuli on the cell surface activate genes inside the nucleus.

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15.1.1

Enzyme-linked receptors

Most enzyme-linked receptors present at the cell membrane have kinase domains, which function as phosphorylating enzymes within the cell. The binding of signal molecules to the receptors induces receptor dimerization, which induces reciprocal tyrosine phosphorylation of the dimerized receptors. Taking the epidermal growth factor (EGF) receptor as an example, this section explains the mechanism of downstream signal transduction after the ligand binds to the cell surface receptor, focusing on the representative proteins and pathways involved (Figure 15-1).

Binding of the EGF ligand to EGF receptors encourages two EGF receptors to come closer to each other and dimerize. During this process, the three-dimensional conformation of the intracellular domain of the EGF receptor changes, thereby activating the tyrosine kinase in the domain, and inducing the two EGF receptors forming the dimers to mutually phosphorylate each other’s tyrosine residues. Adapter proteins and other mediators in signal transduction bind to the activated EGF receptor dimers to form a receptor signal-transduction complex. Some of these adapter proteins have an SH2 (Src Homology 2) domain (see Column at the bottom) that recognizes the phosphorylated tyrosine on the receptor and specifically binds to activated proteins.

Fig. 15.1 Enzyme-linked receptors: EGF receptor signaling pathways

After signal molecules bind to EGF receptors, these receptors form dimers, are activated, and phosphorylate each other as tyrosine kinases. When the cytoplasm end of the receptors is phosphorylated, the adapter protein Grb2 binds to them and activates Ras via Sos (a type of GEF [see Chapter 14]). The activated Ras then activates the serine/threonine kinase Raf, which in turn activates MAP kinase cascade. The activated MAP kinase (ERK) activates transcription factors and promotes protein synthesis.

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Domains

A domain generally consists of 40–100 amino acid residues. Some domains have specific enzyme activities (e.g., kinase domain), whereas others recognize specific amino acid sequences (e.g., SH2 domains). When a protein is phosphorylated, other proteins with domains that recognize such phosphorylation bind to the phosphorylated protein. Signaling molecules are made up of several domains and enzyme activity sites, and bind to phosphorylated amino acid sequences, thereby activating further downstream signal-transduction pathways.
1) Peptide-binding domain
<(1)SH2 (Src Homology 2) domain>
This is a sequence found in the Src protein, a non-receptor tyrosine kinase. SH2 consists of approximately 100 residues and binds to phosphorylated tyrosines.
<(2)SH3 domain>
This sequence, also found in Src, binds to sequences (approximately 8–10 amino acids in length) that are abundant in proline.
2) Protein- and lipid-binding domain
<PH domain>
Consisting of approximately 100 amino acid residues, the PH domain has been found in more than 600 types of proteins. It is involved in the interactions between proteins, and between protein and lipids.
3) Ca2+-binding domain
<C2 domain>
Located in the C-kinase, this C2 domain binds to Ca2+.
4) Kinase domain
The catalytic activities of kinases are based on the kinase domain. One typical example is the A-kinase, which consists of a small domain of approximately 100 amino acids at the N-terminus, and ATP-binding site at the C-terminus.

Column Fig 15.1 Examples of protein with domains

Not all of the proteins shown here are discussed in the text. They are all functional proteins with domains, as shown in the figure.

Some activated complexes activate a series of signal cascades in which the Ras small G protein is involved. Then finally, the activated Ras regulates the expression of proteins through the mitogen-activated protein (MAP) kinase cascade (see Chapter 14). Activated extracellular signal-regulated kinases (ERKs or MAP kinases) dimerize and enter the nucleus. Within the nucleus, ERKs phosphorylate transcription factors and promote the transcription of genes such as c-fos.
Some enzyme-linked receptors have domains that phosphorylate serine or threonine in addition to tyrosine phosphorylation domains. On the other hand, some proteins have domains with phosphatase activity that removes phosphate groups. These proteins also play an important role in signal transduction.

Fig. 15.2 G protein-coupled receptors: adrenergic receptor signaling pathways

There are different types of adrenergic receptors and their coupling G proteins. Downstream signaling pathways differ according to specific type (these differences are omitted in the figure). One of the main signaling pathways activated by adrenergic receptors involves the production of cAMP (left side of figure). Another is the pathway using inositol triphosphate and Ca2+ (right side of figure).

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15.1.2

G protein-coupled receptors

G protein-coupled receptors make up the largest family of receptor proteins in humans. The human genome encodes a number of G protein-coupled receptors that are involved in the reception of various information, ranging from the detection of stimuli such as light and smell to the actions of blood pressure-regulating hormones. As shown in Figure 15-2, these receptor proteins posses seven membrane-spanning domains. They are named “G protein-coupled proteins” because a trimeric G protein (see Chapter 14) is bound on the cytoplasmic end of these receptors.

An example of a signal molecule that binds to a G protein-coupled receptor is adrenaline, a hormone that triggers various reactions (e.g., increased blood pressure, increased pulse rate, pupil dilation, increased blood sugar levels) in an organism when it is engaged in a fight or running away from danger. This section discusses signal transduction via G protein-coupled receptors by using adrenergic receptors*1 as an example.

A signaling pathway that is activated when adrenaline binds to its receptor results in the production of cyclic adenosine 3′,5′-monophosphate (cAMP). Specifically, when an adrenergic receptor is activated by adrenaline binding, the guanosine diphosphate (GDP) bound to the α subunit of the trimeric G protein coupled to the adrenergic receptor, is replaced with guanosine triphosphate (GTP). This releases the α subunit, which in turn activates adenylate cyclase bound to the cell membrane. Adenylate cyclase then converts adenosine 5′-triphosphate (ATP) in the cytoplasm to cAMP, which activates A-kinase through the process explained in Chapter 14. A-kinase in turn activates a target protein, phosphorylase kinase, which then activates glycogen phosphorylase, an enzyme that cleaves glucose molecules one by one from the glycogen chain by phosphorolysis. Activated glycogen phosphorylase produces glucose-1-phosphate from the glycogen stored in cells, and eventually raises blood sugar.

Another important downstream signaling event induced by adrenaline increases intracellular calcium ion (Ca2+) concentrations. The activated adrenergic receptor activates the trimeric G protein and the α subunit activates the enzyme phospholipase C (PLC), which in turn hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2), a phospholipid component of the cell membrane, into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) (See right side of Figure 15-2). IP3 then dissociates from the cell membrane and diffuses into the cytoplasm, binds to the Ca2+ channel on the endoplasmic reticulum and opens the channel, thereby releasing Ca2+ into the cytoplasm. As explained in Chapter 14, Ca2+ functions as a second messenger and transmits signals downstream. For instance, intracellular Ca2+ triggers contraction in smooth muscle cells (see Chapter 17 for details on the mechanism of muscle contraction). Meanwhile, DAG, the other hydrolysis product of PIP2, activates the protein kinase C (C-kinase). Dependent on DAG, C-kinase is involved in various signaling pathways such as activation of MAP kinases.

*1 Adrenergic receptors are divided into three groups (α1, α2, and β) according to their distribution in tissue and their actions. These groups are subdivided into α1B…, α2A…, and β1… For simplification, these differences are omitted in the text.

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15.1.3

Ion channel-linked receptors

Fig. 15-3 Acetylcholine receptors

Channels are normally closed (left). The binding of acetylcholine to two sites of a receptor opens the channel, thereby changing the conformation of the receptor to allow Na+ to pass through (right).

Ion channels, found on the cell and endoplasmic reticulum membranes, serve to regulate the movement of ions across the membranes (see column Selection 1 of Chapter 11). In particular, those receptors that open and close channels in response to the binding of ligands are called ion-channel-linked receptors. Such receptors on the cell or endoplasmic reticulum membranes are opened when signal molecules bind to them, and the resultant changes in the intracellular ion concentration transduce the signals. Figure 15-2 (lower right) illustrates an ion-channel-linked receptor that opens and releases Ca2+ following IP3 binding.

Figure 15-3 is an illustrative model of the acetylcholine receptor, a commonly studied ion channel-linked receptor (see column Selection 3 of Chapter 17). Acetylcholine is an intercellular signal transducer (first messenger). The acetylcholine receptor consists of five subunits, all of which span the cell membrane and form a channel. The binding of signal molecules to a channel receptor triggers a conformational change in the receptor, opens the channel, and allows Na+ to pass through the channel surrounded by the five subunits into the cell.

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Discovery of calcium ions as second messengers

Sydney Ringer was a British physician working at the University College Hospital in London. While treating patients and instructing young doctors, he was also engaged in pharmacological study. During these studies, he discovered that by administering sodium chloride, the beating of frog hearts can be maintained for a while even after being isolated from their body. This beating, however, could only be maintained for a short period of time.

However, one day an isolated frog heart used in an experiment continued beating for several hours. His laboratory assistant had decided to cut corners and prepared the sodium chloride solution using tap water instead of distilled water, because he did not understand why he had to spend so much time on the distillation process. When Ringer found out about this, instead of rebuking the assistant, he examined the composition of the tap water and discovered that it contained a small amount of Ca2+, which allowed the heart to continue beating for long periods of time. He reported these findings in 1883.

In 1943, Takeo Kamata and others found that Ca2+ injected into muscle fiber caused muscle contraction. However, because Japan did not have any means of international publishing owing to the war at the time, it is generally thought that the fact was first discovered by Lewis Heilbrunn and his colleagues 4 years later. Heilbrunn did not focus much on the actions of calcium on proteins; however, Otto Loewi, who had heard Heilbrunn and his colleagues present their findings at the New York Academy, thought “calcium is the key to everything.” He was partly correct, because it is now known that cAMP, G proteins, and kinases are also involved in this process. If cAMP is the first discovered second messenger, then Ca2+ is the second.

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15.1.4

Receptors/transcription factors and intranuclear receptors

The previous discussions are premised on signal transduction that is made possible by the binding of first messenger signal molecules to receptors on the cell surface. The assumption is established because most of the signal molecules are water soluble and are hence unable to penetrate the cell membrane in the native form. However, there are substances that are able to directly cross the cell membrane and reach the nucleus without the use of a special process. If these substances are involved as first messengers, it does not necessarily mean that the start of the signaling pathway is always on the cell membrane.

Many lipid-soluble steroid hormones, such as adrenocortical hormones and male and female sex hormones (see Chapters 5 and 14 for details on hormones), and lipid-soluble signal molecules, such as vitamins A and D, are able to cross the cell membrane and enter the cell. Inside cells and the nucleus, there are receptors/transcription factors that specifically bind to these signal molecules. These are also called (intra) nuclear receptors, and are a particular type of transcription factor that contains special protein motifs known as zinc fingers, which are required for DNA binding (see Chapter 8).

One example of this signaling pathway uses glucocorticoid (type of steroid hormone) as the signal molecule (Figure 15-4). Fat-soluble glucocorticoid is able to penetrate the cell membrane by diffusion. Normally, glucocorticoid receptors (GRs) form complexes with multiple proteins inside the cytoplasm and remain inactivated. However, when glucocorticoids enter the cytoplasm and bind to GR complexes, the GR dissociates from other proteins, and forms a dimer with another GR that is bound to a glucocorticoid. Glucocorticoid-bound GR dimers are then able to translocate to the nucleus, where they regulate the transcription of a specific DNA sequence. GRs have a domain that promotes the transcription of target genes.

Figure 15-4 Receptors/transcription factors: glucocorticoid receptor signaling pathways

Glucocorticoid receptors (GR) form complexes with proteins in cytoplasm and are inactivated. When glucocorticoid binds to the GR complex within the cytoplasm, the GR complex forms an activated dimer, translocates into the nucleus, and promotes transcription.

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15.1.5

Cross talk in signal transduction

Figure 15-5 is a schematic illustration of the receptors described in the above sections. The mechanisms by which cells process various signals and respond to extracellular stimuli have broadly been discussed. In many cases, different signal transduction pathways share the same messengers (proteins and other molecules). Second messengers such as Ca2+ and cAMP are typical of such examples. These are used as second messengers for transducing signals from different signal molecules, but as seen in Figure 15-5, once they enter a common signal pathway, they seem to pass on the same signals downstream, regardless of the difference of the original signal molecule. However, in actual cells, not just one type but different types of signals are continuously transduced at various times. Furthermore, signals that are generated during signal transduction in turn generate and transduce new signals themselves. Gene expression occurring as a result of signal transduction can also affect signal transduction. There are many more substances involved in signal transduction than those discussed here, and in fact, as many as several hundred protein kinases are present in a single mammalian cell. Signaling pathways use their mutual relations and cross-points to combine multiple pieces of information, thereby responding to such combined inputs. Proteins that have multiple phosphorylation sites that are phosphorylated by different protein kinases function as an “integrating apparatuses” in the signaling pathways. There are also pathways in which several signals are converted into one signal. Thus, the flow of various information results in complex cell responses.

Fig. 15.5 Cross talks in intracellular signaling pathways

In this figure, target proteins and genes are regulated by four kinases. In the actual cell, signaling pathways affect each other

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