9.2Intracellular Signal Transduction

Intracellular signal transduction is a system in which signaling molecules bind to the receptors on the cell surface, triggering signaling within a cell. In intracellular signal transduction, information is transduced mainly by protein phosphorylation, G proteins and second messengers. This section discusses three basic mechanisms of signal transduction that are common to many organisms.

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Phosphorylation and Dephosphorylation of Proteins

The most important among the intracellular signal transduction mechanisms is the phosphorylation of the side chains of tyrosine, serine and threonine in proteins. As shown in Figure. 9-2A, phosphorylation is one of the most effective ways of changing the structure of proteins due to the large size and negative charge of the phosphate group; for the same reason, it is also effective as a recognition marker for other proteins. The enzymes that perform phosphorylation are called protein kinases (referred to simply as “kinases” in this chapter), and many types exist. In signal transduction, a reaction that removes the phosphate group takes place. This reaction is called dephosphorylation, and the enzymes that perform it are called protein phosphatases (referred to as “phosphatases” in this chapter) (see Chapter 7).

Fig. 9-2A. Signal transduction by protein phosphorylation

Phosphorylation of amino acids - serine, threonine and tyrosine.

For signal transduction by phosphorylation to take place, kinases need to be activated, as shown in Figure. 9-2B. First, receptors themselves may have kinase activity. In such cases, receptors on the plasma membrane surface become bound with a signaling molecule (i.e., a first messenger) and form dimers, thereby activating the kinase domain of the receptor proteins within the cell (Fig. 9-2B(a)). A kinase may also bind with a second messenger located in the cell and become activated (Fig. 9-2B(b)).
Kinases in the cytoplasm may also be activated. A-kinase, which is activated by cAMP (a second messenger), is normally deactivated when it binds to an A-kinase inhibitory protein. When cAMP binds to this kind of protein, the kinase and the inhibitor are separated from each other, thereby activating A-kinase and thus phosphorylating the target proteins.
Kinases activated in the cell may induce chain reactions (Fig. 9-2B(c)). This phenomenon is called a kinase cascade, since a series of reactions occur in the same way as a waterfall flowing over a cliff. A well-known example is that of MAPK (mitogen-activated protein kinase), which is activated by extracellular stimuli. MAP kinase kinase kinase (or MAPKKK), when activated by stimuli, phosphorylates MAP kinase kinase (MAPKK), which activates MAP kinase by phosphorylation. The activated MAP kinase activates genes by phosphorylating various transcription factors.

Fig. 9-2B. Signal transduction by protein phosphorylation

The three activation patterns of kinase: (a) activation of receptor kinases by first messengers (growth factors, hormones, etc.), (b) activation of kinases by (intracellular) second messengers, and (c) chain reactions of kinases (the Figure shows the MAPK cascade)

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G Proteins

G proteins are a family of intracellular proteins bound with GDP or GTP (see Fig. 2-5 in Chapter 2). They function by alternating between an inactive GDP-bound state and an active GTP-bound state.
There are two G protein groups. One is low-molecular-weight G proteins, which act as monomers (molecular weight: 20,000 - 30,000). As shown in Figure. 9-3A, low-molecular-weight G proteins exist normally as an inactive GDP-bound form, are activated by a factor that exchanges GDP with GTP in response to signal transduction from receptors, and on completion of their roles are inactivated by RGS and GAP - GTPase activating proteins. Low-molecular-weight G proteins are used to transfer information from one place to another within a cell.
The other G protein type is trimetric G proteins, which consist of three subunits - Gα, Gβ and Gγ (Fig. 9-3B). A trimetric G protein binds to a G protein-coupled receptor - a characteristic receptor that penetrates the plasma membrane seven times - in which GDP binds to Gα, thereby inactivating the protein. When a signaling molecule binds to the receptor protein, the Gα subunit releases GDP and is bound instead with GTP, thereby becoming activated. This activated Gα transduces signals to target factors through protein-protein interaction. This active state is brought back to an inactive state through the hydrolysis of GTP by RGS and GAP activity. Gβ and Gγ transfer signals in some cases.

Fig. 9-3. G protein functioning cycle

A) G proteins alternate between an active GTP-bound state and an inactive GDP-bound state. There are activating factors that release GDP and exchange it with GTP located in the cytoplasm, and inactivating factors (RGS and GAP) that promote GTPase activity in Gα subunits and shift the protein to an inactive state.
B) Binding of a signaling molecule to the receptor transforms the Gα subunit to a GTP-bound form, which releases the subunit as well as Gβ and Gγ from the receptor. When the GTP of the Gα subunit is hydrolyzed into GDP, the subunits are returned to their original position.

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Low-molecular-weight Second Messengers

As discussed in the section on phosphorylation, in intracellular signal transduction, not only proteins but also low-molecular substances and ions (such as cAMP, inositol trisphosphate (IP3) and Ca2+) are second messengers that play important roles. These low-molecular substances transduce information by diffusing within the cell.
Figure 9-4 shows how the concentration of Ca2+, a second messenger, increases. The Ca2+ level inside a cell is normally significantly lower than that outside it. As shown in Figure. 9-4B, the Ca2+ level is high in an endoplasmic reticulum; if a channel that releases Ca2+ from the endoplasmic reticulum opens in response to a signal from the receptor, the intracellular Ca2+ level increases.

Fig. 9-4. Signal transduction by Ca2+

A) Increase in the Ca2+ level in the cell: the wave of Ca2+ that occurs at the moment of fertilization can be observed by adding a Ca2+-sensitive fluorescent dye to a cell.
B) The Ca2+ level in a cell is low, whereas that in an endoplasmic reticulum is relatively high. Once a stimulus is applied, therefore, the Ca2+ channel of the endoplasmic reticulum opens, supplying a large amount of Ca2+ to the cytoplasm. (Photo provided by Professor Katsuhiko Mikoshiba of the University of Tokyo)


Relationship between Receptors and Signaling Molecules

It is known that hormones, which are major signaling molecules in the human body, function by binding to receptors at a low concentration of around 10-10 mol/l (6 x 1013 molecules/l). This underlines the high affinity of hormones to receptors. However, hormones (H) and receptors (R) do not form covalent bonds; rather, they bind reversibly.

H + R ⇄ HR

When hormone-receptor binding is in equilibrium, the rates of the normal and reverse reactions are the same:

kon [H][R] = koff [HR]

where kon is the binding rate constant, koff is the dissociation rate constant, [H] is the concentration of free hormones that are not bound with receptors, [R] is the concentration of free receptors, and [HR] is the concentration of hormones bound with receptors.

This can be changed to:

Kd = [H][R]/[HR] = koff/kon (Equation 9-1)

where Kd is the dissociation constant.

For a simple system in which one receptor has a single binding site, the total concentration of receptors in a cell, R, can be expressed as follows:

[R] = Rtot - [HR]

By assigning this to Equation 9-1:

[HR] = Rtot [H]/(Kd + [H])

In an actual experiment, hormones labeled by a radioisotope are added to a cell culture, and after equilibrium is reached, the cells are washed to separate bound hormones from free hormones. Kd is determined by measuring the concentration of hormones bound to 50% of the receptors, as illustrated in Column Figure. 9-1A. Column Figure 9-1B uses a semi-logarithmic scale as the concentration range extends over multiple digits. Using this, the total concentration of receptors (i.e., the total number of receptors to which hormones can bind) and Kd (the concentration at which 50% of receptors are occupied) are calculated.

Column Fig. 9-1. Saturation binding

Direct binding curves that use (A) the linear scale and (B) the semi-logarithmic scale.

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