1.

1- PI-3K/AKT Pathway
A second major strategy that RTKs use to relay intracellular signal depends on a quite different
intracellular relay mechanism that includes activation of phosphoinositide 3-kinase (PI3-
kinase) as well as phospholipase C-γ. The pathway is present in all cells of high eukaryotes and
is highly conserved. The PI3-kinase is a plasma membrane-bound enzyme that binds to the
intracellular tail of RTK molecules and can be activated by both RTKs and GPCRs. It mainly
phosphorylates a membrane lipid, rather than proteins. When activated, PI3-kinase catalyzes
phosphorylation of a membrane lipid called, phosphatidyl inositol, to generate a variety of
phosphorylated lipids called phosphoinositides. Among these lipid molecules, the production of
PI(3,4,5)P3 (phosphatidylinositol (3,4,5)-trisphosphate) is of most importance. This is because it
can serve as a docking site for various intracellular signaling proteins that relay the signal from
the cytosolic face of the plasma membrane to downstream transducer proteins (Figure 11). It
should be noted too that there is a difference between the use of phosphoinositides in the
different signaling pathways. For example, they are cleaved by PLC-β (in the case of GPCRs)
or PLC-γ (in the case of RTKs) to generate soluble inositol trisphosphate (IP3) and
membrane-bound diacylglycerol (Figures 4). By contrast, they are phosphorylated by PI3-
kinase to produce phosphoinositide triphosphate, which then remain in the plasma membrane
until specific phosphoinositide phosphatases (such as PTEN phosphatase) dephosphorylate
them. When ligands, such as insulin and insulin-like growth factor (IGF), bind their specific
RTKs, the receptors activate PI3-kinase to generate the second messenger, phosphoinositide
triphosphate. Eventually, two protein kinases called Akt and phosphoinositide-dependent
protein kinase 1 (PDK1), recognizes this second messenger and dock to it via a specific
interaction domains, called PH (for Pleckstrin Homology) domain. The PDK1 phosphorylates,
and in turn, activates Akt. Once activated, Akt phosphorylates various intracellular proteins
provoking downstream responses such as cell survival, growth, proliferation, cell migration and
angiogenesis (Figure 11).
1.2- Phospholipase C-γ Pathway
RTKs can also relay intracellular signal through activation of the plasma membrane-bound
enzyme, phospholipase C-γ (PLC-γ). Signaling by nerve growth factor receptors (NGFRs) is
an illustrative example for activation of PLC-γ by RTKs. The NGFRs are primarily involved in
the regulation of growth, maintenance, proliferation, and survival of certain nerve cells as well as

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non-neuronal cells. The common ligands of these receptors are neurotrophins (nerve growth
factors). When NGF binds to the NGFRs, it drives the homodimerization of the receptor, which
in turn causes the autophosphorylation of the tyrosine kinase domain. This leads to the activation
of PI3-kinase, Ras, and PLC signaling pathways. Activation of PLC-γ results in hydrolysis of
phosphoinositides to form second messengers including diacylglycerol (DAG) and inositol
trisphosphate (IP3). These two second messengers appear to target many of the same
downstream effectors as Ras. Thus, IP3 induces release of Ca2+ from the endoplasmic reticulum,
resulting in the activation of various protein kinases and phosphatases, whilst DAG activates
protein kinase C isoforms (PKC) (Figure 12). These PKCs are a family of intracellular serine-
threonine kinases; once activated, they phosphorylates target proteins that vary depending on the
cell type. For example, they can activate Raf-MAPK cascade (Figure 9) regulating cell survival
or neuronal differentiation.
1.3- RTKs and GPCRs Activate Overlapping Signaling Pathways
The RTKs and GPCRs activate some of the same intracellular signaling pathways; for example,
both can activate the inositol triphosphate pathway triggered by phospholipase C. Moreover,
even when they activate different pathways, the different pathways can converge on the same
target proteins. Figure 13 illustrates five parallel intracellular signaling pathways that are
overlapped by RTKs and GPCRs systems, one triggered by GPCRs, two triggered by RTKs,
and two triggered by both kinds of receptors. Interactions among these pathways allow different
extracellular signal molecules to modulate and coordinate each other’s effects.
1- Tyrosine Kinase-Associated Receptors
Many cell-surface receptors depend on tyrosine phosphorylation for their activity and yet lack a
tyrosine kinase domain. These receptors act through cytoplasmic tyrosine kinases, which are
associated with the receptors and phosphorylate various target proteins, often including the
receptors themselves, when the receptors bind their ligand. These tyrosine kinase-associated
receptors (TKAR) thus function in much the same way as RTKs, except that their kinase domain
is encoded by a separate gene and is noncovalently associated with the receptor polypeptide
chain. In other words, whether the signal is transmitted by RTKs or by TKARs, the outcome is
similar; a protein that is positioned at the top of an intracellular signaling pathway is activated
(Figure 14A). A variety of receptor classes belong to this category, including the receptors for
antigen and interleukins on lymphocytes, integrins, and receptors for various cytokines and some

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hormones. These receptors are able to regulate several cellular processes, such as cellular
adhesion, division, proliferation and survival, gene expression, and immune response, among
others. As with RTKs, many of these receptors are either preformed dimers or are cross-linked
into dimers by ligand binding. Some of these receptors depend on members of the largest family
of mammalian cytoplasmic tyrosine kinases such as the Src family to relay their signals down to
effector proteins. The Src family protein kinases all contain SH2 and SH3 domains and are
located on the cytoplasmic side of the plasma membrane, held there by their interaction with
transmembrane receptor proteins. Different family members of cytoplasmic tyrosine kinases are
associated with different receptors and phosphorylate overlapping but distinct sets of target
proteins. In each case, the kinase is activated when an extracellular ligand binds to the
appropriate receptor protein. Src itself, as well as several other family members, can also bind to
activated RTKs; in these cases, the receptor and cytoplasmic kinases mutually stimulate each
other’s catalytic activity, thereby strengthening and prolonging the signal. There are even some
G proteins that can activate Src, which is one way that the activation of GPCRs can lead to
tyrosine phosphorylation of intracellular signaling proteins and effector proteins (Figure 14B).
2.1- Integrin Receptor Activates Focal Adhesion Kinase Signaling Pathway
One of the most prominent TKAR-signaling pathways is the Integrins-FAK pathway. Integrins
are transmembrane proteins that link between extracellular matrix and cytoskeletal structures.
They serve both as adhesive receptors and also direct intracellular signaling events. The
extracellular matrix substances provide cells with a structural, chemical and mechanical substrate
that is essential for normal development and responses to pathophysiological signals. Integrins
orchestrate multiple functions in the intact organism including organogenesis, regulation of gene
expression, cell proliferation, differentiation, migration and death. The binding of matrix
components to integrins activates intracellular signaling pathways that influence the behavior of
the cell. When integrins cluster at sites of matrix contact, they trigger the assembly of cell-
matrix junctions called focal adhesions. Among the many proteins recruited into these junctions
is the cytoplasmic tyrosine kinase called focal adhesion kinase (FAK), which binds to the
cytosolic tail of one of the integrin subunits with the assistance of other proteins. The clustered
FAK molecules phosphorylate each other, creating phosphotyrosine docking sites where the Src
kinase can bind. Src and FAK then phosphorylate each other and other proteins that assemble in
the junction, including many of the signaling proteins used by RTKs. In this way, the two

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tyrosine kinases signal to the cell that it has adhered to a suitable substratum, where the cell can
now survive, grow, divide, migrate, and so on (Figure 15).
2.2- Cytokine Receptors Activate the JAK–STAT Signaling Pathway
The largest and most diverse class of receptors that rely on cytoplasmic tyrosine kinases to relay
signals into the cell is the class of cytokine receptors. These large family includes receptors for
many kinds of local mediators (collectively called cytokines), as well as receptors for some
hormones, such as growth hormone and prolactin. These receptors are stably associated with
cytoplasmic tyrosine kinases called Janus kinases (JAKs), which phosphorylate and activate
transcription regulators called STATs (Signal Transducers and Activators of Transcription).
STAT proteins are located in the cytosol and are referred to as latent transcription regulators
because they migrate into the nucleus and regulate gene transcription only after they are
activated (Figure 16). Cytokine receptors are dimers or trimers and are stably associated with
one or two of the four known JAKs (JAK1, JAK2, JAK3, and Tyk2). Cytokine binding alters the
arrangement so as to bring two JAKs into close proximity so that they phosphorylate each other,
as well as specific tyrosine residues on the cytoplasmic tails of cytokine receptors, creating
phosphotyrosine docking sites for STATs (Figure 16). Some adaptor proteins can also bind to
some of these sites and couple cytokine receptors to the Ras-MAPK signaling pathway. JAKs
also phosphorylate the STATs on tyrosines, causing the STATs to dissociate from the receptors.
There are at least six STATs in mammals; the phosphorylated STATs associate as a homodimer
or a heterodimer and translocate to the nucleus, where they bind to a specific cis-regulatory
sequence in various genes and stimulates their transcription (Figure 16). For example, in
response to the hormone prolactin, which stimulates breast cells to produce milk, activated
STAT5 stimulates the transcription of genes that encode milk proteins.
Negative feedback regulates the responses mediated by the JAK/STAT pathway. In
addition to activating genes that encode proteins mediating the cytokine-induced response, the
STAT dimers can also activate genes that encode inhibitory proteins that help shut off the
response. Some of these proteins bind to and inactivate phosphorylated JAKs and their
associated phosphorylated receptors; others bind to phosphorylated STAT dimers and prevent
them from binding to their DNA targets. In addition, protein phosphatases inactivate the
activated JAKs and STATs through dephosphorylation of their phosphotyrosines.
2- Receptor Serine/Threonine Kinases-Dependent Signaling Pathway

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Receptor serine/threonine kinases (RSTKs) are transmembrane proteins with extracellular
ligand-binding domains and cytoplasmic kinase domains. All RSTKs of the human tissue act as
signaling receptors for secreted polypeptide members of the transforming growth factor β
(TGF-β) superfamily. The superfamily consists of the TGFβ/activin family and the bone
morphogenetic protein (BMP) family. The TGFβ ligands act either as hormones or, more
commonly, as local mediators to regulate a wide range of biological functions in all animals.
During early embryogenesis, they regulate pattern formation, proliferation, specification and
differentiation, extracellular matrix production, and cell death. In adults, they are involved in
tissue repair and in immune regulation, as well as in many other processes.
There are two classes of receptor serine/threonine kinases, type I and type II, which are
structurally similar homodimers. Each member of the TGFβ superfamily binds to a characteristic
combination of type-I and type-II receptor dimers, bringing the kinase domains together so that
the type-II receptor can phosphorylate and activate the type-I receptor, forming an active
tetrameric receptor complex. The activated type-I receptor directly binds and phosphorylates a
latent transcription regulators of the Smad family called R-Smads that include (Smad-1, -2, -3, -
5 and -8). Once one of these receptor-activated Smads has been phosphorylated, it dissociates
from the receptor and binds to Smad4 (called a co-Smad), which can form a complex with any
of the five R-Smads. The Smad complex then translocates into the nucleus, where it associates
with other transcription regulators and controls the transcription of specific target genes (Figure
17). During the signaling response, the Smads shuttle continuously between the cytoplasm and
the nucleus: they are dephosphorylated in the nucleus and exported to the cytoplasm, where they
can be rephosphorylated by activated receptors. Although RSTKs operate mainly through the
Smad pathway, they can also stimulate other intracellular signaling proteins such as MAPKs
and PI 3-kinase. Conversely, signaling proteins in other pathways can phosphorylate Smads and
thereby influence signaling along the Smad pathway.
Activated TGFβ receptors and their bound ligand are endocytosed by two distinct routes,
one leading to further activation and the other leading to inactivation. The activation route
depends on clathrin-coated vesicles and leads to early endosomes, where most of the Smad
activation occurs. An anchoring protein called SARA (for Smad Anchor for Receptor
Activation) has an important role in this pathway; it is concentrated in early endosomes and
binds to both activated TGFβ receptors and Smads, increasing the efficiency of receptor-

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mediated Smad phosphorylation. The inactivation route depends on caveolae and leads to
receptor ubiquitylation and degradation in proteasomes (Figure 18).
As in other signaling systems, negative feedback regulates the Smad pathway. Among the
target genes activated by Smad complexes are those that encode inhibitory Smads, either
Smad6 or Smad7. Smad7 (and possibly Smad6) binds to the cytosolic tail of the activated
receptor and inhibits its signaling ability in at least three ways. For example, (1) it competes with
R-Smads for binding sites on the receptor, decreasing R-Smad phosphorylation; (2) it recruits a
ubiquitin ligase called Smurf, which ubiquitylates the receptor, leading to receptor
internalization and degradation; and (3) it recruits a protein phosphatase that dephosphorylates
and inactivates the receptor (Figure 18).
3- Receptor-Like Tyrosine Phosphatases
In all signaling pathways that use tyrosine phosphorylation, the tyrosine phosphorylations are
reversed by protein tyrosine phosphatases. These phosphatases are as important in the signaling
process as the protein tyrosine kinases that add the phosphates. Most tyrosine phosphatases
display exquisite specificity for their substrates, removing phosphate groups from only selected
phosphotyrosines on a subset of proteins. These phosphatases ensure that phosphorylation
processes are short-lived and that the level of phosphorylation in resting cells is very low. They
do not, however, simply continuously reverse the effects of protein tyrosine kinases; they are
often regulated to act only at the appropriate time and place.
Like tyrosine kinases, the tyrosine phosphatases occur in both cytoplasmic and
transmembrane forms. Two cytoplasmic tyrosine phosphatases in vertebrates have SH2 domains
and are therefore called SHP-1 and SHP-2. Both SHP-1 and SHP-2 also help terminate
responses mediated by some receptor tyrosine kinases (Figure 15-64). SHP-1 helps to terminate
some cytokine responses in blood cells by dephosphorylating activated JAKs: mutant
erythropoietin receptors that cannot recruit SHP-1, for example, activate JAK2 for much longer
than normal. Moreover, SHP-1-deficient mice have abnormalities in almost all blood cell
lineages, emphasizing the importance of SHP-1 in blood cell development.
There are a large number of transmembrane protein tyrosine phosphatases, but the
functions of most of them are unknown. At least some are thought to function as receptors; as
this has not been directly demonstrated, however, they are referred to as receptor-like tyrosine
phosphatases. They all have a single transmembrane segment and usually possess two tyrosine

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phosphatase domains on the cytosolic side of the plasma membrane. An important example is the
CD45 protein, which is found on the surface of all white blood cells and has an essential role in
the activation of both T and B lymphocytes by foreign antigens. The ligand that is presumed to
bind to the extracellular domain of the CD45 protein has not been identified.
4- Receptor Guanylyl Cyclases (The Natriuretic Peptide Family Receptors)
Receptor guanylyl cyclases are single-pass transmembrane proteins with an extracellular binding
site for a signal molecule and an intracellular guanylyl cyclase catalytic domain. These receptors
are unique because they synthesize their own second messengers upon agonist binding. The
binding of the signal molecule activates the cyclase domain to produce cyclic GMP, which in
turn binds to and activates a cyclic GMP-dependent protein kinase (PKG), which
phosphorylates specific proteins on serine or threonine. Thus, receptor guanylyl cyclases use
cyclic GMP as an intracellular mediator in the same way that some G-protein-linked receptors
use cyclic AMP, except that the linkage between ligand binding and cyclase activity is a direct
one. Among the signal molecules that use receptor guanylyl cyclase receptors are the natriuretic
peptides (NPs), a family of structurally related secreted signal peptides that regulate salt and
water balance and dilate blood vessels. Muscle cells in the atrium of the heart secrete atrial
natriuretic peptide (ANP) when blood pressure rises. The ANP stimulates the kidneys to
secrete Na+ and water and induces the smooth muscle cells in blood vessels walls to relax. Both
of these effects tend to lower the blood pressure. When gene targeting is used to inactivate the
ANP receptor guanylyl cyclase in mice, the mice have chronically elevated blood pressure,
resulting in progressive heart enlargement. An increasing number of receptor guanylyl cyclases
are being discovered, but in most cases they are orphan receptors, where the ligand that normally
activates them is unknown.
In addition to this particulate guanylate cyclase (the membrane form of the enzyme), an
intracellular soluble form exists. It is a heterodimer consisting of α- and β-subunits, both of
which are necessary for enzyme activity, and is expressed in most tissues, though not
uniformly. It is activated by intermediate substances derived from the biosynthesis of
eicosanoids (prostaglandins and leukotrienes) and by NO. Guanylate cyclases and cGMP-
mediated signaling cascades play a central role in the regulation of diverse pathophysiologic
processes, including vascular smooth muscle motility, intestinal fluid and electrolyte
homeostasis, and retinal phototransduction.