Cell Signalling ©Debajyoti Chakraborty

Cell signaling is part of a complex system of communication that governs basic cellular activities and
coordinates cell actions. All living organisms are constantly receiving and interpreting signals from their
environment in the form of light, heat, odours, touch or sound. These signals are important to keep cells alive
and functioning as well as to stimulate important events such as cell division and differentiation, development,
tissue repair, immunity as well as normal tissue homeostasis. Errors in cellular information processing are
responsible for diseases such as cancer, autoimmunity, and diabetes. Cell signaling pathway involves
three main steps:

1. Reception:
Cells communicate by means of extracellular signaling molecules called ligands that are produced and
released by signaling cells, which are detected by a specific protein molecules called receptors on the
surface or inside the recipient cell.
2. Signal Transduction:
The signal is converted in a single step or multiple steps into different molecules called (relay
molecules) that can bring specific cellular response.
3. Response:
The signal finally triggers specific cellular responses via changing the metabolism of the cell or result
in a change in gene expression (transcription) within the nucleus of the cell or both.

Categories of Cell Signaling:

Depending on the distance that the signaling molecule has to travel, there are five categories of chemical
signaling found in multicellular organisms:
A. Paracrine signaling:
Signals act locally between cells that are close together by diffusion signals through the
extracellular matrix. Paracrine signaling usually elicit quick responses that last for a short time
because ligand molecules are quickly degraded by enzymes or removed by neighboring cells to
reestablish their concentration gradient for allowing them to quickly diffuse through the intracellular
space if released again. One example of paracrine signaling is the transfer of signals across synapses
between nerve cells. The neurotransmitters are transported across the very small distances between
nerve cells, which allows the signal to travel quickly; this enables an immediate response.

1
Cell Signalling ©Debajyoti Chakraborty

B. Endocrine signaling
The signaling molecules (ligands) are called hormones
that travel a large distances from signaling cells
(endocrine gland) via the bloodstream until reach their
target cells found in other body regions some distance
away. Endocrine signaling usually produce a slower
response, but have a longer-lasting effect because during
transport, hormones get diluted and are present in low
concentrations when they act on their target cells. This is
different from paracrine signaling in which local
concentrations of ligands can be very high.

C. Autocrine signaling
Both ligands and their specific receptors are produced by the
same cells. This type of signaling often occurs during the
early development of an organism to ensure that cells
develop into the correct tissues and take on the proper
function, also regulates pain sensation and inflammatory
responses. Further, if a cell is infected with a virus, the cell
can signal itself to undergo programmed cell death and
eliminate virus.
D. Juxtacrine signaling
It is a type of cell-cell or cell-extracellular matrix signaling in multicellular organisms that requires
close contact, so it is also known as contact-dependent signaling. Juxtacrine signaling include some
growth factors, cytokine and chemokine cellular signals, playing an important role in the immune
response. Also direct signaling can occur by transferring signaling molecules across gap junctions
between neighbouring cells.

2
 Cell Signalling ©Debajyoti Chakraborty

E. Intracrine signaling
If signaling molecules function within the intracellular space, they are termed intracrines that are
associated with a wide variety of peptides/proteins including hormones, growth factors, cytokines,
enzymes, and DNA-binding proteins among others. Intracrine factors are involved in the related
phenomena of senescence, apoptosis, and regulation of stem cell proliferation and differentiation.
There are several types of intracrines; some of them can function in their cells of synthesis without
secretion or after secretion and reuptake. While others can function after release in micro-vesicles and
internalization by target cells following fusion of the micro- vesicles with the membranes of those cells
or can transit between cells via nanotubes and function in the recipient cells.

Types of Ligands
Signaling molecules (also known as first messenger) can belong to several chemical classes:
1. Hormones:
They are signaling molecules of the endocrine system and can be grouped into two categories:
a) Non-steroid hormones are water soluble (hydrophilic) hormones bind to receptors on the plasma
membrane which are either proteins (e.g. insulin, glucagon), or peptides (e.g. vasopressin-ADH), or
amino acid derivatives (e.g. histamine, epinephrine). They do not enter the cell but bind to plasma
membrane receptors, generating a chemical signal known as second messenger inside the target cell
that activate other intracellular chemicals to produce the target cell response.

b) Steroid hormones are small lipid soluble (lipophilic) hormones can diffuse through the cell
membrane to reach cytosolic or nuclear receptors, such as progesterone, estrogen, testosterone, and
thyroid hormones, which are generally regulate transcription.

3
Cell Signalling ©Debajyoti Chakraborty

2. Neurotransmitters:
They are signaling molecules of the nervous system, some can function as both a hormone and a
neurotransmitter, such as epinephrine and norepinephrine can function as hormones when released
from the adrenal gland into the blood stream, and also be produced by neurons to function as a
neurotransmitter.

3. Cytokines:
They are signaling molecules of the immune system that function nearly via all categories of cell
signaling system, but have a strong presence in the circulation with systemic effect such as altering iron
metabolism or body temperature.

4. Growth Factors:
They act as messengers in addition to nutrients because cells often need growth factors to grow, thus
these signaling molecules are very important in embryonic & adult development of different tissues
such as muscle, cartilage, bone and blood cells. There are several types of growth factors including:
Platelet-derived growth factor (PDGF), insulin-like growth factor 1 (IGF-1), fibroblast growth factor
(FGF), epidermal growth factor (EGF) and nerve growth factor (NGF).

4
 Cell Signalling ©Debajyoti Chakraborty

5. Gases:
The simple gas nitric oxide (NO) is a major paracrine signaling molecule in the nervous, immune, and
circulatory systems. Like the steroid hormones, NO is able to diffuse directly across the plasma
membrane of its target cells. The molecular basis of NO action, however, is distinct from that of steroid
action; rather than binding to a receptor that regulates transcription, NO alters the activity of
intracellular target enzymes.

Another simple gas, carbon monoxide (CO), also functions as a signaling molecule in the nervous
system. CO is closely related to NO and appears to act similarly as a
neurotransmitter and mediator of blood vessel dilation. The synthesis of CO in brain cells, like that of NO,
is stimulated by neurotransmitters.

Types of Receptors
A ligand binds its receptor through weak non-covalent bonds by fitting into a specific binding site or "pocket".
Low concentrations of a ligand will result in binding of most of the receptors, because their affinity is high.
However, low receptor affinity occurs when a high concentration of the ligand is required for most receptors
to be occupied. With prolonged exposure to a ligand (and occupation of the receptor) cells often become
desensitized and may lead to tolerance. Desensitization of the cell to a ligand, depends upon receptor down-
regulation by either Removal of the receptor from the cell surface (receptor-mediated endocytosis), or by
receptor inactivation via lowering their affinity to ligand or blocking the subsequent signaling cascade.

5
Cell Signalling ©Debajyoti Chakraborty

According to their location, receptors are classified into two groups:

A. Intracellular Receptors
They are protein found in the cytosol or nucleus of target cells. Small or hydrophobic chemical
messengers such as steroid and thyroid hormones can readily cross the membrane and activate
receptors. An activated hormone-receptor complex can act as a transcription factor, turning on specific
genes.

B. Cell surface receptors

These receptors bind signal molecules, including neurotransmitters and signal proteins, on their
surface. This binding of a ligand is then converted into intracellular signals to alter the behavior of the
target cell. There are three main classes of cell surface receptors:
1) Ion-channel-linked receptors
Ion-channel-linked receptors also called (ligand-gated channels or fast ionotropic receptors) are
cell membrane bound receptors act through synaptic signaling on electrically excitable cells and
convert chemical signals (ligand) to electrical ones that is essential in neuronal activities. The
molecules that pass through these receptors are often ions, such as (Na+) or (K+). The ion channels
are opened only for a short time, after which the ligand dissociates from the receptor and the
receptor is available once again for a new ligand to bind.

2) G protein- coupled receptor (GPCR)

Many different mammalian cell-surface receptors are coupled to a Heterotrimeric G protein,

covalently linked to a lipid in the membrane. All G protein-coupled receptors (GPCRs) are
ligand specific and differ in their extracellular surface and involved in a range of signaling
pathways, including detection of light, odor, certain hormones and neurotransmitters.

6
Cell Signalling ©Debajyoti Chakraborty

3) Enzyme-linked receptor:
They are integral membrane proteins that have two important domains, an extra- cellular
ligand binding domain and an intracellular domain, which has a Catalytic function. The
signaling molecule binds to the receptor outside of the cell and causes a conformational change
on the Catalytic function located on the receptor inside of the cell. These receptors fall into two
subgroups: a) Receptor Tyrosine Kinases (RTKs) with high-affinity for many polypeptide
growth factors, cytokines, and hormones, that act as key regulators of normal cellular
processes and also play a critical role in the development and progression of many types of
cancer; b) Receptor Serine/Threonine Kinases (RS/TK) that play a role in the regulation of cell
proliferation, programmed cell death (apoptosis), cell differentiation, and embryonic
development.

7
 Cell Signalling ©Debajyoti Chakraborty

Signal Transduction
Ligand-receptor complex activates series of events known as signal transduction that relays the signal to the
interior of the cell. Many intracellular signaling proteins behave as molecular switches and fall into two main
classes:

1. G-proteins:
The main feature of G proteins is that they are regulated to either turn (On or off) based on their binding to
guanosine diphosphate (GDP) or guanosine triphosphate (GTP). Firstly, inactive G protein is normally
bound to GDP and waiting for the reception of a signal on the surface of the cell, which will cause the G
protein to exchange GDP for GTP and become active. Once this occurs the G protein will begin interacting
with other signaling components to transmit a signal. Both features of a G protein (i.e. their ability to
exchange GDP for GTP and also degrade GTP to GDP) require the use of numerous other signaling
proteins. These additional signaling proteins help the cell keep tight control over when a G protein switches
on or off. These associated proteins include GTPases-activating proteins (GAPs), guanine exchange factors
(GEFs) and guanine-dissociation inhibitors (GDIs).

All G proteins fall into two major groups:

A. Heterotrimeric G proteins
These proteins are composed of three subunits; an alpha (α) subunit that is the activator portion of the G-
protein, and beta (β) and gamma (γ) subunits that are attached to the alpha component and also to the
inside of the cell membrane adjacent to the receptor protein. When no stimulus is present, α subunit is
bound to GDP and complexes to the β/γ subunits. A signal binding to the receptor promotes the
dissociation of bound GDP from α subunit of the G protein and stimulates the association of GTP and the
alpha subunit of the G-protein separates from the beta and gamma portions and may either be directly
coupled to an ion channel or may trigger the activation or inhibition through a secondary messenger
system. Both α and βγ subunits may then interact with other proteins to transmit the signal. The ending of
the response occurs when GTP is hydrolyzed the Gα subunit rebinds to Gβγ, returning the complex to its
inactivated state. In a cell, this all occurs within a few seconds.

8
Cell Signalling ©Debajyoti Chakraborty

Some times this system become unregulated as in certain diseases like Cholera which is caused by a
bacterium that multiples in the intestine, where it produces a protein called cholera toxin. This protein
enters the cells lining the intestine and modifies α subunit of a G protein so that it is no longer able to
convert GTP back to GDP. And thus once activated, this G protein cannot be turn off and continues to
transmit its message to the cellular machinery. In this case, adenylate cyclase is dramatically increased and
causes the intestinal cells to continually expel sodium and water into the gut, resulting in diarrhea and
dehydration. This can result in death if steps are not taken to replace water and salts.

B. Monomeric G proteins:
Monomeric G-protein comprised of a single unit and binds GTP and at the same time has GTPase activity
(often called GTPases) to distinguish them from heterotrimeric G proteins. They are resemble α subunit
of heterotrimeric G proteins and function as a molecular switch in a similar way. Monomeric G-proteins
play important role in regulation of growth, morphogenesis, cell motility, and cytokinesis. Normally they
are bound to GDP and inactive, upon the reception of a signal, they exchange GDP for GTP and become
active. Once in the active state, the GTPase will initiate a cascade in which a series of proteins will
phosphorylate each other to transmit the signal. They differ in their mode of activation by not being
directly associated with the receptor. Instead, GTPases are several steps away from the receptor (often
termed an enzyme-linked

9
 Cell Signalling ©Debajyoti Chakraborty

receptor) that ultimately cause activation a variety of adaptor proteins. These proteins include 5 families;
Ras, Rho, Arf, Rab, and Ran proteins which are anchored to lipid membrane. One of the most famous
small monomeric G proteins is Ras, which was first discovered as a cellular copy of a viral gene that
causes cancer in mammals (the name 'Ras' is an abbreviation of Rat sarcoma). It was later discovered that
Ras genes are proto-oncogenes, thus mutation in these genes induce pathologic proliferation and anti-
apoptosis. About 30% of all human tumors involve cells expressing mutated Ras oncogene.

2. Protein Kinases
Protein kinases are enzymes that modify their target proteins by transferring phosphate groups from ATP
(phosphorylation) resulting in a signaling cascade. A cascade can be stopped by removal of phosphate
groups by protein phosphatases, which can remove phosphate groups from kinase substrates. As many as
30% of all human proteins are modified by phosphorylation, making kinases extremely important in
cellular regulation.

10
 Cell Signalling ©Debajyoti Chakraborty

Protein kinases can be broadly divided into two groups based on the amino acids to which they add
phosphate groups: serine/threonine (Ser / Thr) kinases that involved in regulation of metabolic and
cytoskeletal activity; and tyrosine (Tyr) kinases that act primarily in downstream signaling from growth
factors. When a signal (growth factor) arrives, binds to two receptors, causes the two tyrosine kinase
domains to come into contact with one another. Each kinase phosphorylates each other on multiple tyrosine
sites. Several other proteins may be involved in the cascade, ultimately activating one or more transcription
factors. The activated transcription factors enter the nucleus where they stimulate the expression of the
genes that are under the control of that factor. This is an example of the RAS pathway, which results in cell
division. Abnormal activity of protein kinases is a frequent cause of disease, particularly cancer, therefore,
protein kinases have emerged as one of the most frequently targeted families of proteins in drug discovery.
The development of small-molecule inhibitors that have the potency and selectivity necessary to be
effective cancer drugs has gained notable successes over the past decades.

Second Messengers
Second messengers are molecules that relay signals from receptors on the cell surface to target molecules
inside the cell (cytoplasm or nucleus). Because the signal from an extracellular molecule (first messenger, e.g.,
a hormone, neurotransmitter or drug) does not cross the cell membrane to enter the cytoplasm, they bound to
the cell's surface receptor which in turn activate another molecules called effectors which bind to specific
receptors to initiate the pathways for the production of 'second messengers to continue the control function
inside the cell. The binding of this effector to receptor is often- mediated by a protein, whose activity depends
on GTP/GDP binding or kinases proteins. Therefore, secondary messengers are a component of signal
transduction cascades that relay and greatly amplify the strength of the signal via binding and activating
protein kinases, ion channels, and other proteins, thus continuing the signaling cascade.

11
 Cell Signalling ©Debajyoti Chakraborty

There are three basic types of 2nd messengers which are chemically diverse:
1. Hydrophobic molecules: They are water-insoluble molecules, like diacylglycerol (DAG),
and phosphatidylinositol (PI), which are membrane-associated and diffuse from the plasma membrane
into the inter membrane space where they can reach and regulate membrane- associated effector
proteins. They reach different targets in the cell depending on whether they are more soluble in the lipid
bilayer or in water.
2. Hydrophilic molecules: They are water-soluble molecules, like cyclic nucleotides (cAMP & cGMP),
inositol triphosphate (IP3), and calcium ions that are located within the cytosol and act globally because
they diffuse rapidly throughout the cytoplasm.
3. Gases: They include nitric oxide (NO), carbon monoxide (CO) and hydrogen sulphide (H2S) which can
diffuse both through cytosol and across cellular membranes.

Cyclic AMP (cAMP):

Cyclic adenosine monophosphate (cAMP) was the first second messenger to be identified and plays
fundamental roles in cellular responses to many hormones and neurotransmitters. It is derived from adenosine
triphosphate (ATP) and used for intracellular signal transduction in many different organisms. Ligands such as
hormones and neurotransmitters require at least four components to regulate the intracellular cAMP
concentration: (1) GPCR, (2) heterotrimeric G protein, (3) adenylyl cyclase (AC), and (4) phosphodiesterase
(PDE). The intracellular levels of cAMP are regulated primarily by the balance between the activities of two
enzymes; (AC) and (PDE). The cAMP is synthesized from ATP by adenylyl cyclase which is large
transmembrane proteins. However, cAMP decomposition into AMP is catalyzed by the enzyme
phosphodiesterase.

12
 Cell Signalling ©Debajyoti Chakraborty

Effectors of cAMP
The cAMP has been shown to be a universal intracellular messenger that mediates a wide variety of biological
responses in almost all tissues in mammals. An elevation of cAMP activates several effectors, the best known
is the protein kinase A (PKA). Not all protein kinases respond to cAMP, for example protein kinase C is not
cAMP-dependent. In eukaryotes, cyclic AMP works by activating PKA which is normally inactive as a
tetrameric holoenzyme, consisting of two catalytic and two regulatory units (C2R2), with the regulatory units
blocking the catalytic centers of the catalytic units. Cyclic AMP binds to specific locations on the regulatory
units and causes dissociation between the regulatory and catalytic subunits, thus enabling those catalytic units
to phosphorylate substrate proteins which may either directly act on the ion channels, or activated / inhibited
enzymes, or bind to promoter regions of DNA causing increased expression of specific genes.

13
 Cell Signalling ©Debajyoti Chakraborty

Example (Glucose Mobilization):

Glucose is stored in animal cells as an insoluble polymer (glycogen). Liver adenylyl cyclase responds more
strongly to glucagon, and muscle adenylyl cyclase responds more strongly to adrenaline. The enzyme
phosphorylase catalyzes glycogen breakdown, while glycogen synthase catalyzes polymerization. Regulation
is achieved by several hormones, such as glucagon (from the pancreas) and epinephrine (from the adrenal
medulla). Binding of epinephrine or glucagon to their receptors activates adenylyl cyclase. The cAMP binds to
the regulatory subunit of protein kinase A (PKA) causing the release of the catalytic subunit which acts on
several targets leading to conversion of glycogen into glucose and release it into blood circulation.

PKA can do that by acting on three different targets:

1) PKA phosphorylates and activates phosphorylase kinase, which activates phosphorylase to breakdown
glycogen into glucose-1-phosphate, then glucose-6-phosphate, and finally to glucose molecules that enters
blood circulation.

14
Cell Signalling ©Debajyoti Chakraborty

2) PKA phosphorylates and inhibits glycogen synthase.

3) PKA also translocates to the nucleus where it phosphorylates the transcription factor CREB
(cAMP response element-binding protein), that binds to certain DNA sequences called cAMP
response elements (CRE), thereby increasing or decreasing the transcription of the downstream genes
involved in gluconeogenesis. CREB was first described in 1987 as a cAMP- responsive transcription
factor regulating the somatostatin gene.

15