ASO (2024) 4 Cell Signaling 1

• Cells grow, divide or differentiate in response to specific signals. Coordination and

regulation of the multiple functions of these cells in order to adapt the environmental
conditions require precise and highly sophisticated integrative systems.
• The nervous system uses electrical impulses to send messages through neurons while
endocrine glands use hormones to send messages to the target cells through the
bloodstream.
• Endocrine responses occur over several seconds at their fastest, to days or weeks at their
slowest.

Signal transduction is the process by which the message carried by signaling molecule is
accepted by specific cell associated molecule called receptor, then transmitted via
intracellular modulators and generate the appropriate responses of the cells.
Signaling Molecules
Signaling molecule can generate specific response in its target cell to adapt the changes in its
environment. They are:
 Hormones
 Growth factors & cytokines
 Cellular antigens
 Special senses (sight, hearing, taste, smell)
 Gases (NO, CO & free radicals)

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Hormone receptors
• Receptors are cell associated recognition molecules that able to recognize and bind
specific ligand leading to signal transduction.
• Receptors have very high specificity and affinity to its hormone.
• Chemical nature; protein or glycoprotein
• All receptors have at least two functional domains.
1. a recognition or ligand-binding or extracellular domain binds the hormones
2. a signal transduction or cytoplasmic or intracellular or catalytic domain that couples
hormone recognition to some intracellular function.
• Many of the receptors for chemical messengers are not static components of the cells.
Their numbers increase and decrease in response to various stimuli and their properties
change with changes in physiologic conditions. Therefore, they can be up or down
regulated. They are also target of certain therapeutic agents.

Types of receptors
(1) The intracellular receptors
• These are receptors for steroid /thyroid hormones (hydrophobic)
• They are found in the cytoplasm or in nucleus.
• They have several functional domains; ligand binding domain, DNA binding domain,
nuclear translocation domain, etc.
• The ligand receptor complex mediates intracellular responses by influencing gene
expression.
• Nuclear receptors are actually dormant transcription regulators. In the absence of signal,
these receptors are in the cytoplasm, complexed with other proteins (Heat Shock Protein;
HSP) and inactive. When a steroid hormone enters the cell and binds the receptor,
receptor undergoes conformational change that makes the release of HSP dissociating
from the HSP. The active hormone receptor complex translocate into the nucleus. In the
nucleus, they regulate the transcription of target genes by binding to their regulatory
sequences (Hormone Response Elements; HRE) and modulate their expression. Because
these responses involve gene expression, they are relatively slow.

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(2) The cell surface receptor

• Water-soluble signaling molecules including neurotransmitters, protein hormones,
peptide hormones and growth factors bind to the cell surface receptor.

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• These receptors act as signal transducers; they bind the ligand with high affinity and
convert this extracellular event into one or more intracellular signals that alter the
behavior of the target cell. They have three functional domains.
(i) Recognition domain that binds the hormone
(ii) Transmembrane domain that spans the plasma membrane
(iii) Catalytic domain or signal transduction domain

Three classes of cell surface receptors defined by the mechanism use are:
(1) Ion channel linked receptors
(2) G protein coupled receptors
(3) Enzyme linked receptors

(1) Ligand gated ion channels

• The simplest and fastest of signal pathways
• made up of multiple transmembrane proteins that create a pore, or channel
• Changes in the interior environment of the cell are thus brought about in microseconds
and in a single step. E.g., acetylcholine and serotonin as neurotransmitters
• When acetylcholine binds to ligand gated ion channels on post-synaptic membrane,
causing it to open and allowing Na+ to enter the cells. This promotes muscle contraction.

(2) G-protein coupled receptors (GPCR)

• act indirectly to regulate the activity of a separate plasma-membrane-bound target
protein, which can be an enzyme or an ion channel.
• belong to a large super family of homologous seven pass transmembrane proteins
(Serpentine receptors).
• The interaction between the receptor and the membrane bound target protein is mediated
by G-protein. E.g. Receptors for catecholamines

(3) Enzyme linked receptors

• either function directly as enzymes or in association with enzymes.
• Most are single pass transmembrane protein with the hormone binding site outside the
cell and their catalytic site inside. E.g. Receptors for insulin and growth factors.

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G-protein
• G-protein is a guanine nucleotide binding regulatory protein that can interact with a G-
protein linked receptor. They are membrane bound proteins associated with the cytosolic
side of the plasma membrane.
• There are many different G-proteins, all of which share a characteristic structure.
• 1. Monomeric G protein
• 2. Trimeric G proteins; Gs, Gi, Gq

Monomeric G protein (Ras)

• Hormone receptor complex activate G protein. Activated Ras triggers a phosphorylation
cascade of three protein kinases, that are members of a group called the MAP kinases
(Mitogen Activated Protein Kinases). The final kinase in this cascade phosphorylates
various target proteins, including enzymes and transcriptional activators.
• Ras involves in cell growth and differentiation and mutation of ras is found in some types
of cancer.
Trimeric G protein
• Heterotrimeric G-proteins composed of three subunits called alpha, beta and gamma
(αβγ).
• In the unstimulated state of the cell, the G-proteins are found in the trimeric form (αβγ
bound together) and the α subunit binds GDP i.e., inactive state.
• When a signal molecule binds to the G-protein linked receptor (GPCR) causes the
conformational change of a G-protein i.e., active state. This has two consequences:
o First, the alpha subunit of the G-protein ejects its GDP and binds a GTP.
o Second, the G-protein breaks up into the GTP-bound α part and the βγ part.

Cell signaling through G-proteins

• That depends on types of G-proteins interact and types of target proteins.
• When a signal (ligand) binds to a GPCR, which activates a G-protein that then binds and
opens the ion channel or increase or decrease the activity of the enzymes.
• Opening of the ion Channels results in the change in the distribution of ions across the
plasma membrane causes a change in the membrane potential.
• Two common enzymes whose activity is regulated by a G-protein are adenylate cyclase
and phospholipase C.

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Activation of adenylate cyclase by G protein

• If hormone receptor complex activates Gs protein, adenylate cyclase is activated and
large amounts of cAMP is produced. If hormone receptor complex activates Gi protein,
adenylate cyclase is inhibited and cAMP production is reduced.
• cAMP molecules bind and activate protein kinase A (PKA) that is composed of two
catalytic and two regulatory subunits that are bound tightly together. Upon binding of
cAMP to regulatory subunits, the catalytic subunits are released from the regulatory
subunits. Then activated PKA phosphorylates other enzymes or transcription factor;
cyclic AMP response element binding protein (CREBP). CREBP binds to CRE of
targeted gene and regulates its gene expression and thus protein synthesis.
• For example, the binding of glucagon to its receptor on the surface of liver cells, activates
the enzyme glycogen phosphorylase by phosphorylation, through the stimulation of G-
proteins, raising the intracellular cAMP level, subsequent activation of PKA. It leads to
the breakdown of glycogen, releasing glucose for use by other cells.
• Mechanisms to turn the pathway off are:
o The alpha subunit hydrolyzes GTP to GDP by its intrinsic GTPase activity.
o cAMP is broken down by phosphodiesterase. When cAMP levels drop, PKA
returns to its inactive state.

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Activation of Phospholipase C (PLC) by a Gq-protein

• When hormone receptor complex activates Gq, activated phospholipase C catalyzes the
production of second messengers; inositol trisphosphate (IP3) and diacylglycerol (DAG)
from phosphatidyl inositol 4,5-bisphosphate (PIP2).
• The IP3 and DAG work together to activate a protein kinase C. IP3 increases the
intracellular calcium concentration by releasing stored calcium from ER and increasing
the entry of extracellular calcium into the cell.
• The increase cytosolic calcium binds with calmodulin and activates a protein kinase
called protein kinase C, together with the DAG. Like PKA, Protein kinase C
phosphorylates a variety of proteins in the cell, altering their activity and thus changing
the state of the cell.
• To terminate the hormone action, IP3 and DAG are degraded, and calcium are
sequestrated into ER.
• Recycling is IP3 to PIP2 is inhibited by lithium that is used in manic-depressive disorder.

Second messengers
1. cyclic AMP
2. cyclic GMP
3. Inositol triphosphate (IP3)
4. Diacyl glycerol (DAG)
5. Calcium

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Receptor Tyrosine Kinase

• They mediate responses to signaling molecules; insulin and growth factors like epidermal
growth factor. It has extracellular ligand binding domain, intracellular catalytic domain
which has a tyrosine kinase activity and a transmembrane alpha helix.
• Insulin binds to alpha subunits of insulin receptor (α2β2, glycoprotein) activating beta
subunit tyrosine kinase activity. This causes autophosphorylation of beta subunits and
phosphorylation of insulin receptor substrate.
• EGF binds to the extracellular domains of EGFR, causing two receptor molecules to
dimerize. This causes activation of tyrosine kinase activity of these tails that then
phosphorylate each other on several tyrosine residues (autophosphorylation).
• The newly phosphorylated tyrosine amino acids serve as binding sites for signaling
proteins and triggers the assembly of an intracellular signaling complex.

Non-receptor tyrosine kinase or tyrosine kinase associated receptor

Intracellular domains of receptors of cytokines, Growth Hormone, prolactin,
Erythropoietin and antigen specific receptors on T and B lymphocytes do not have protein
kinase activity. But it is associated with an intracellular protein which has tyrosine kinase
activity.
Hormone receptor interaction promotes the binding and activation of cytoplasmic
protein tyrosine kinases which phosphorylates intracellular proteins containing SH2 domain.
Then phosphorylation mechanism is propagated. Most of the target proteins are DNA binding
proteins or transcription factors. Phosphorylated transcriptional factors become dimerized and
translocated to nucleus.

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Signaling Mechanism via receptor guanylyl cyclase

• Atrial natriuretic peptide and nitric oxide binds to cell surface receptor with guanylyl
cyclase activity and the soluble intracellular guanylyl cyclase receptively. Activated
guanylyl cyclase catalyzes GTP to 2nd messenger cGMP. cGMP activates protein kinase
G (PKG) which in turn phosphorylate the cellular proteins such as cytoskeletons. The
cellular responses are vasodilation or smooth muscle relaxation.
• ANP causes natriuresis, diuresis, vasodilation & inhibition of aldosterone secretion.
Nitroprusside, nitroglycerine, sodium nitrite & sodium azide are potent vasodilators by
relaxation of smooth muscles.

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Eukaryotic Cell Cycle

• The cell cycle is an ordered series of events involving cell growth and cell division that
produces two new genetically identical daughter cells via mitosis.
• The cell cycle has two major phases: interphase (G1, S, and G2) and the mitotic phase.
During interphase, the cell grows, and DNA is replicated. During the mitotic phase, the
replicated DNA and cytoplasmic contents are separated and the cell divides.
• The length of the cell cycle is highly variable even within the cells of an individual
organism. In humans, the frequency of cell turnover ranges from a few hours in early
embryonic development to an average of two to five days for epithelial cells, or to an
entire human lifetime spent without dividing in specialized cells such as cortical neurons
or cardiac muscle cells.
• There is also variation in the time that a cell spends in each phase of the cell cycle. The
timing of events in the cell cycle is controlled by mechanisms that are both internal and
external to the cell.

G1 Phase (First Gap)

• From a microscopic aspect, little change is visible and quite active at the biochemical
level. The cell is accumulating the building blocks of chromosomal DNA and the
associated proteins as well as accumulating sufficient energy reserves to complete the
task of replicating each chromosome in the nucleus.
S Phase (Synthesis of DNA)
• It occurs only once in the nucleus of the cell. DNA replication results in the formation of
identical pairs of DNA molecules; sister chromatids that are firmly attached to the
centromeric region. Thus, the amount of DNA doubles.
G2 Phase (Second Gap)
• In the G2 phase, the cell replenishes its energy stores and synthesizes proteins necessary
for chromosome manipulation. Some cell organelles are duplicated, and the cytoskeleton
is disassembled to provide resources for the mitotic phase.
• There may be additional cell growth during G2. DNA repair is also taking place. The final
preparations for the mitotic phase must be completed before the cell is able to enter the
first stage of mitosis.

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The Mitotic Phase

• It is a multistep process during which the duplicated chromosomes are aligned, separated,
and moved to opposite poles of the cell, and then the cell is divided into two new identical
daughter cells.
• 1st portion = Mitosis, is composed of five stages, which accomplish nuclear division.
• 2nd portion called cytokinesis, is the physical separation of the cytoplasmic components.
G0 Phase
• Cells in the G0 phase are not actively preparing to divide and are in a quiescent (inactive)
stage. Some cells enter G0 temporarily until an external signal trigger the onset of G1 e.g.,
liver cells. Other cells that never or rarely divide, such as mature cardiac muscle and
nerve cells, remain in G0 permanently

Mechanism of Cell cycle progress

• All eukaryotic cells have gene products (proteins) that govern the transition from one
phase of the cell cycle to another e.g., cyclin. Growth factor stimulation results in
induction of genes producing proteins e.g. cyclin D. Cyclins concentration increases and
decreases at specific times and are abruptly destroyed during mitosis. Four types of
cyclins, 5 types of cyclin-dependent kinase (CDK) regulate cell cycle transition points.
• Cyclin activates specific cyclin dependent protein kinases (CDK) that in turn
phosphorylates many proteins for progression through the cell cycle. When cell is
exposed to mitogen in G1 phase, cyclin D level becomes rise. By activating CDK4 and
CDK6, cyclin D induces the synthesis of
cyclin E. cyclin E and CDK2 make the cell
to pass G1 checkpoint and initiation of
DNA synthesis in early S phase. Cyclin A,
CDK1 and CDK2 bring the cell through S
phase and remain active through G2 phase.
Cyclin B and CDK1 derive the transition
from G2 to M phase.

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Regulation of cell cycle progress

• The important checks occur in 3 stages; at G1-S transition, during S phase or at G2 –M
boundary. Of this G1 phase check point is more complex and is under strict control.
• In quiescent (resting) cells, retinoblastoma (Rb) protein which is a cell cycle regulator
binds and inactivates transcription factor E2F. E2F is necessary for the transcription of
certain genes e.g. histones genes, DNA replication proteins. In growth factor activation,
cyclin D, CDK4 and CDK6 complex phosphorylate Rb causing release of E2F and cell
cycle progression occurs.
• Cyclin-CDK complex is rapidly inhibited by CDK inhibitor protein,
desensitization of the complex and degradation of cyclins.
• DNA damage increases the synthesis and activation of tumor suppressor
protein, p53. It is a transcription factor that induces the expression of G1-CDK inhibitor
protein: p21, allowing the cell to repair its DNA before proceeding into S phase. If the
damage is too extensive, the p53 level highly increases which initiate programmed cell
death called apoptosis.

Biomedical importance
• Mutation of retinoblastoma genes can cause Retinoblastoma. Certain tumor antigens
derived from viruses such as SV40, HSV, HPV may combine with Rb. Then Rb cannot
inhibit cell cycle, leading to continuous cell division and cancer.
• P53 involves in cell cycle regulation and apoptosis. It is mutated or lost in 50% of human
cancers.

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