CELL

SIGNALING

Galih Samodra
260120150515

Program Pendidikan Magister Farmasi

Universitas Padjadjaran
2016
Overview

G Protein-Coupled Receptor (GPCR) : Receptor Tyrosine

Kinase
Receptor Guanylyl Cyclase : gated ion chanel
Adhesion receptor, nuclear receptor
G Protein Coupled Receptors (GPCR)
A G-Protein-Coupled Receptor
Or G Protein-linked Receptor
7 transmembrane domains
The disassembly of G-Protein
upon stimulation
Spontaneous deactivation is
very fast, in minutes.
However, with the help of
RGS (regulator of G protein
signaling, a GAP for unit),
signals can be shut off even
faster
The Activation
cycle of G-
Protein
Enzyme-Linked Cell Surface
Receptors

*Receptor Tyrosine Kinase

*Tyrosine kinase associated receptors
*Receptor-like tyrosine phosphatase
*Receptor serine/threonine kinase
Receptor guanylyl cyclase
Histidine like associated receptor
Receptor Tyrosine Kinases (RTKs)
Seven subfamilies of receptor tyrosine kinases
Three ways in which signaling proteins can cross-link
receptor chains
1. dimer, 2. monomer but brought together by
proteoglycan, 3. cluster on membrane
The importance of receptor
oligomerization
The docking of signaling molecules at RTK
The binding of SH2-containing intracellular
signaling proteins to an activated PDGF receptor
The structural view of SH2 domain
RTK Signaling: Ras Pathway
The regulation of Ras activity, a famous
downstream molecule of RTK responsible for
cancer development
The activation of Ras by RTK signaling
The MAP-kinase regulated by Ras
RTK Signaling: PI3K Pathway
The inositol phospholipids generated by PI3K
The recruitment of
signaling molecules with
PH domains to the plasma
membrane during B cell
activation
One PI3K pathway

PH domain: pleckstrin
homology domain
Another PI3K pathway to regulate cell survival
 Another PI3K pathway to regulate cell migration

PI3K->PIP3->GEF->Rac->Wave->Arp2/3->Actin polymerization
Intracellular Signaling Pathways activated by RTKs and GPCRs
 Tyrosine kinase associated receptors

Integrins: cell-extracellular matrix adhesion

Binding to Src and FAK
 Receptorlike tyrosine phosphatases
Intracellular protein and receptor
Receptor Serine/threonine kinase
Transforming growth factor (TGF-b) and
Smad signaling pathway
Protein kinase Summary
Enzyme-linked Receptor Signaling
Summary

1.receptor types
2. RTK and its signaling: Ras and PI3K
3.Tyrosine kinase associated receptors
and Receptor-like tyrosine phosphatase
4. Receptor serine/threonine kinase, TGF-
and Smad
 Atrial natriuretic peptide (ANP), atrial natriuretic factor
(ANF), atrial natriuretic hormone (ANH), Cardionatrine,
Cardiodilatine (CDD) or atriopeptin, is a powerful
vasodilator, and a protein (polypeptide) hormone secreted
by heart muscle cells.[1][2][3] It is involved in the
homeostatic control of body water, sodium, potassium and
fat (adipose tissue). It is released by muscle cells in the
upper chambers (atria) of the heart (atrial myocytes) in
response to high blood volume. ANP acts to reduce the
water, sodium and adipose loads on the circulatory system,
thereby reducing blood pressure.[1] ANP has exactly the
opposite function of the aldosterone secreted by the zona
glomerulosa in regard to its effect on sodium in the kidney
that is, aldosterone stimulates sodium retention and ANP
generates sodium loss
 Nitric oxide has been shown to be important in the
following cellular activities:

Help memory and behavior by transmitting

information between nerve cells in the brain
Assist the immune system at fighting off bacteria and
defending against tumors
Regulate blood pressure by dilating arteries
Reduce inflammation
Improve sleep quality
Increase your recognition of sense (i.e. smell)
Increase endurance and strength
Assist in gastric motility
 nitroglycerin acts by releasing nitric oxide which relaxes
narrowed blood vessels, increasing oxygen and blood
flow.
Three different guanylyl cyclase cell receptors are known, but others
will likely be discovered within the next few years. The general function
ofthese receptorsappeartorelatetotheregulation of fluid volume or fluid
movement. New receptors, or possibly the currently known receptors,
therefore, may be discovered in areas of the body where fluid volume
regulation is important. Such fluids whose volume or composition might
be regulated by guanylyl cyclase receptors include synovial fluid,
uterine/oviductal luminal fluid, follicular fluid, aqueous humor, cerebral
spinal fluid, seminferous tubule luminal fluid, epididymal luminal fluid,
seminal plasma, and airway luminal fluid. The function of the
heterodimeric forms of guanylyl cyclase appear to relate to a primary
regulation ofnitric oxide (or similar molecules) concentrations, which
are in turn regulated by a Ca2"/calmodulin-sensitive nitric oxide
synthase.
Nicotinic receptor
 Nicotinic acetylcholine receptors, or nAChRs, are
neuron receptor proteins that respond to the
neurotransmitter acetylcholine. Nicotonic receptors also
respond to drugs, including the nicotinic receptor
agonist nicotine. They are found in the central nervous
system of humans, and also play two important roles in
the peripheral nervous system: (1) they transmit
outgoing signals from the presynaptic to the
postsynaptic cells within the sympathetic and
parasympathetic nervous system, and (2) they are the
receptors found on skeletal muscle that receive
acetylcholine released to signal for muscular
contraction.
 GABA (Gamma Amino Butiric Acid)Merupakan
neurotransmitter inhibitor, artinya akan menghalangi
penghantaran impuls di serabut saraf.GABA akan
membuka gerbang ion chlorine yang bermuatan
negative sehingga serabut saraf akan bermuatan sangat
negative. Dengan begitu impuls sulit untuk dihantarkan
melalui serabut saraf
ATP receptor
P2X7

ligand-gated ion channel

ATP

Essential Cell Biology, 2nd

Edition (Alberts et al.,
2004)
 Purinergic receptors, also known as purinoceptors, are
a family of plasma membrane molecules that are found
in almost all mammalian tissues. Within the field of
purinergic signalling, these receptors have been
implicated in learning and memory, locomotor and
feeding behavior, and sleep
Ion channel receptor (Ligand
gated ion channels
1. General structure
2. Structure of protein subunits (4-TM receptor subunits)
3. Detailed structure of ion channel
4. Gating
Receptor superfamilies
Ion Channel structure

General structure
Ion channel receptors

Gating
 Gating
Cell adhesion receptors and the
control of cell cycle
1. Cell adhesion impact the regulatory elements of cell
cycle .
2. Cell adhesion regulate the transfer of cell cycle
information .
3. Adhesion disorders result in cell cycle out of control .
1.1 cell adhesion can induce the expression of cyclin
D1 mRNA

Someone detect the levels of cyclin D1

Mrna in adhesion state and suspended
state,and find that the adhesion state is
three to five times higher than the
suspended state. late G1 cell cant express
in the suspension state but express positive
in the adhesion state.
 In order to study relation of cyclin D1 and Rb
phosphorylated from G1 to S phase, Biologists take
the retrovirus transfected NIH - 3T3 cells with cyclin
D1 gene to express cyclin D1 sustained. Even leaving
the transfection of NIH - 3T3 cells under suspension
state, forced expressive cyclin D1 can keep
phosphorylation of Rb and overcome the G1 to S
phase retardarce caused by the loss of adhesion. the
expression of adhesion dependence cyclin D1 can
induce continued activity of extracellar signal-
regulated kinase, but growth factor only induce ERK
activity moment.
1.2 cell adhesion can
activate the activity of
cyclin E-CDK2
Under the state of Suspended, the activity of
cyclin E-CDK2 kinase is Significantly lower
than the state of adhesion. The effects can
not be interpreted by cyclin E or complex
changes because the expressions of cyclin E
and CDK2 is independent adhesion, but
combination with CKI: P21 and P27 are
affected by adhesion. Western blot shows
that under the state of suspended ,P21, P27
are three times and two times higher than
adhesion. Therefore, the activity of cyclin-
dependent adhesion E-CDK2 is regulated
through the realization of P21 and P27 .
 1.3 the expression of
cyclin mRNA depend cell
adhesion
Under the state of suspended, NRK, NIH - 3T3 and embryonic fibroblast cells cant
express cyclin A, so the cyclin A of S phase also need cell adhesion to express. In normal
cells, the expression of cyclin A begins after the expression of cyclin D, E and CDK,
mRNA and protein of cyclin A begin to appear in late G1. Cyclin A gene promoter
contains the regulation of cell cycle (E2F binding sites) and its expression phosphorylate
Rb, release E2F, and induct E2F gene transcription .
 2. cell adhesion regulate the
transfer of cell cycle
information
When the cell adhesion links with ECM, integrin clustering gather to join the ECM with
cell cytoskeletal proteins , phosphorylate FAK, and produce a series of regulatory
protein about SH2 region, e.g. Paxillin, Crk, Grb-2 etc. Grb-2 and Crk can activate the
pathway of mitogen-activated protein kinase, and then transcript and translate cyclin
D1 mRNA .
3. adhesion disorders result
in cell cycle out of control
Epidermal cell needs to adhere to ECM to survive. Once break away the ECM,
it will apoptosis. Frisch names this apoptosis Anoikis (homeless). The cell
which phosphorylated sustained by FAK is the key of adhesion to inhibit
apoptosis. Tumor-derived growth of cells perform Non-growth factor and
adhesion dependence.
The protein which Cancer gene encoded is one of normal cell growth control
pathways, but its over-expression or mutation lead the pathway irreversible
activation. These proteins which tumor gene encoded activate the pathway of
Integrin-mediated and transform the cell to adhesion-independent, such as Ibc,
dbl. The down regulation of Drs (one of tumor suppressor genes) connect with
Tumor-derived independent adhesion, and result in the abnormal expression of
cyclinA.
 In the field of molecular biology, nuclear receptors are a
class of proteins found within cells that are responsible for
sensing steroid and thyroid hormones and certain other
molecules. In response, these receptors work with other
proteins to regulate the expression of specific genes,
thereby controlling the development, homeostasis, and
metabolism of the organism.
Nuclear receptors have the ability to directly bind to DNA
and regulate the expression of adjacent genes, hence these
receptors are classified as transcription factors.[2][3] The
regulation of gene expression by nuclear receptors
generally only happens when a ligand a molecule that
affects the receptor's behavior is present. More
specifically, ligand binding to a nuclear receptor results in a
conformational change in the receptor, which, in turn,
activates the receptor, resulting in up- or down-regulation
of gene expression.
Nuclear Receptor
 Most nuclear receptors have molecular masses between 50,000 and
100,000 daltons.
Nuclear receptors are modular in structure and contain the
following domains:[16][17]
(A-B) N-terminal regulatory domain: Contains the activation
function 1 (AF-1) whose action is independent of the presence of
ligand.[18] The transcriptional activation of AF-1 is normally very
weak, but it does synergize with AF-2 in the E-domain (see below)
to produce a more robust upregulation of gene expression. TheA-B
domain is highly variable in sequence between various nuclear
receptors.
 (C)DNA-binding domain (DBD): Highly conserved domain containing two
zinc fingers that binds to specific sequences of DNA called hormone
response elements (HRE).
(D)Hinge region: Thought to be a flexible domain that connects the DBD
with the LBD. Influences intracellular trafficking and subcellular
distribution.
(E)Ligand binding domain (LBD): Moderately conserved in sequence and
highly conserved in structure between the various nuclear receptors.
The structure of the LBD is referred to as an alpha helical sandwich fold
in which three anti parallel alpha helices (the "sandwich filling") are
flanked by two alpha helices on one side and three on the other (the
"bread"). The ligand binding cavity is within the interior of the LBD and
just below three anti parallel alpha helical sandwich "filling". Along with
the DBD, the LBD contributes to the dimerization interface of the
receptor and in addition, binds coactivator and corepressor proteins.
The LBD also contains the activation function 2 (AF-2) whose action is
dependent on the presence of bound ligand.[18]
(F) C-terminal domain: Highly variable in sequence between various
nuclear receptors.
Mechanism of action

Nuclear receptors are multifunctional proteins that

transduce signals of their cognate ligands. Nuclear
receptors (NRs) may be classified into two broad classes
according to their mechanism of action and subcellular
distribution in the absence of ligand.
Small lipophilic substances such as natural hormones
diffuse through the cell membrane and bind to nuclear
receptors located in the cytosol (type I NR) or nucleus
(type II NR) of the cell. Binding causes a conformational
change in the receptor which, depending on the class of
receptor, triggers a cascade of downstream events that
direct the NRs to DNA transcription regulation sites which
result in up or down-regulation of gene expression. In
addition, two additional classes, type III which are a variant
of type I, and type IV that bind DNA as monomers have also
been identified
 Heat shock proteins (HSP) are a family of proteins that are produced by cells
in response to exposure to stressful conditions. They were first described in
relation to heat shock,[1] but are now known to also be expressed during other
stresses including exposure to cold,[2] UV light,[3] and during wound healing or
tissue remodeling.[4] Many members of this group perform chaperone function
by stabilizing new proteins to ensure correct folding or by helping to refold
proteins that were damaged by the cell stress.[5] This increase in expression is
transcriptionally regulated. The dramatic upregulation of the heat shock
proteins is a key part of the heat shock response and is induced primarily by
heat shock factor (HSF).[6] HSPs are found in virtually all living organisms, from
bacteria to humans.
Heat-shock proteins are named according to their molecular weight. For
example, Hsp60, Hsp70 and Hsp90 (the most widely-studied HSPs) refer to
families of heat shock proteins on the order of 60, 70, and 90 kilodaltons in
size, respectively.[7] The small 8-kilodalton protein ubiquitin, which marks
proteins for degradation, also has features of a heat shock protein
Type of Nuclear Receptor
 Type I

Type II

Type III Type IV

Type I
Ligand binding to type I nuclear receptors in the cytosol
results in the dissociation of heat shock proteins, homo-
dimerization, translocation (i.e., active transport) from
the cytoplasm into the cell nucleus, and binding to
specific sequences of DNA known as hormone response
elements (HREs). Type I nuclear receptors bind to HREs
consisting of two half-sites separated by a variable
length of DNA, and the second half-site has a sequence
inverted from the first (inverted repeat). Type I nuclear
receptors include members of subfamily 3, such as the
androgen receptor, estrogen receptors, glucocorticoid
receptor, and progesterone recep
Type II
Type II receptors, in contrast to type I, are retained in
the nucleus regardless of the ligand binding status and
in addition bind as hetero-dimers (usually with RXR) to
DNA. In the absence of ligand, type II nuclear receptors
are often complexed with corepressor proteins. Ligand
binding to the nuclear receptor causes dissociation of
corepressor and recruitment of coactivator proteins.
Additional proteins including RNA polymerase are then
recruited to the NR/DNA complex that transcribe DNA
into messenger RNA.
Type III
Type III nuclear receptors (principally NR subfamily 2)
are similar to type I receptors in that both classes bind
to DNA as homodimers. However, type III nuclear
receptors, in contrast to type I, bind to direct repeat
instead of inverted repeat HREs
Type IV
Type IV nuclear receptors bind either as monomers or
dimers, but only a single DNA binding domain of the
receptor binds to a single half site HRE. Examples of
type IV receptors are found in most of the NR
subfamilies
Coregulatory proteins

Coactivators
Binding of agonist ligands (see section below) to nuclear
receptors induces a conformation of the receptor that
preferentially binds coactivator proteins. These proteins often
have an intrinsic histone acetyltransferase (HAT) activity,
which weakens the association of histones to DNA, and
therefore promotes gene transcription.
Corepressors
Binding of antagonist ligands to nuclear receptors in contrast
induces a conformation of the receptor that preferentially
binds corepressor proteins. These proteins, in turn, recruit
histone deacetylases (HDACs), which strengthens the
association of histones to DNA, and therefore represses gene
transcription.
 Acetylation of histones unwinds DNA
loosely packed = transcription
= genes turned on
attachment of acetyl groups (COCH3) to histones
conformational change in histone proteins
transcription factors have easier access to genes
Agonism vs antagonism
Agonists
The activity of endogenous ligands (such as the hormones
estradiol and testosterone) when bound to their cognate
nuclear receptors is normally to upregulate gene
expression. This stimulation of gene expression by the
ligand is referred to as an agonist response. The agonistic
effects of endogenous hormones can also be mimicked by
certain synthetic ligands, for example, the glucocorticoid
receptor anti-inflammatory drug dexamethasone. Agonist
ligands work by inducing a conformation of the receptor
which favors coactivator binding
 Vitamin D refers to a group of fat-soluble secosteroids
responsible for enhancing intestinal absorption of
calcium, iron, magnesium, phosphate and zinc