Cell-cell interactions

Cell Communication

Different Receptor Types

Ion channel-linked receptors
GPCR signaling
Enzyme linked receptors
Regulated proteolysis
INTRACELLULAR SIGNAL TRANSDUCTION:
A Journey from the Plasma Membrane to the Nucleus
INTRACELLULAR SIGNALING:
Signal Transduction

Cell membranes, as well as the cell cytoplasm and even
the cell nucleus, contain cell-specific receptors for which
are involved in outside-inside signaling,
i.e. signal transduction.

Ligands include hormones, growth factors, cytokines,
prostaglandins and proteases

Hormones are involved in a variety of metabolic
processes that maintain homeostasis
e.g. fuel metabolism. Particularly noteworthy in that
regard are glucagon, insulin and the catecholamines
(epinephrine and norepinephrine).

Growth factors are involved in mitogenesis, whereas
cytokines play critical roles in the differentiation,
proliferation and function of various cell lineages

Interaction of such ligands with their membrane, cell-
specific receptors or intracellular receptors causes
conformational changes in the receptor and, in many
instances receptor-associated cytoplasmic proteins.

Such events result in the initiation of a cascade of
important, but as yet incompletely understood, events
leading to e.g. enzyme activation, differentiation and/or
cell division

Extracellular signaling
molecules released by
cells occurs over distances
from a few microns -
autocrine (c) and paracrine
(b) signaling to several
meters in endocrine (a)
signaling. In some
instances, receptor proteins
attached to the membrane
of one cell interact directly
with receptors on an
adjacent cell (d).


FORCES DRIVING SELECTION OF CURRENT
MECHANISMS

Need for coordinated intercellular communication



Need to translate extracellular signals into series of
intracellular events, while allowing for specificity




Specificity Determinants:
1. Specific receptors on or in the target cells
recognize an appropriate ligand.
2. Specific response to receptor occupancy - effector
pathways





Diversity of intercellular communication is achieved
with hundreds of signaling molecules


Proteins, small peptides, amino acids, nucleotides,
steroids, fatty acid derivatives, and even dissolved
gases such as NO and CO

Intracellular receptors:
- signaling molecules include
steroid hormones, retinoids,
thyroxine, etc
- receptor-hormone complex
acts as a transcription factor
to alter transcription of certain
genes

Cell surface receptors:
- signaling molecules include
peptide hormones, catechol-
amines, insulin, growth
factors, cytokines, etc
binding, and subsequent events,
triggers an | or | in the
cytosolic concentration of
a second messenger; or the
activated, bound receptor acts
as a scaffold to recruit and
activate other intracellular
proteins

1. Each cell is programmed to
respond to specific
combinations of signaling
molecules.

2. Different cells can respond
differently to the same
chemical signal.
HORMONES - First class of signaling molecules
defined

Secreted from endocrine cells - specialized signaling cells that
control the behavior of an organism as a whole:

1.Differ from other intracellular mediators

2. Usually stimulate metabolic activities in tissues remote from the
secretory organ

3. Active at very low concentrations (pM - uM)

4. Response to hormonal signal comes as a direct and rapid result
of its secretion

5. Metabolized rapidly so effects are, in most instances, short-
lived, leading to rapid adaptations to metabolic changes

Categories:


1. Peptides or polypeptides - insulin, glucagon, growth
hormone, insulin-like growth factors, vasopressin,
prolactin.

2. Glycoproteins - follicle stimulating hormone (FSH),
thyroid stimulating hormone (TSH)

3. Steroids - glucocorticoids (aldosterone, cortisol),
steroids (progesterone,testosterone), retinoic acid

4. Amino acid derivatives - epinephrine,
norepinephrine, thyroxine, triidothyronine



AGONISTS vs. ANTAGONISTS

Agonist mimics a hormone in binding productively to
a receptor

Antagonist mimics a hormone stereochemically, but binds to the
receptor non-productively, inhibiting the action.
natural hormone

Agonist
Hormone
Antagonist
RECEPTOR CHARACTERISTICS
1. Participates in transduction of the signal from the
external messenger to some component of the
metabolic machinery

2. Has at least one additional functional site which is
altered by ligand binding (allosteric site)

3. Ligand binding to receptors is saturable, resembling
Michaelis-Menten kinetics


4. Ligand-receptor interaction characterized by tight
binding (Kd = pM - uM)


Intracellular Signaling Mediated by G protein-linked
Membrane Receptors
e.g. Glucagon, Epinephrine and Thrombin as signaling
molecules
1. Activates a chain of events alterations in
concentrations of signaling molecules; elaborate sets
of interacting molecules that can relay signals from
cell surface to the nucleus

2. Components: Receptor; Transducer (G protein):
Effector (membrane-bound enzyme);
Second messenger (e.g. cAMP); Response (cascade
of highly-regulated protein phosphorylations

H
3
N
+
C COO

CH
2
OH
H

serine (Ser)
H
3
N
+
C COO

CH OH
CH
3
H

threonine (Thr)
Many enzymes are regulated by covalent attachment
of phosphate, in ester linkage, to the side-chain
hydroxyl group of a particular amino acid residue
(serine, threonine, or tyrosine)
Protein OH + ATP Protein O P
O
O

+ ADP
P
i
H
2
O
Protein Kinase




Protein Phosphatase
A protein kinase transfers the terminal
phosphate of ATP to a hydroxyl group on a
protein.
A protein phosphatase catalyzes removal of
the Pi by hydrolysis
Phosphorylation may directly alter activity of an
enzyme, e.g., by promoting a conformational change.

Alternatively, altered activity may result from binding
another protein that specifically recognizes a
phosphorylated domain.

E.g., 14-3-3 proteins bind to domains that include
phosphorylated Ser or Thr in the sequence
RXXX[pS/pT]XP, where X can be different amino
acids.

Binding to 14-3-3 is a mechanism by which some
proteins (e.g., transcription factors) may be retained
in the cytosol, & prevented from entering the
nucleus
Protein kinases and phosphatases are
themselves regulated by complex signal
cascades. For example:
Some protein kinases are activated by Ca++-
calmodulin.
Protein Kinase A is activated by cyclic-AMP
(cAMP).


N
N
N
N
NH
2
O
OH
O
H
H
H
H
2
C
H
O
P
O
O-
1'
3'
5'
4'
2'
cAMP
Adenylate Cyclase
(Adenylyl Cyclase)
catalyzes:
ATP cAMP + PPi
Binding of certain hormones
(e.g., epinephrine) to the
outer surface of a cell
activates Adenylate Cyclase
to form cAMP within the
cell.
Cyclic AMP is thus
considered to be a second
messenger.


N
N
N
N
NH
2
O
OH
O
H
H
H
H
2
C
H
O
P
O
O-
1'
3'
5'
4'
2'
cAMP
Phosphodiesterase
enzymes catalyze:
cAMP + H2O AMP
The phosphodiesterase
that cleaves cAMP is
activated by
phosphorylation
catalyzed by Protein
Kinase A.
Thus cAMP stimulates its
own degradation, leading
to rapid turnoff of a
cAMP signal.
RECEPTOR: typically a seven transmembrane domain, integral
membrane protein

|-adrenergic receptor
All G protein-coupled receptors resemble the |-adrenergic receptor
in their amino acid sequence and membrane topography.

Signal Transduction Pathway Activation of Adenylate Cyclase by Gs


Receptor: 7 TM domain integral membrane protein

Transducer: Gs

Effector: adenylate
cyclase
Second messenger:
cAMP
Response: PKA
regulated
protein
phosphorylations
Cycle of G protein dissociation/association:

Activation of G proteins involves GTP
displacement of GDP bound to the o subunit
and dissociation of the complex from the |
subunits, an exchange facilitated by a GEF
protein (guanine nucleotide exchange factor)
specific for Gs.

The active o subunit binds to and activates/
stimulates membrane associated adenylate
cyclase which catalyzes the conversion of ATP to
cyclic AMP (cAMP).

The GTPase activity associated with the o subunit
slowly hydrolyzes the bound GTP to GDP which is
facilitated by a GAP (GTPase activating protein)
specific for Gs.

The o subunit with GDP bound reassociates
with the | subunits, and membrane
associated adenylate cyclase returns to its
basal activity level

G-protein-Coupled Receptors may dimerize or form
oligomeric complexes within the membrane.

Ligand binding may promote oligomerization, which may
in turn affect activity of the receptor.

Various GPCR-interacting proteins (GIPs) modulate
receptor function. Effects of GIPs may include:altered
ligand affinity receptor dimerization or oligomerization
control of receptor localization, including transfer to or
removal from the plasma membrane promoting close
association with other signal proteins

Characteristics of G proteins

1. G protein is an o| trimeric protein which binds guanine
nucleotides.
2. They function to couple integral membrane receptors to target
membrane-bound enzymes.
3. They can be considered molecular switches wherein
o|
GDP
(inactive) o
GTP
(active) + |
4. The dissociated o subunit expresses GTPase activity
5. GTPS blocks GTPase activity of oGTP
o
i
| with
GDP bound
Region that changes
conformation when
GTP is hydrolyzed
Activation of cAMP-dependent protein kinase (PKA)

cAMP binds to the PKA regulatory subunits conformational changes, which
causes their dissociation from the catalytic subunits kinase activation.
Release of the catalytic subunits requires the binding of more than two cyclic
AMP molecules greatly sharpening the response of the kinase to changes in
[cAMP].
PKA is a Ser/Thr kinase with discrete substrate specificity, thus facilitating a
cascade of highly regulated protein phosphorylations.


The Whole Signaling
Network related to cAMP
Signal Transduction Pathway Activation of
Adenylate Cyclase by Gs

Down stream effects:

1. Phosphorylation of regulatory enzymes of
metabolic pathways.


2. Increase transcriptionof certain proteins via
phosphorylation of CREB leading to | protein syn-
thesis of target proteins.



Activated PKA enters the
nucleus and phosphorylates
CREB (cAMP Response
Element Binding protein).
Once phosphorylated, CREB
recruits the coactivator CBP
(CREB Binding Protein).
This complex binds to the
CREB-binding element to
stimulate gene transcription
How Increased Intracellular cAMP Leads to Increased Gene Transcription
Signal Transduction Pathway Activation of Adenylate Cyclase by Gs

Early off signals:
1. Gso-associated GTPase
activity GTP hydrolysis
to GDP leading to re-
association of GsGDP
and dissociation of the
hormone/receptor complex

Down stream off signals:

1. cAMP hydrolyzed
by phosphodiesterase

2. Ser/Thr phosphatases
dephosphorylate the
phosphorylated target
proteins.

Desensitization or Endocytosis of GPCRs Effected by Phosphorylation

1. The ligand activated receptor can be phosphorylated on select Ser/Thr
2. residues by GRK (e.g. BARK - | adrenergic receptor kinase). These
phosphorylated residues
provide a docking site for arrestin resulting in inactivation/desensitization.
2. In some instances, arrestin binding targets the receptor for clathrin-dependent
endocytosis.
3. In addition, if the occupied GPCR leads to | cAMP, the receptor can also be
phosphorylated by PKA leading to its inactivation/densensitization.


Clathrin-dependent Receptor-mediated endocytosis

Gs vs. Gi
Regulation of Adenylate Cyclase Activity

Gs stimulates adenylate cyclase

Gi inhibits adenylate cyclase

e.g. epinephrine can increase or decrease
intracellular cAMP concentrations,depending
upon the receptor to which it binds

| adrenergic receptors couple to Gs, whereas
o2 adrenergic receptors couple to Gi



Vasopressin binding to its GPCR activates Gs | cAMP and activation
of PKA.
PKA phosphorylates various proteins the ultimate aggregation of
microtubular subunits, which insert as water channels in the luminal
plasma membrane to increase the reabsorption of water by free diffusion.

Inhibition of Gs and Gi by Bacterial Toxins
Cholera toxin effects on Gs:
ADP ribosylation of an Arg
residue in the os subunit of
Gs inhibition of associated
GTPase activity

Pertussis toxin effects on Gi:
ADP ribosylation of a Cys
residue in the oi subunit of Gi
an inability to inhibit
adenylate cyclase activity.


Thus, both toxins cause
increased intracellular cAMP
concentrations!


Gs vs Gi vs Gq




Gs and Gi coupled to adenylate cyclase A
[cAMP]

Gq coupled to phospholipase C A [Ca2+]

INTRACELLULAR Ca2+ AS A SECOND MESSENGER

Cells must work very hard to maintain low intracellular [Ca2+]
in order for| Ca2+ to result in effective intracellular signaling.

Cellular mechanisms that maintain very low intracellular Ca2+
concentrations
A: Ca2+ is actively pumped out of the cytosol.
B: Ca2+ is pumped into the ER and mitochondria.

Three Types of Inositol phospholipids
PI, PI(4)P, PI(4,5)P2
Phospholipase C- |
(PLC-|) Produces
DAG
(diacylglycerol) and
IP3 (inositol 1,4,5-
trisphosphate (IP3))

Gq->PLC-|
Gq signaling pathways and Calcium
Phospholipase C| (PLC|)-catalyzed hydrolysis of PIP2 Formation of 2
second messengers
IP3 release of Ca2+ from intracellular stores by binding to an IP3-gated
Ca2+ channel in the endoplasmic reticulum.
DAG with released Ca2+ and membrane-associated phosphatidylserine
activates Protein Kinase C (PKC - a Ser/Thr kinase). PKC directly
phosphorylates intracellular proteins some of which | gene transcription
Phosphoinositide-activated Second Messenger System
| Intracellular [Ca2+]


Ca2+ Second Messenger System: Release from
intracellular stores subsequent to PLC|-catalyzed hydrolysis
of PIP2

1)Must be distinguished from cAMP-induced effects where
cAMP activates a variety of Ca2+ channels | Ca2+ influx
from extracellular milieu (e.g. |-adrenergic receptor
occupancy in muscle cells | Ca2+ influx | rate and
force of heart beat

2)Increased intracellular [Ca2+] - Third messenger: Immediate
vs. Sustained responses Ca2+ binds to its ubiquitous
intracellular receptor, calmodulin, thereby a) activating
Ca2+/calmodulin-dependent kinases (CaM kinases), Ser/Thr
kinases b) that have their own sets of substrate proteins,
some of which when phosphorylated can | or | gene
transcription.





Increased Intracellular Ca2+: A Third Messenger

Calmodulin in a ubiquitous intracellular Ca2+ receptor
Ca2+/calmodulin (CaM) complexes activate CaM-dependent
kinases (e.g. CaM-kinase II)


Fertilization of an egg by a sperm triggering an increase in
cytosolic Calcium
3 major types of calcium channels:
Voltage dependent Ca channels on plasma membrane
IP3-gated Ca release channels on ER membrane
Ryanodine receptor on ER membrane

Calcium uptake and deprivation
Na/Ca exchanger on plasma membrane, 2. Ca pump on ER
membrane, 3. Ca binding molecules, 4. Ca pump on Mitochondia

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

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

Intracellular Signaling Pathways activated by RTKs
and GPCRs

1. 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-b
and Smad

Notch and Delta interaction
Lateral inhibition

The inhibitory pathway of Notch
Proteolysis-mediated
Summary

1. GPCR signaling: PKA and Calcium
2. Enzyme-linked Receptor signaling: RTK->Ras
and PI3K
3. Proteolysis-mediated signaling pathways