Transcript Presentation (PowerPoint File) - IPAM

"Receptor-ligand interactions cell signaling, adhesion, motility,
cell migration"
Patricia Zuk, PhD
Research Director
Regenerative Bioengineering and Repair (REBAR) Lab
Department of Surgery
David Geffen School of Medicine at UCLA
No cell lives in isolation
• survival depends on an elaborate
intercellular communication network that
coordinates growth, differentiation and
metabolism
• cells adjacent to one another frequently
communicate through cell-cell contact
• other forms of communication cover larger
distances = extracellular signaling
molecules
Extracellular Signalling
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signaling molecules are released by signaling cells
the signal is called the ligand
the ligand binds to its specific receptor on a target cell
this ligand-receptor interaction induces a conformational
or shape-change in the receptor
• produces a specific response - called the cellular
response
• can include a vast array of compounds
– e.g. small amino acid derivatives, small peptides, proteins
Cell-to-cell communication by extracellular
signaling usually involves six steps
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(1) synthesis of the signaling molecule by the signaling cell
(2) release of the signaling molecule by the signaling cell
(3) transport of the signal to the target cell
(4) detection of the signal by a specific receptor protein –
receptor-ligand specificity
• (5) a change in cellular metabolism, function, or
development = cellular response
– triggered by the receptor-ligand complex – specific to the ligandreceptor complex
• (6) removal of the signal, which usually terminates the
cellular response – degredation of ligand
Signaling molecules operate over various distances in
animals
-extracellular signaling can occur over:
1. large distances or endocrine signaling – signaling molecules are called
hormones
- act on target cells distant from their site of synthesis
-usually carried through the bloodstream
2. short distances or paracrine signaling – affects target cells within proximity
to the cell that synthesized the molecule
-usually mediated by neurotransmitters and some growth
factors
Signaling molecules operate over various
distances in animals
-extracellular signaling can occur over:
3. no distance or autocrine signaling – the signal feeds-back and affects
itself
-action of many growth factors
-these compounds generally act on themselves to
regulate proliferation
-seen frequently in tumor cells
-many compounds can act through two or even three types of cell signaling
e.g. growth factors (e.g. EGF) – paracrine and autocrine and endocrine
-epinephrine – endocrine and paracrine
Circulating & Local Hormones
• Circulating
hormones
– act on distant targets
– travel in blood
– endocrine hormones
• Local hormones
– paracrine hormones
& autocrine
hormones
Hormones
• two types
– lipid soluble
– water soluble
Lipid-Soluble Hormones
-lipid-soluble hormones can easily
enter a cell by diffusing through the
plasma membrane
-PROBLEM: how do they travel in the
water-based blood??
-SOLUTION: they are carried by
carrier-proteins
-these hormones then enter their
target cell where they result in a
specific cellular effect or response
Water-soluble Hormones
-water soluble hormones can easily travel within the blood
-PROBLEM: how do they enter a cell and result in a cellular response??
-SOLUTION: binding to specific cell-surface receptors
-this binding activates the receptor and results in a series of cellular events called
the second messenger system
Lipid-soluble Hormones
• Steroids
– lipids derived from cholesterol in SER
– different functional groups attached
to core of structure provide
uniqueness
– interact with specific intracellular
receptors (within the cell) to turn
specific genes on or off
– effective for hours or days
• Thyroid hormones
– tyrosine ring plus attached iodines
are lipid-soluble
– activate enzymes involved in the
catabolism of fats and glucose
– help set our basal metabolic rate
• Retinoids
– vitamin A derivatives
– have dramatic effects on proliferation
and differentiation plus cellular death
(i.e. apoptosis)
Water-soluble Hormones
• Amino acid derivatives,
small peptides and protein
hormones
– modified amino acids or amino
acids put together
• serotonin, melatonin, histamine,
epinephrine
– larger peptide hormones
• insulin and glucagon
• Eicosanoids
– derived from arachidonic acid
(fatty acid)
– prostaglandins or leukotrienes
– prostaglandins despite being
lipidphilic – bind to cell surface
Action of Lipid-Soluble Hormones
• Hormone diffuses
through phospholipid
bilayer & into cell
• Binds to receptor
turning on/off specific
genes
• New mRNA is formed
& directs synthesis of
new proteins
• New protein alters
cell’s activity
Action of Water-Soluble Hormones
• Can not diffuse through plasma
membrane
• Hormone receptors are integral
membrane proteins
– act as first messenger
• Receptor protein activates G-protein in
membrane
• G-protein activates adenylate cyclase to
convert ATP to cAMP in the cytosol
• Cyclic AMP is the 2nd messenger
• Activates kinases in the cytosol to
speed up/slow down physiological
responses
• Phosphodiesterase inactivates cAMP
quickly
• Cell response is turned off unless
new hormone molecules arrive
Cell-surface receptors belong to four major classes
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GPCRs are involved in a range of signaling pathways, including
light detection, odorant detection, and detection of certain
hormones and neurotransmitters
Many different mammalian cell-surface receptors including
GPCRs are coupled to a trimeric signal-transducing G protein
– made of an alpha, beta and gamma subunit complex
Ligand binding activates the receptor, which activates the G
protein, which activates an effector enzyme to generate an
intracellular second messenger
– e.g. adenylyl cyclase – converts ATP to cAMP
depending on regulation at the effector enzyme – this pathway
can be either activated or inhibited
– by the type of G protein activated by the hormone-receptor
complex
– Gs proteins result in stimulation of the effector enzyme
– Gi proteins result in inhibition of the effector enzyme
adenylyl cyclase (AC)
Four classes of cell-surface
receptors
-ligand binding changes the confirmation of the receptor so that specific ions flow
through it
-the resultant ion movement alters the electric potential across the plasma
membrane
-found in high numbers on neuronal plasma membranes
e.g. ligand-gated channels for sodium and potassium
-also found on the plasma membrane of muscle cells
-binding of acetylcholine results in ion movement and
eventual contraction of muscle
-lack intrinsic catalytic activity
-binding of the ligand results in the formation of a receptor dimer (2 receptors)
-this dimer than activates a class of protein called tyrosine kinases
-this activation results in the phosphorylation of downstream targets by
these tyrosine kinases (stick phosphate groups onto tyrosines within
the target protein)
-receptors for cytokines such as XXXX, interferons (XXXXXXX)
Signal
transduction
Cascade
-also called receptor tyrosine kinases OR ligand-triggered protein kinases
-similar to tyrosine-linked receptors - ligand binding results in formation of a dimer
-BUT: they differ from tyrosine-linked receptors – intrinsic catalytic activity
-means that ligand binding activates it and the activated receptor acts as a
kinase
-recognize soluble or membrane bound peptide/protein hormones that act as
growth factors
e.g. NGF, PDGF, insulin
-binding of the ligand stimulates the receptor’s tyrosine kinase activity,
-results in phosphorylation of multiple amino acid residues within its target
– such as serine and threonine residues
-this phosphorylation activates downstream targets
-its targets are generally other protein kinases –which phosphorylate their
own downstream targets (other kinases??)
Signal transduction cascades
Signal
• -the successive
phosphorylation/activation of multiple
kinases results in a cascade of
phosphorylation/activation
• -this cascade is frequently called a
signal-transduction cascade
• -this cascade eventually leads to a
specific cellular response
•
e.g. changes in cell physiology
and/or patterns of gene
expression
• -RTK pathways are involved in
regulation of cell proliferation and
differentiation, promotion of cell
survival, and
• modulation of cellular metabolism
p
KINASE #1
p
KINASE #2
p
KINASE #3
TARGET
EFFECT
p
Second messengers
• produced by the activation of GPCRs and RTKs
• Hormone stimulation of Gs protein-coupled receptors leads to activation
of adenylyl cyclase and synthesis of the second messenger cAMP
– most commonly studied second messenger
– cAMP does not function in signal pathways initiated by RTKs
– cAMP and other second messengers activate specific protein kinases
(cAMP-dependent protein kinases or PKAs)
• cAMP has a wide variety of effects depending on the cell type and the
downstream PKAs and other kinases
– in adipocytes, increased cAMP activates a PKA that stimulates production of
fatty acids
– in ovarian cells another PKA will respond to cAMP by increase estrogen
synthesis
• second messenger systems allow for amplification of an extracellular
signal
– one epinephine molecule can bind one GPCR – this can result in the
synthesis of multiple cAMP molecules which can go on to activate and
amplified number of PKAs
• a blood level as low as 10-10M epinephrine can raise blood glucose levels by 50%
Second messengers
• other second messengers include:
– IP3 and DAG – breakdown products of phosphotidylinositol (PI)
• produced upon activation of multiple hormone receptor types
(GPCRs and RTKs)
– calcium – IP3 production results in the opening of calciumchannels on the plasma membrane of the ER – release of
calcium
• a rise in calcium in pancreatic beta cells triggers the exocytosis of
insulin
• a rise in intracellular calcium also triggers contraction of muscle
cells
• much study has been done on the binding of calcium to a protein
called calmodulin and the effect of this complex on gene
expression
MAP kinase pathways
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best characterized signal transduction pathway
activation of RTKs by growth factors, hormones etc…..
result in activation of an adaptor protein called Ras GTPase
ras induces a kinase signal cascade that starts with a kinase called rac and
culminates in activation of a MAP kinase (MAPK)
in between are a series of kinases that are part of the cascade
MAPK activation results in translocation into the nucleus and phosphorylate
many different proteins, including transcription factors that regulate gene
expression
MAPK kinase paths
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Stress, cytokines, hormones & mitogens signal
through cdc42/rac
cdc42/rac
– then activates one of three MAPK paths:
p38MAPK: stress
Stress
Cytokines
Stress
Hormones
Cytokines
Mitogens
Hormones
response & apoptosis (MAPKAP-
2, HSP27)
JNK:
cdc42/rac
stress response & proliferation (jun)
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Activation of ras/MEK/ERK path: proliferation &
differentiation
grb2
MAPK kinase
transcription
factors (nucleus)
MEKKs
MEKKs
MEK 3/6
JNKK 1/2
p38MAPK
MAPKAP-2
HSP27
JNK 1/2
ATF
ATF
elk
jun
elk
ras
raf-1
MEK 1/2
ERK 1/2
elk
RSK
fos
sos
Common signaling pathways are
initiated by different receptors in a class
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The effects of activation of GPCRs
and RTKs is more complicated than
a simple step-by-step cascade
Stimulation of either GPCRs or
RTKs often leads to production of
multiple second messengers, and
both types of receptors promote or
inhibit production of many of the
same second messengers
in addition, RTKs can promote a
signal transduction cascade that
eventually acts on the same target
as the GPCR
therefore the same cellular
response may be induced by
multiple signaling pathways by
distinct mechanisms
Interaction of different signaling
pathways permits fine-tuning of
cellular activities
Integrating Cells into Tissues:
Cell-Cell Adhesion and Communication
-a key event in the evolution of multicellularity is the ability for cells to adhere to one
another and be able to communicate with each other
-evolved a series of cell-adhesion molecules or CAMs that allow interaction with
each other and with the surrounding extracellular matrix (ECM)
-this results in coordinated functioning of tissues
-HOW??
-these interactions result in the activation of specific signal transduction
cascades eventually resulting in the desired cellular effect
-therefore the physical interaction of CAMs with the ECM can turn pathways on or
off – cellular effect
e.g. cellular interactions with the adhesion protein b1-integrin can result in
activation of the MAPK cascade
-various classes of CAMs found on cells
1. cadherins – cell-cell adhesion
-calcium dependent
e.g, E-cadherin, P-cadherin
2. Ig superfamily of CAMs – cell-cell adhesion
e.g. N-CAM, V-CAM
-calcium-independent
-some are found enriched on specific cell types – N-CAM
3. Integrins – major cell-matrix adhesion molecule
e.g. a1 integrin, b1 integrin
Cell-Cell adhesion: Cadherin-containing
junctions
1. adherens junction – continuous band of
cadherins found in epithelial cells
-connects cells tightly and interact
with the actin portion of the cytoskeleton
2. desmosomes – also found in high #s in
epithelial tissues
-cells attach via cadherins – connect
to protein “plaques” that attach to the
plasma membrane and to intermediate
filaments within the cytoskeleton
-cadherins are a family of calcium-dependent
CAMs
-major molecules of cell-cell adhesion (homophilic)
-over 40 different are known
e.g. E or epithelial cadherin
N or neural cadherin
-tissues have specialized junctions made of cadherins
1. Adherens junction
2. Desmosome
Cell Matrix
• three components to the ECM
– insoluble collagens – structural framework of the ECM, provides
strength and resiliency
– proteoglycans – cushions cells
– adhesive matrix proteins – bind these components to receptors
on the cell surface
– different combinations result in unique ECM compositions which
can directly affect the activity of the cells
– the interaction of ECM components with specific CAMs (i.e. ECM
receptors) can turn certain signaling paths ON or OFF
– also the ECM is capable of sequestering growth factors and
hormones by binding them and making them unavailable to turn
cellular signaling on or off
Cell-matrix interactions: integrins
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integrins allow connection between the extracellular matrix with the cytoskeleton of the cell
also can mediate cell-cell interactions (weak)
made of an alpha and a beta subunit – 17 types of a chains, 8 types of b chains
these ab complexes act as receptors to specific matrix components
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e.g. a1b1 – collagen and laminin
a5 b1 - fibronectin
cells will express multiple integrin types
attachment can be regulated by up- or down-regulated expression of these integrins
integrins can also form very specific adhesive junctions
– focal adhesions (FN to actin) – complex of more than 20 proteins
• can increase and decrease based on physical stress
– hemidesmosomes (intermediate filaments to collagens and laminins) – epithelial cells
•activation of these integrins can promote cell signaling
•e.g. formation and activation of focal adhesions triggers a cascade initaited by a
Focal Adhesion Kinase (FAK) – modulates cell growth and motility
Cell-Cell Communication: Gap
Junctions
-almost all cells have specialized junctions
that allow the free passage of materials
(e.g. signaling molecules) back and forth
-these junctions = gap junctions
-made of a channel protein called connexon
-connexons interact to form channels between
two cells
-one important compound small enough to
traverse this junction is the second messenger
cAMP
-also allows passage of calcium ions and other
ions crucial to signaling (sodium, phosphate)
-these gaps are not free-swinging gates
-they actually open and close in
response to ion concentrations
thereby restricting the flow of too
many ions
-they also exclude materials based
on size
Cell-Cell and Cell-Matrix interactions can
activate signal transduction cascades
•
J Biomed Sci. 2006 Feb 23; Crosstalk between hepatocyte growth factor and integrin
signaling pathways. Chan PC, Chen SY, Chen CH, Chen HC
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J Neurochem. 2006 Jan;96(1):148-59
Fibronectin promotes brain capillary endothelial cell survival and proliferation through
alpha5beta1 and alphavbeta3 integrins via MAP kinase signalling. Wang J, Milner R.
•
Immunopharmacol Immunotoxicol. 2005;27(3):371-93.
Interleukin-3, -5, and granulocyte macrophage colony-stimulating factor induce adhesion
and chemotaxis of human eosinophils via p38 mitogen-activated protein kinase and
nuclear factor kappaB. Ip WK, Wong CK, Wang CB, Tian YP, Lam CW.
•
Cell Commun Adhes. 2004 Sep-Dec;11(5-6):137-53.
ERK signaling pathways regulate the osteogenic differentiation of human mesenchymal
stem cells on collagen I and vitronectin. Salasznyk RM, Klees RF, Hughlock MK, Plopper
GE.
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Microsc Microanal. 2005 Jun;11(3):200-8.
Cross talk between cell-cell and cell-matrix adhesion signaling pathways during heart
organogenesis: implications for cardiac birth defects.Linask KK, Manisastry S, Han M.