Cell Signaling I:

Signal Transduction & Short-

term Cellular Responses
Reminder of structure of Biomembranes

• Hydrophobic core: impermeable barrier

– Prevents H20 soluble (hydrophilic) solutes across the membrane
– Membrane proteins modulate this barrier by mediating transport
of specific molecules across
• Strength of bilayer
– from hydrophobic & van der Waals interactions b/w lipid chains
 General Principles of Signaling by Cell
Surface Receptors

• Hydrophilic chemical signals

that bind to cell-surface
receptors activate signal-
transduction pathways
– Ligand binding to the
receptor
– Transduction via 2nd
messengers, cascade
– response(s)
 Types of Signals

• Act outside the organism

– Pheromones: a signaling molecule released by an
individual that can alter the behavior or gene expression
of another individual of the same species
• Yeast mating-type factors

• Act within the organism

– Extracellular signaling molecules that control
• metabolic processes within cells
• growth & differentiation of tissues
• synthesis and secretion of proteins
• composition of intracellular and extracellular fluids
 Schemes of Intercellular Signaling
• Endocrine: act on target cells at a
long distance from their site of
synthesis

• Paracrine: signaling molecules

released by a cell affect target
cells in close proximity

Note: some molecules can serve as both

paracrine & endocrine signals
(eg. epinephrine as neurotransmitter &
systemic hormone)

• Autocrine: cells respond to

substances that they themselves
release

• Juxtacrine: some membrane-

bound proteins act as signals
How do Signaling Molecules Work?

1. Change the activity or function of a pre-existing

protein.

2. Change the amount of protein in a cell by modifying

transcription factors, leading to activation or
suppression of gene transcription.

Response 1 generally occurs more rapidly. Why?

General Steps of Extracellular Signaling

1. Synthesis of signaling molecule

2. Release of signaling molecule
3. Transport of signaling molecule to target cell
4. Binding of signal by a receptor leading to its activation
5. Initiation of intracellular signal-transduction pathways
by the activated receptor
6. Specific changes in cellular function, metabolism, or
development
7. Removal of the signal
 Common Properties of Cell-Surface
Receptors

• All receptors are proteins

• Receptors have high ligand binding specificity
– Ability to distinguish closely related substances
• Ligands, on the other hand, exhibit binding versatility
– one type of ligand may bind to different types of receptors to
activate different pathways
• Receptor-ligand complex exhibits effector specificity
– a specific cellular response is turned on
Interaction between Receptor and Ligand
• Binding of ligand to receptor is by weak, non-covalent
interactions (ionic, hydrophobic, van der Waals)
– molecular complementarity b/w interacting surfaces of a
receptor and ligand

Patches of amino
acids in receptors and
ligands determine
their highly specific
mutual interaction
 Common Properties of Cell-Surface
Receptors
• Ligand binding can be viewed as a simple reversible
reaction:
k
R + L k RL
on

off

• KD (Dissociation constant) = [R][L]/[RL]

– KD is the concentration of ligand at which half of its
receptors are occupied
– measures the affinity of the receptor for the
ligand/substrate
– The lower the KD value, the higher the affinity of a
receptor for its ligand
 Common Properties of Cell-Surface
Receptors
• Sensitivity of a cell to a ligand depends on the number of
surface receptors and the number of RL complexes.
– The fewer the receptors present on the cell surface, the less
sensitive the cell is to that ligand.
• Maximal cellular response to a signaling molecule may
not require activation of all receptors present.
– In fact, the maximal response of a cell to a particular ligand
generally occurs at a ligand concentration at which most of
its receptors are still not occupied.
– Example: Erythroid progenitor cell has 1000 surface
receptors for erythropoietin (Epo)
• Epo induces progenitor cells to proliferate and
differentiate into rbc
• However, only 100 receptors need to bind to ligand to
induce division of target cell.
 Receptors & Associated Signal-
Transduction Proteins may be Localized
• Some receptors may be uniformly distributed on the cell
surface; however, others are localized to particular regions.
• Clustering may be mediated by adapter domains of particular
cytosolic proteins (PDZ, SH3, etc)
– PDZ domain is a common element in several cytosolic proteins
that bind to integral plasma membrane proteins
• Small domain, 90 aa residues, that binds to sequences at the C-
term of target proteins
• Example: Proteins containing PDZ domains organize and localize
receptors in the plasma membrane of the post-synaptic cell
– Most cell surface receptors contain multiple subunits, each of
which can bind to a PDZ domain
– Many cytosolic proteins contain multiple PDZ domains that
participate in multiple protein-protein interactions simultaneously
Multiple Protein-protein Interactions:
Membrane Protein Clustering

• PDZ domain binds to

certain C-term sequences
• SH3 domain binds to
proline rich sequences

• A single actin filament can

bind many these clusters,
so large #s of pm proteins
can be specifically
clustered together
• Result: many receptors
binding same or different
ligands are localized to a
specific region of the pm
 Highly Conserved Components of
Intracellular Signal-transduction Pathways

1. GTP-binding switch proteins

2. Protein kinases
3. Protein phosphatases
4. 2nd messengers
 GTPase Switch Proteins

• Belong to GTPase superfamily

• Guanine nucleotide-binding proteins
– are “on” when GTP bound
– are “off” when GDP bound
• After activation, switch 1 & II segments
remove P from GTP, cause inactivation
• Signal induced conversion of inactive to active state is mediated by
GEF (G nt-exchange factor), release of GDP, allowing GTP to bind
• Classes of GTPase Switch Proteins
– Trimeric: directly bind & are activated by cell surface receptors
– Monomeric: indirectly linked to receptors via adaptor proteins
• eg. Ras
 Kinases & Phosphatases

• Protein kinases and phosphatases are employed virtually all

signaling pathways
– Human genome currently has 600 kinases and 100
phosphatases known

• Protein Kinases: enzymes that phosphorylate other proteins

– Animals cells contain 2 types:
• one phosphorylates the -OH group of tyrosine
• one that phosphorylates the -OH group of serine, threonine,
or both
• Protein Phosphatases: Enzymes that remove phosphate
groups (dephosphorylate) from other proteins
 Second Messengers Carry & Amplify
Signals from Many Receptors
• Binding of ligands to cell-surface receptors leads to the
activation of non-protein intracellular molecules called second
messengers that transduce signals
• cAMP (cyclic AMP): activates protein kinase A
• cGMP (cyclic GMP): activates protein kinase G
• DAG (Diacylglycerol): activates protein kinase C
• IP3 (inositol triphosphate): causes Ca2+ release from ER
• Ca2+: activates a variety of proteins that cause cellular
responses, such as:
– Muscle contraction in muscle cells
– Release of neurotransmitters (vesicle exocytosis in nerve cells)
– Release of hormones (exocytosis in endocrine cells)
Common Intracellular Second
Messengers
 G Protein Coupled Receptor Systems
• Activation of receptor (GPCR) by its ligand will activate the
coupled trimeric G protein which interacts with downstream
signal transduction proteins
• 4 elements:
– GPCR
– Coupled trimeric G protein switch
– Membrane bound effector protein
– Feedback regulation & desensitization
• GPCR have short term effects in cell that quickly modify
existing proteins
– Eg. Enzymes or ion channels
• Examples of GPCR family members:
– Some hormone & neurotransmitter receptors
– Light activated receptors in the eye (rhodopsin)
– Odorant receptors in the nose
 G-Protein Coupled Receptors (GPCR)
• N-term in the exoplasmic face
• C-term in the cytosolic face
• 7 membrane-spanning regions, H1-H7
• 4 extracellular segments, E1-E4
• 4 cytosolic segments, C1-C4
– C3 and C4 domains interact with trimeric G-proteins
 Structure of Trimeric G Protein

• 3 subunits: G, G, and G

• G and G subunits remain together
– referred to as the G subunit
• G subunit is the GTPase
– Switch protein that alternates b/w
on (active) & off (inactive)
 Multiple G Proteins Encoded in
Eukaryotic Genome

• Most effector proteins are activated by G-GTP, but some

are inhibited
• In some cells, the G subunit signals to the effector protein
• The activity of several different effector proteins is controlled
by different GPCR-ligand complexes.
• The different G subunits function similarly, where as the
G class show functional differences
 Ligand Induced Activation of Effector
Proteins through GPCR

Stimulation Activation of Modulation of Activity of

of GPCR G protein associated effectors protein
Examples of Effector proteins:
•Membrane bound ion channels
•Enzymes that catalyze
formation of 2nd messengers
GPCR activate exchange of GTP (for GDP) on  subunit of trimeric G protein
General Mechanism of GPCR Activation
of Effector Proteins
 Some GPCR Regulate Ion Channels

• Effector proteins are some are Na+ or K+ channels

– Binding of ligand to GPCR will result in
• activation of 2nd messengers
• subsequently the associated ion channel will open or close
• leads to changes in membrane potential
• Examples include:
– Some neurotransmitter receptors
– Cardiac muscarinic acetylcholine receptors
– Odorant receptors in nose
– Photoreceptors in eye
 Muscarinic Acetylcholine Receptors in
Heart Muscle
• Binding of acetylcholine to muscarinic acetylcholine receptors in
cardiac muscles are inhibitory (coupled to a Gi protein)
– Slows the rate of heart muscle contraction
• acetylcholine binding causes
receptor to bind to Giα subunit
• GDP is replaced by GTP
• Activated Giα subunit
dissociates from Gβγ subunit
• Gβγ subunit binds to K+ channel
(effector protein)
• K+ channel opens
• K+ flow out of the cell causing
hyper-polarization
• frequency of heart muscle
contraction decreases
2 General Types of GPCR that Activate or
Inhibit Adenylyl Cyclase
β-andrenergic receptors: α-andrenergic receptors:
• these are coupled to • these are coupled to an
stimulatory Gs protein inhibitory Gi protein
• stimulate adenylyl cyclase • inhibit adenylyl cyclase
• increase [cAMP] • decrease [cAMP]
 Activation of Adenylyl Cyclase
• Signal binds to receptor, receptor undergoes conformational
change, becomes active, activated receptor binds to G subunit
• G subunit undergoes a conformational change
– GDP dissociates & GTP binds, G dissociates from G subunit
• G binds to effector protein, adenylyl cyclase, thereby activating it
• Hydrolysis of GTP to GDP within a few seconds inactivates G, it
re-associates w/ G
– this terminates effector activation (adenylyl cyclase inactivated)
 Function of Adenylyl Cyclase
• Adenylyl cyclase catalyzes synthesis of cAMP from ATP
• cAMP activates Protein Kinase A (PKA) by releasing catalytic
subunits
• PKA activates additional proteins, leads to a cellular response
– cAMP-activated PKA mediates various responses in different cells
 Epinephrine
• Hormone that is important in mediating body’s response to
stress & heavy exercise when tissues need to produce more
energy (ATP) from glucose catabolism
• Liberation of glucose can be triggered by epinephrine binding
to their receptors
• All epinephrine receptors are GPCRs
– Examples:
• Epinephrine binds to β-andrenergic receptors on liver cells
• Epinephrine binds to α-andrenergic receptors on smooth
muscle cells lining blood vessels of intestinal tract
– Different types of receptors are coupled to different G
proteins and stimulate different intracellular signaling
pathways
 Glycogen Metabolism
• Glycogen = large glucose polymer
– major glucose storage form in animals
– synthesized by one set of enzymes, degraded by another

} Incorporation
of glucose

} Removal of
glucose units
(Glucose 1-P)
 Glycogen Metabolism
• Glycogen metabolism is regulated by epinephrine, which
increases cAMP, and induces activation of PKA
• Epinephrine binding to β-andrenergic GPCRs in muscle and
liver cells leads to increased cAMP production.
– In both muscle and liver cells, glycogen is broken down to
Glucose-1 P, and converted to Glucose-6 P.
– In muscle cells: Glucose-6 P enters glycolysis for
production of ATP used to power muscle contraction.
– In liver cells: the Glucose-6 P is hydrolyzed to glucose
which is exported from these cells by a plasma membrane
glucose transporter (GLUT2).

• If epinephrine is removed, the level of cAMP drops, PKA is

inactivated
– Mediated by phosphoprotein phosphatase (PP) which reverses
the PKA effects.
 Role of PKA in Glycogen Metabolism
PKA enhances glycogen metabolism in 2 ways:

• Directly: PKA inhibits glycogen synthase

– PKA phosphorylates and inactivates glycogen synthase
– Stops glucose incorporation into building glycogen

• Indirectly: stimulates glycogen phosphorylase

– Stimulates degradation of glycogen by
• phophorylating and activating glycogen phosphorylase kinase
(GPK)
• GPK then phosphorylates and activates glycogen
phosphorylase
 Regulation of Glycogen Metabolism by cAMP
1. Direct (3. At high [cAMP]
2. Indirect PKA phosphorylates
inhibitor)

* *

* *

*
*
* = active
* *

*
 Signal Amplification
• activation of higher numbers of molecules downstream from receptor
– especially common with cell surface receptors
– signaling molecules involving 2nd messengers and kinase cascades
amplify an external signal tremendously

} Amplification
of 104
 GPCRs that Activate Phospholipase C
• Involve 2nd messengers that have inositol, which can be
reversibly phosphorylated
• Example: PIP2= PI 4,5-bisphosphate (membrane-bound)
– binds many cytosolic proteins to the plasma membrane

• PLC = phospholipase C
– Plasma membrane associated
enzyme
– Cleaves PIP2 to generate two
important 2nd messengers:
• DAG: 1,2-diacylglycerol, lipophilic
molecule, remains in membrane
• IP3: inositol 1,4,5-triphosphate
which diffuses in the cytoplasm
 IP3/DAG Pathway & Release of ER Ca2+

• Ligand binding to GPCR causes

activation of PLC
• IP3 diffuses thru cytosol, binds IP3-
gated Ca2+ channel on smooth ER
• Ca2+ released, causes PKC
recruitment to plasma membrane,
where it interacts with DAG and is
activated.

• PKC phosphorylates and activates cellular enzymes & receptors

• leads to various cellular response in different cells, playing key
roles in different aspects of cellular growth & metabolism
– eg. PKC regulates glycogen metabolism by phosphorylating &
inhibiting glycogen synthase
 Ca2+/Calmodulin Complex
• Release of Ca2+ into the cytosol from IP3-mediated processes
can lead to a variety of cellular responses
– Localized increases in cytosolic Ca2+ in specific cell types
are critical to its function as a 2nd messenger
– eg. Acetylcholine stimulation of GPCR in secretory pancreas
cells causes IP3-mediated Ca2+ rise, triggering fusion of
secretory vesicles with plasma membrane & content release

• Calmodulin: a small cytosolic Ca2+ binding protein functioning as

a multipurpose switch protein mediating multiple Ca2+ cellular
effects
– binds to 4 Ca2+ ions to form a complex that interacts w/ &
modulates the activity of many other proteins & enzymes
 Various Processes are Activated by the
Ca2+/Calmodulin Complex
1. Activates protein kinases, which phosphorylate TS factors,
which modifies their activity and regulate gene expression
2. Activates phosphatases that dephosphorylate TS factors
– Example: NFAT= nuclear factor of activated T cells
• Essential TS factor enhanced by Ca2+ ions
• In unstimulated cells, phosphorylated NFAT in cytosol
• After stimulation, cytosolic [Ca2+] increased, Ca2+/Calmodulin
complex binds to and activates calcineurin (a phosphatase)
• Activated calcineurin dephosphorylates NFAT, exposing NLS
• NFAT moves into the nucleus, stimulates expression of
genes essential for T cell activation
3. Plays a key role in controlling diameter of blood vessels