Ch 16: Cell Signaling

Extracellular signal molecules are used by cells to

communicate with each other
signals can be proteins, peptides, amino acids, nucleotides, steroids...

Endocrine cells produce

hormones

Hormones "broadcast" signal throughout

the whole body (enter the bloodstream)

Paracrine signaling - rather than entering the bloodstream, the signal molecules diffuse

locally through extracellular fluid
act as local mediators on nearby cells

Autocrine signaling

acting on itself

Neuronal signaling

nerve cells can deliver messages over long distances
signals are delivered quickly and specifically to individual target cells
extracellular signal molecule is a neurotransmitter

Contact dependent signaling

allows adjacent cells that are initially similar to become specialized to form different cell types

4 types of cell signaling:

endocrine
paracrine
neuronal
contact-dependent

Whether a cell responds to a signaling molecule depends on

whether it has a receptor for that signal

Each receptor is usually activated by

only one type of signal

Two types of extracellular signaling molecules: (depending on the type of receptor they interact with)

1. molecules that are too large or too hydrophilic to cross the membrane of the target cell
- these signal molecules rely on receptors on the surface of the target cell to relay their message across the membrane
2. molecules that are small enough or hydrophobic enough to cross the membrane and into the cytosol of the target cell, where they will bind to intracellular receptor proteins

Effector proteins

have a direct effect on the behavior of the target cell

Extracellular signal molecules bind either to

cell surface receptors or to intracellular enzymes or receptors

Different types of cells respond to the same signal in different ways

ex: acetylcholine binds to similar receptors on heart pacemaker cells, salivary gland cell, and skeletal muscle cell
evokes different responses in each cell type

A cell's response to a signal can be

fast or slow

Extracellular signals can act slowly or rapidly

changes in gene expression = slowly (cell growth and division)
changes in cell movement, secretion, or metabolism = quickly

An animal cell depends on multiple extracellular signals

signals to survive, grow and divide, differentiate, or die

Cell surface receptors relay extracellular signals via

intracellular signaling pathways

Majority of extracellular signal molecules are

proteins, peptides, or small hydrophilic molecules that bind to cell surface receptors

Transmembrane receptors detect a signal on the outside and

relay a message in a new form across the membrane into the interior of the cell

Many extracellular signals activate intracellular signaling pathways to change the behavior of the target cell

1. extracellular signal molecule
2. receptor protein
3. intracellular signaling molecules
4. effector proteins (ex metabolic enzymes, cytoskeletal proteins, transcription regulators)
5. target cell responses (ex altered metabolism, altered cell shape or movement, altered gene expression)

The components of intracellular signaling pathways perform one or more crucial functions:

1. they can relay the signal onward and thereby help spread it through the cell
2. they can amplify the signal received, making it stronger, so that a few extracellular signal molecules are enough to evoke a large intracellular response
3. they can detect signals from more than one intracellular signaling pathway and integrate them before relaying a signal onward
4. they can distribute the signal to more than one effector protein, creating branches in the information flow diagram and evoking a complex response
5. they can modulate the response to the signal by regulating the activity of components upstream in the signaling pathway = feedback

The activity of monomeric GTPases is controlled by two types of regulatory proteins

1. guanine nucleotide exchange factors (GEFs) promote the exchange of GDP for GTP = switches the protein on
2. GTPase activating proteins (GAPs) stimulate the hydrolysis of GTP to GDP = switches the protein off

Many switch proteins controlled by phosphorylation are often organized into

phosphorylation cascades
transmits signal onward and amplifies, distributes, and regulates

Two main types of protein kinases operate in intracellular signaling pathways:

serine/theronine kinasees - phosphorylates serines or threonines
tyrosine kinases - phosphorylate proteins on tyrosines

Other class of switch proteins

GTP-binding proteins
- toggle between active and inactive state depending on whether GTP or GDP is bound
- GTP hydrolyzying (GTPase) activity shuts themselves off by hydrolyzing their bound GTP to GDP

Two main types of GTP binding proteins participate in intracellular signaling:

1. large, trimeric GTP binding proteins (G proteins) - relay messages from G protein coupled receptors
2. small, monomeric GTPases - helps relay signals
- generally aided by two sets of regulatory proteins that help them bind and hydrolyze GTP: guanine nucleotide exchange factors (GEFs) active the switches by promoting the exchange of GDP to GTP
- GTPase activating proteins (GAPs) turn them off by promoting GTP hydrolysis

Feedback regulation can occur anywhere in the signaling pathway

can either boost or weaken response to the signal

In positive feedback

a component downstream in the pathway acts on an earlier component in the same pathway to enhance the response to the initial signal
positive feedback can generate all or none switch like responses

In negative feedback

a downstream component acts to inhibit an earlier component in the pathway to diminish the response to the initial signal
negative feedback can generate responses that oscillate on and off as the activities or concentrations of the inhibitory components rise and fall

Feedback regulation within an

intracellular signaling pathway can adjust the response to an extracellular signal
intracellular signaling proteins can relay, amplify, integrate, distribute, and modulate via feedback

Some intracellular signaling proteins act as

molecular switches

Molecular switches receive a signal that causes them to switch from an

inactive to an active state
once activated, these proteins can stimulate or suppress other proteins in the signaling pathway
stay in an active state until some other process switches them off again
(many intracellular signaling proteins act as molecular switches)

Two classes of switch proteins:

signaling by protein phosphorylation
signaling by GTP binding proteins
- a GTP binding protein is activated when it exchanges its bound GDP for GTP. The protein then switches itself off by hydrolyzing it's bound to GTP to GDP.
- the phosphate is added covalently via protein kinase, which transfers the terminal phosphate group from ATP to the signaling proteins

The biggest class of molecular switches consists of

proteins that are activated or inactivated by phosphorylation

Protein kinase covalently attaches a

phosphate group onto the switch protein and in the opposite direction a protein phosphatase takes the phosphate off again

Cell surface receptors fall into three main classes

ion channel coupled receptors
G protein coupled receptors
enzyme coupled receptors

Ion channel coupled receptors change

the permeability of the plasma membrane to select ions which alter the membrane potential and produce an electrical current

G protein coupled receptors activate

membrane bound trimeric GTP binding proteins, which then activate or inhibit an enzyme or an ion channel in the plasma membrane
initiates in intracellular signaling cascade

Enzyme coupled receptors either act as enzymes or are associated with enzymes inside the cell

when stimulated, the enzymes can activate a wide variety of intracellular signaling pathways

An ion channel coupled receptor opens in response to

binding an extra cellular signal molecule

When a G protein coupled receptor binds its extracellular signal molecule

the activated receptor signals to a trimeric G protein on the cytosolic side of the plasma membrane, which then turns on or off an enzyme in the same membrane

When an enzyme coupled receptor binds its enzyme or single molecule

an enzyme activity is switched on at the other end of the receptor inside the cell

Ion channel coupled receptors convert chemical signals into

electrical ones

Ion channel coupled receptors function in the simplest and most direct way

transduce chemical signal in the form of a pulse of secreted neurotransmitter molecules delivered to a target cell, directly into an electrical signal in the form of a change in voltage across the target cells plasma membrane
- when the neurotransmitter binds to ion channel coupled receptors on the surface of a target cell, the receptor alters its confirmation to open the channel in the target cell membrane

GPCRs form the

largest family of cells surface receptors
- these receptors mediate responses to an enormous diversity of extracellular signal molecules, including hormones, local mediators, and neurotransmitters

There are several different kinds of G proteins and each is specific for

a particular set of receptors in for a particular set of target enzymes or ion channels in the plasma membrane

GPCRs are composed of three protein subunits

alpha, beta, and gamma
two are tethered to the plasma membrane by short lipid tails

In the unstimulated state of GPCRs, the alpha subunit has

GDP bound to it and the G protein is idle

When an extracellular signal molecule binds to its receptor, (in GPCRs)

the altered receptor activates a G protein by causing the alpha subunit to decrease its affinity for GDP, which is exchanged for a molecule of GTP
- in some cases, this activation breaks up the G proteins subunits, so that the activated alpha subunit holding its GTP, detaches from the B-Y complex, which is also activated
- the two activated parts of the G protein can then interact directly with target proteins in the plasma membrane

The longer target proteins remain bound to an alpha subunit or a beta gamma complex,

the more prolonged the relayed signal will be

The amount of time the alpha subunit and beta gamma complex remain switched on also determines

how long a response lasts
- this timing is controlled by the behavior of the alpha subunit
- the other subunit has an intrinsic GTPase activity, and it eventually hydrolyzes its bound GTP to GDP, returning the whole G protein to its original inactive confirmation
- the inactive G protein is now ready to be reactivated by another activated receptor

An activated GPCR activates G proteins by

encouraging the alpha subunit to expel its GDP and pick up GTP

Binding of an extracellular signal molecule to the receptor changes (in GPCRs)

the confirmation of the receptor, which alters the confirmation of the boundary protein

The alteration of the other subunit of the G protein allows it to exchange its GDP for GTP

this exchange triggers in additional conformational change that activates both the alpha subunit and a beta gamma complex, which dissociate to interact with their preferred target proteins in the plasma membrane
- the receptor stays active as long as the external signal molecule is bound to it, and it can therefore activate many molecules of G protein

Both the alpha and gamma subunits of the G protein have covalently attached

lipid molecules that help anchor the subunits to the plasma membrane

The G proteins of a subunit switches itself off by

hydrolyzing its bound GTP to GDP

When an activated alpha subunit interacts with its target proteins, it activates

that target protein for as long as the to remain in contact
- the alpha subunit then hydrolyzes its bound GTP to GDP
- the hydrolysis of GTP inactivates the alpha subunit, which dissociates from its target protein and re-associates with a beta gamma complex to reform an in active G protein
- both the activated alpha subunit and activated beta gamma complex can interact with target proteins in the plasma membrane

Some G proteins directly regulate

ion channels

A Gi protein directly couples receptor activation to the opening of K+ channels in

the plasma membrane of heart pacemaker cells
- binding of the neurotransmitter acetylcholine to its G protein coupled receptor on the heart cells results in the activation of the G protein
- activated B-Y complex directly opens a K+ channel in the plasma membrane, increasing its permeability to K+ and making the membrane harder to activate and slowing the heart rate
- inactivation of the alpha subunit by hydrolysis of bound GTP returns the G protein to its inactive state, allowing the K+ channel to close

Enzymes activated by G proteins increase the concentrations of

small intracellular signaling molecules
- the signal is greatly amplified at this step in the pathway because each activated enzyme generates many molecules of second messengers

Cholera - caused by a protein called cholera toxin

- the protein enters the cells that line the intestine and modifies the alpha subunit of a G protein Gs
- the modification prevents Gs from hydrolyzing its bound GTP and locks the G protein in the active state, continuously stimulating adenylyl cyclase
- this constant stimulation causes a prolonged and excessive outflow of Cl- and water into the gut, resulting in catastrophic diarrhea and dehydration

Gs stimulates the enzyme

adenylyl cyclase

Gi inhibits

adenylyl cyclase

Whooping cough - caused by protein called pertussis toxin

protein alters the alpha subunit of a G protein Gi
- toxin disables the G protein by locking it in its inactive GDP bound state
- results in prolonged activation of adenylyl cyclase
stimulates coughing

When G proteins interact with ion channels they cause

an immediate change in the state and behavior of the cell

The interaction of activated G proteins with enzymes produces interactions that are

less rapid and more complex, as they lead to the production of additional intracellular signaling molecules
the small molecules generated by these enzymes are often called second messengers
(once activated, the enzymes generate large quantities of second messengers, which rapidly diffuse away from their source, thereby amplifying and spreading the intracellular signal)

The two most frequent target enzymes for G proteins are

adenylyl cyclase - produces cyclic AMP
phospholipase C - generates small molecules called inositol triphosphate (IP3) and diacylglycerol (DAG)

IP3 promotes the accumulation of

cytosolic Ca2+

Cyclic AMP is synthesized by

adenylyl cyclase
degraded by cAMP phosphodiesterase

Many extracellular signals acting via GPCRs affect the activity of the enzyme adenylyl cyclase and thus alter the

intracellular concentration of the second messenger molecule cAMP

The activated G protein alpha subunit switches on the adenylyl cyclase, causing a dramatic and sudden increase in the synthesis of

cAMP from ATP
- to help terminate the signal, a second enzyme called cyclic AMP phosphodiesterase rapidly converts cAMP to ordinary AMP

A nerve cell in culture responds to the binding of the neurotransmitter serotonin to a GPCR by synthesizing

cyclic AMP

Cyclic AMP phosphodiesterase is continuously active inside the cell

Because it eliminates cAMP so quickly, the cytosolic concentration of this messenger can change rapidly in response to extracellular signals

Cyclic AMP exerts most of its effects by activating the enzyme

cyclic AMP dependent protein kinase
- the binding of cyclic AMP to the regulatory proteins forces a conformational change that releases the inhibition and unleashes the active kinase
- activated PKA then catalyze the phosphorylation of particular serines or threonines on specific intracellular proteins, thus altering the activity of these target proteins

In skeletal muscle, epinephrine increases intracellular

cyclic AMP, causing the breakdown of glycogen
- It does this by activating PKA, which leads to the to both the activation of an enzyme that promotes glycogen breakdown and the inhibition of an enzyme that drives glycogen synthesis
- By stimulating glycogen breakdown and inhibiting its synthesis, the increase in cyclic AMP maximizes the amount of glucose available as fuel for muscle activity

The effects of increasing cyclic AMP are rapid in some cases, and in other cases cyclic AMP responses involve changes in gene expression that are slower

In these slow responses, PKA typically phosphorylates transcription regulators

Epinephrine stimulates glycogen breakdown in skeletal muscle cells

- the hormone activates a GPCR, which turns on a G protein Gs that activates adenylyl cyclase to boost the production of cAMP
- the increase in cAMP activates PKA, which phosphorylates and activates an enzyme called phosphorylase kinase
- this kinase activates glycogen phosphorylase, which breaks down glycogen

A rise in intracellular cyclic AMP can activate

gene transcription
PKA, activated by a rise in intracellular cyclic AMP, can enter the nucleus and phosphorylate specific transcription regulators
- once phosphorylated, these proteins stimulate the transcription of a whole set of target genes
- activated PKA can also phosphorylate and regulate other proteins and enzymes in the cytosol

The inositol phospholipid pathway triggers a rise in intracellular Ca2+

GPCR activates Gq protein
Gq activates phospholipase C
phospholipase C activates IP3 and DAG

some GPCRs exert their effects through a G protein called Gq, which activates

the membrane bound enzyme phospholipase C
once activated, phospholipase C propagates the signal by cleaving a lipid molecule that is a component of the plasma membrane (inositol phospholipid)

The cleavage of a membrane inositol phospholipid by phospholipase C generates two second messenger molecules:

inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG)

IP3 is released into the cytosol

there it binds to opened calcium channels that are embedded in the ER membrane
calcium stored inside the ER rushes out into the cytosol through these open channels, causing a sharp rise in the cytosol and concentration of free calcium, which is kept very low

Diacylglycerol is a lipid that remains embedded in the plasma membrane after it is produced by

phospholipase C
- It helps recruit and activate a protein kinase, which translocates from the cytosol to the plasma membrane
- This enzyme is called protein kinase C Because it also needs to bind calcium to become active

Phospholipase C activates two signaling pathways

Two messenger molecules are produced when a membrane inositol phospholipid is hydrolyzed by activated phospholipase C
- IP3 diffuses through the cytosol and triggers the release of calcium from the ER by binding to an opening special calcium channels in the ER membrane
- Diacylglycerol remains in the plasma membrane and together with calcium helps activate the enzyme protein kinase C, which is recruited from the side result of the cytosolic face of the plasma membrane
- Phospholipase C then phosphorylates its own set of intracellular proteins
- At the start of the pathway, both the alpha subunit and the beta gamma complex of the G protein Gq are involved in activating phospholipase C

Protein kinase C must bind to

Ca2+ to become active

Fertilization of an egg by sperm triggers an increase in

cytosolic calcium in the egg
- when a sperm enters the egg, a wave of cytosolic calcium released from the ER sweeps across the egg from the site of sperm entry
- its calcium wave provokes a change in the egg surface, preventing entry of other sperm and it also initiates embryonic development

Calmodulin

calcium binding protein
when Ca2+ binds to calmodulin, the protein undergoes a conformational change that enables it to interact with a wide range of target proteins in the cell

Ca2+/calmodulin-dependent protein kinases (CaM-kinases)

when these kinases are activated by binding to calmodulin complexed with Ca2+, they influence other processes in the cell by phosphorylating selected proteins

Calcium binding changes the shape of the calmodulin protein

each of the calmodulin ends have two Ca binding sites

cAMP and calcium - hydrophilic molecules; generally act within

the cell they are produced

Nitric Oxide (NO)

signaling molecule, gas
diffuses readily
triggers smooth muscle relaxation in blood vessel wall

Nitric Oxide triggers

smooth muscle relaxation in blood vessel wall
- acetylcholine causes the blood vessel to dilate by binding to a GPCR on the surface of the endothelial cells, thereby inactivating a G protein (Gq) to trigger Ca2+ release
- Ca2+ activated NO synthase, stimulating the production of NO
- NO then diffuses out the endothelial cells and into adjacent smooth muscle cells, where it regulates the activity of specific proteins, causing the muscle cells to relax
- one target protein that can be activated by NO in smooth muscle cells is guanylyl cyclase - catalyzes the production of cyclic GMP from GTP

Endothelial cells release NO in response to

acetylcholine secreted by nearby nerve endings

Acetylcholine binds to a GPCR on the endothelial cell surface

- activates Gq and then releases Ca2+ inside the cell
- Ca2+ then stimulates nitric oxide synthase, which produces NO from the amino acid arginine
- NO diffuses into smooth muscle cells in the adjacent vessel wall, causing the cells to relax and allows vessel to dilate so that blood flows more freely

Inside many target cells, NO binds to and activates the enzyme

guanylyl cyclase, stimulating the formation of cyclic GMP from GTP
cyclic GMP = second messenger, key link to NO signaling chain

In a photoreceptor cell, light is sensed by

rhodopsin - G protein coupled receptor
- when stimulated by light, rhodopsin activates a G protein - transducin
- activated alpha subunit of transducin then activates an intracellular signaling cascade that causes cation channels to close in the plasma membrane of the photoreceptor cell
- this produces a change in voltage across the membrane, which alters neurotransmitter release and leads to a nerve impulse that is sent to the brain
- This signal is repeatedly amplified as it is relayed along this intracellular signaling pathway

Bright vs dim lighting conditions

When lighting conditions are dim, the amplification is enormous
In bright sunlight, the signaling cascade undergoes a form of adaptation, stepping down the amplification so that the photoreceptor cells are not overwhelmed and can still register increases/decreases in strong light
- The adaptation depends on feedback: an intense response in the photoreceptor cell decreases the cytosolic Ca2+ concentration, inhibiting the enzymes responsible for signal amplification

Adaptation frequently occurs in intracellular signaling pathways that respond to extracellular signal molecules, allowing cells to respond to fluctuations in the concentration of such molecules... takes advantage of

positive/negative feedback, allowing cells to respond equally well to contrasting signals

A rod photoreceptor cell from the retina is exquisitely sensitive to light

- when the rod cell is stimulated by light, a signal is relayed from the rhodopsin molecules in the discs, through the cytosol, to ion channels that allow positive ions to flow through the plasma membrane of the outer segment
- cation channels close in response to the cytosolic signal, producing a change in the membrane potential of the rod cell