Lecture 22: Cell Signaling

Question 1

What is the importance of signaling

Answer 1

Living organisms constantly receive and interpret signals from their
environment.

Question 2

Signal transduction

Answer 2

is the process of converting external signals into a cellular response through (often) transmembrane receptors

Question 3

What does signal transduction allow for?

Answer 3

allows for the alteration of gene expression and protein activity in response to environmental signals.
• It allows for communication between
the different cells of an organism
• It allows for cells to adapt to their
environment

Question 4

• Cells of multi-cellular organisms….

Answer 4

receive signals from other cells,
including signals for cell division and differentiation. Most cells in our
bodies must constantly receive signals that keep them alive and
functioning.

Question 5

Protein kinases

Answer 5

transfer a phosphate from ATP to a protein.
This usually activates the protein

can act as cascades, where
one type of kinase activates the
next ‘step in the cascade.

Question 6

Protein phosphatases

Answer 6

remove the phosphate, reversing the ‘switch’.

- inactivating

Question 7

Approx_________of all proteins are regulated by ___________!

Answer 7

Approx 50% of all proteins are regulated by phosphorylation!

Question 8

Phosphorylase

Answer 8

enzyme adds a P to a substrate using inorganic phosphate (not ATP)

Answer 9

These can target ion channels,
transcription factors and other
regulatory proteins. In the case of
signal transduction cascades, the
substrate for the phosphorylases
and kinases are often also
phosphorylases and kinases. This
causes a chain reaction that
ultimately leads to a cellular
response.

Question 10

Give examples of post-translational

modification (PMT) of proteins

Answer 10

phosphorylation, ubituitinylation, and acylation as
covalent attachments to proteins. also cleaving off proteins
- attaching some molecule covalently

Question 11

Phosphorylation can affect proteins in different ways:

Answer 11

– Activate or inactivate an enzyme (or other protein function)

– Target protein for degradation (via initiating ubiquitinylization)

– Allow movement from one cellular compartment to another

– Increase or decrease protein-protein interactions

Question 12

Phosphoproteomics

Answer 12

new type of proteomics that quantifies not only all the proteins in a cell,
but which proteins are phosphorylated, and at what amino acid

Question 13

autocrine

Answer 13

the cell has receptors on its surface
that respond to an extracellular messenger it releases.

cell releases molecules and it is bound back to the same cell

Question 14

paracrine signaling

Answer 14

the extracellular messenger travel
short distances to nearby cells through the extracellular
space (example: nerve cell releasing acetylcholine to
trigger muscle contraction).

Question 15

endocrine

Answer 15

extracellular messengers (i.e.
hormones) can travel long distances through the
bloodstream and target distal cells.

Question 16

Explain the general idea of cell signalling

Answer 16

1. A ligand binds to a receptor on the PM. This
   causes a conformational change in the receptor
   on the cytosolic side (a process called signal
   transduction).
   receptor
2. This triggers a cascade of effects in the
   cytosol. These cascades amplify the signal
   inside the cell.
3. The final ‘layer’ of the proteins in the
   cascade trigger effects in the cell. These
   can include alteration of transcription of
   genes, or changes in the activity/function
   of proteins.

Gene transcription
Protein activity changes
(enzymes, cytoskeletal,
Ion channels, etc.)

Question 17

Why and how are signals amplified?

Answer 17

Amplification of the signal permits
- the initial signaling molecule
(hormone) to be in limited
concentrations and still be effective
– one hormone to activate numerous
enzymes
• e.g. each protein kinase (a second
messenger) can activate several
MORE kinase molecules.
– coordination of several different
pathways simultaneously, as all are
induced by a single signa

Question 18

Describe the two main ways signaling (in general) works

Answer 18

A:

Extracellular signaling molecule (1st messenger)

The activation of the receptor activates an
effector protein (4).

The effector makes a soluble second
messenger (5) which diffuses into the cell.

The second messenger triggers the signal
cascade leading to cell effects.

Our example: glucagon
signaling

B: The activation of the receptor forms a
‘recruiting station’ that in turn recruits other
proteins.

These proteins trigger the signal cascade.
Our example: MAP
kinase cascade

Question 19

Ligand

Answer 19

any molecule that binds to a receptor that triggers signaling

Question 20

Hydrophobic ligands

Answer 20

• Often made from cholesterol
• can cross the cell membrane
• Thus, the receptors are inside the cell
• We`ve already seen an example of this
(PEPCK activation by glucocorticoid in
the transcription factor lecture)• Often made from cholesterol
• can cross the cell membrane

Question 21

Hydrophilic ligands

Answer 21

Many types (proteins, peptides, amino
acids, small molecules,
• Can`t cross the cell membrane
• Bind to integral membrane receptors

Question 22

Cyclic AMP (cAMP)

Answer 22

Second messenger

Made by the effector adenylyl cyclase

cAMP can readily diffuse into the
cytosol and trigger downstream
effects

Question 23

adenylyl cyclase

Answer 23

effector makes cAMP
is an integral membrane
protein

Question 24

Inositol phosphates and diacylglycerol (DAG)

Answer 24

Derived from phosphatidylinositol
• The inositol portion can be phosphorylated by kinases
• Seven different possible P patterns

PIP2 is the substrate for the effector

• The main lipid second messengers are produced by the
effector phosphatidylinositol-specific phospholipase C.
– abbreviated PI-PLC
– produces two signal molecules: • Diacylglycerol (DAG) Inositol triphosphate (IP3),

Question 25

phosphatidylinositol-specific phospholipase C.

Answer 25

effector produces two signal molecules: Diacylglycerol (DAG), Inositol triphosphate (IP3), cuts the PIP2 in 1/2

Question 26

• Diacylglycerol (DAG),

Answer 26

2nd messanger | which stays within membrane

Question 27

Inositol triphosphate (IP3)

Answer 27

highly soluble, enters | cytoplasm

Question 28

G protein

Answer 28

G-proteins are inactive when bound to GDP. The GEF enzyme swaps the GDP for a GTP, activating the G-protein.

Question 29

What are the 2 ways s G-proteins switch ‘off’

Answer 29

They have a slow intrinsic GTPase activity and will self-hydrolyze the GTP to GDP. If the proper GAP is present, the GAP will greatly speed up the process. So, G-proteins are more like ‘timers’ than ‘switches’.

Question 30

Why are G proteins more | like ‘timers’ than ‘switches’

Answer 30

B/c eventually they will all shut off on their own b/c of GTPase activity; can be sped up by GAPS

Question 31

Describe a G-Protein Couple Receptor’s structure and function

Answer 31

A family of integral proteins, all have seven transmembrane a-helix segments • All work in the same way – through the heterotrimeric Gproteins with α, β, γ subunits * Ga and Gy are lipid anchored ``` – Which then turn on an effector molecule which makes the second messenger • e.g. epinephrine and glucagon turn on adenylyl cyclase to make cAMP (2nd messenger). • Other ligands (e.g. acetylcholine also activates GPCRs) use phosphoinositol and DAG second messengers • others (e.g. photoreceptors) use cyclic GMP ```

Question 32

Describe how GPCRs induce G-protein and second messengers

Answer 32

1. When receptor combines with ligand, receptor changes shape and binds the a subunit of the G protein ``` 2. Activation of the G protein: a subunit then exchanges a GDP for a GTP, entering activated state - the receptor/ligand can activate several G proteins, as long as ligand is bound ``` ``` 3. Relay: the a subunit dissociates from b,g and associates with effector, producing second message - b,g stay together - second message is made for duration of binding ``` 4. Activated effector produces second messenger (eg. adenylyl cyclase makes cyclic AMP = cAMP) 5. a subunit hydrolyzes GTP into GDP, thereby deactivating itself 6. a subunit binds other two subunits - now inactive

Question 33

How does GPCR-mediated signaling stop?

Answer 33

``` To prevent overstimulation, activated receptors can be blocked from interacting with G-proteins 7. G-protein-coupled receptor kinase (GRK) phosphorylates receptor ``` 8. Arrestin protein binds to phosphorylated receptor to prevent G-proteins from binding – “desensitization”

Question 34

– Gsα

Answer 34

stimulates adenylyl cyclase

Question 35

– Gqa

Answer 35

activates phospholipase C

Question 36

Gia

Answer 36

inactivates adenylyl cyclase

Question 37

Provide an example of a disease state brought about by G-protein mis-regulation:

Answer 37

Cholera toxin specifically binds Gsα in intestinal epithelial cells Locks it into the active state by adding an ADP-ribose to the G-protein Adenylyl cyclase remains active all the time and makes gobs of cAMP cAMP induces chloride channels to always remain open, and water follows by osmosis. Severe dehydration can be fatal

Question 38

Utilization of glucose:

Answer 38

– primary energy source (of course) – stored as the polymer glycogen in liver and muscle – glycogen conversion to glucose is promoted by hormones:

Question 39

glucagon

Answer 39

(released from pancreas), boosts | blood glucose when blood glucose drops

Question 40

epinephrine

Answer 40

(adrenal gland), boosts blood | glucose during stress

Question 41

To get more glucose into the bloodstream:

Answer 41

Promote breakdown of glycogen to glucose-1- phosphate (first step in catabolism). This glucose is either catabolized or sent to bloodstream for delivery to other places. Inhibit glycogen synthase: this enzyme makes glycogen, so it has to be turned off in order for the cells to release or burn glucose. Promote gluconeogenesis to make glucose from smaller molecules

Question 42

In liver cells, glucagon and epinephrine (adrenaline).....

Answer 42

In liver cells, glucagon and epinephrine (adrenaline) bind to different GPCRs, but the GPCRs then both activate Gsα which activates adenylyl cyclase

Question 43

Outline the signaling pathway following glucagon reception that results in glucose production

Answer 43

1. Hormone binds to receptor which binds to G-protein 2. Activation of effector: Adenylyl cyclase, formation of cAMP, diffuses into cytoplasm ``` 3. cAMP then binds to Protein kinase A (PKA) & activates it ``` 4. PKA phosphorylates glycogen synthase, inactivating it. Glycogen no longer produced 5. At same time, PKA phosphorylates the enzyme Phosphorylase kinase, activating it 6. the phosphorylase kinase then phosphorylates its target enzyme: Glycogen phosphorylase, activating it,' 7. phosphorylase catalyzes glycogen break-down, glucose-1-phosphate is released ``` 8. also at same time, in the nucleus, PKA phosphorylates transcription factor: cyclic AMP response element binding protein (CREB) ``` ``` 9. phosphorylated CREB dimerizes and binds to cAMP response element (CRE), turning on PEPCK gene, gluconeogenesis increases to produce even more glucose ```

Question 44

Outline how the cell terminates all components of glucagon-induced signaling

Answer 44

1. cAMP is broken down by phosphodiesterase ``` 2. Phosphatases reverse the phosphorylation of the three proteins: 1. phosphorylase kinase 2. glycogen synthase and 3. phosphorylase ``` 3. Adenylyl cyclase is deactivated when the α subunit of the G-protein hydrolyzes the GTP back to GDP (α subunit re-associates with γ and β) – this hydrolysis is a timed event - aided by another protein: RSG (regulator of G protein signaling)… a bit like GAP 4. inactivation of the receptor, a 2-step process of desensitization. This means that the cell stops responding, even when ligand is still present around the cell a. Phosphorylation of the receptor by G-protein receptor kinase (GPRK), inactivates the receptor b. The phosphorylated receptor binds another protein called arrestin, which acts as adaptor for clathrin, allowing receptors to be internalized by endocytosis, thus further desensitizing the cell

Question 45

Which is a faster way to yield glucose?

Answer 45

. The cytoplasmic conversion of glycogen to glucose

Question 46

cAMP then binds to Protein kinase A (PKA) & activates it – how?

Answer 46

``` PKA is a tetramer with 2 regulatory subunits and 2 catalytic subunits; cAMP removes the inhibitory regulatory subunits ```

Question 47

Describe how GPCRs are involved in light perception

Answer 47

Rhodopsin is one of a few GPCR in rod cells that make up your retina Retinal is a small molecule cofactor (made from vitamin A) that is bound to the receptor ``` An incoming photon is absorbed by the retinal, which causes the retinal to go from cis to trans conformation, which then causes the GPCR to change shape and start signal transduction inside of the cell. The signals initiate nerve signaling by opening ion channels, and these signals are (somehow!) interpreted by your brain as an image ```