T5: Cell signaling and communication

Question 1

4 types of signalling

Answer 1

1. endocrine: release signals into bloodstream (hormones)
2. paracrine: molecule released in intercellular space (now called local mediator) as the 2 cells are relatively close
3. neuronal: neurotransmitter released from presynaptic neuron into synaptic cleft
4. contact dependent: signal molecule is a membrane bound molecule because the two cells are in contact

Question 2

general pattern of signal transduction

Answer 2

SRIER:
1. Stimulus
2. receptor
3. intracellular signaling proteins
4. effector proteins
5. response

Question 3

slow vs rapid responses used in cell signaling

Answer 3

FAST RESPONSE: modulates proteins already present in the cell cytoplasm

SLOW RESPONSE: changes the protein synthesis and regulation of gene expression

Both these responses alter cell cytoplasmic machinery which then changes cell response

Question 4

General roles of ACh (3)

Answer 4

Muscarinic receptor: G protein
-cardiomyocytes: decreases contraction force and frequency
-salivary glands: induces release of saliva

Nicotinic receptor: Ion channel
-skeletal muscle: opening of Ca channels to increase contraction

Question 5

types of messenger molecules

Answer 5

1. amino acids/derivatives
2. steroids
3. gases
4. eicosanoids
5. polypeptides/proteins

Question 6

Action of lipophilic receptors

Answer 6

CYTOPLASMIC:
-found in their inactive form when there is 0 stimulation
-when the signaling molecule is bound to the ligand domain, the inhibitory protein is detached from the complex
-this allows translocation of the mediator receptor complex from the cytoplasm to nucleus
-binding to the DNA domain in nucleus (promotor region of target genes)
-change in expression (slow response)

Question 7

role of NO in vasodilation

Answer 7

NO is a small molecule that can diffuse through the plasma membrane, hence follows the lipophilic mechanism of action
-ACh binds to muscarinic receptor on epithelial cell
-This stimulates synthesis of NO from arginine
-NO diffuses out of epithelial cells and into smooth muscle cells
-Binds to the enzyme guanylyl cyclase which produces cGMP from GTP
-this induces the relaxation of the smooth muscle which causes vasodilation

Question 8

Molecular switches: ways of activating molecules (2)

Answer 8

1. Phosphorylation activates the substrate (done by protein kinases). Dephosphorylation switches the molecule off (done by protein phosphatases)
2. GDP/GTP switching:
   -GDP bound molecules are inactive.
   -GDP detaches and swaps to GTP bound to activate.
   -GTP is hydrolyzed by the molecule and is inactivated again.

Question 9

Classes of membrane receptors (3) and description

Answer 9

1. Ion channels: binding of signal molecule induces a conformational change which opens the channel and allows ions to move according to their electrochemical gradient.
2. G-protein coupled receptors (GPCR): Interact with trimeric G protein complexes to activate downstream action of molecules
3. Enzyme-coupled receptors (ECR): activated upon binding to signal molecule due to enzymatic activity in their intracellular portion. Amino acid residues are phosphorylated to cause activation of downstream signaling molecules

Question 10

Structure of GPCRs

Answer 10

-7 transmembrane domains
-Amino terminal faces the extracellular space and contains ligand binding site
-Carboxyl terminal faces the cytoplasm and interacts with trimetric G protein complex
-intracellular loops joining the 5th and 6th transmembrane domains contain amino acids responsible for the interaction with trimeric G protein complexes

Question 11

General activation mechanism for GPCR and G protein complex

Answer 11

AT REST: Both GPCR and G complex is inactive (containing 3 subunits: a,b,c joined together)

ACTIVATION:
1. signal binds to ligand domain of GPCR - activates receptor
2. Receptor interacts with trimeric G protein complex
3. GDP bound to the a subunit is replaced to GTP and the trimeric G protein is activated
4. activation causes the a subunit to dissociate from the b/c subunits
5. Induces downstream signaling effects depending on the specific G protein complex used

!!ACTION CAN BE EITHER A OR B/C MEDIATED

6. When the signal needs to be stopped, enzymes are desensitized either by kinases of GPCR or b-arrestins

Question 12

G proteins family one

Answer 12

1. Gs A: activates adenylyl cyclase and activates Ca channels
2. Golf A: activates adenylyl cyclase in olfactory sensory neurones

Question 13

G proteins family two

Answer 13

1. Gi (inhibitory) A: inhibits adenylyl cyclase
   Gi (B/C): activates K channels
2. Go (B/C): activates K channels and inactivates Ca channels
   Go (A and B/C) : activates PLC-b
3. Gt (transducin) A: activates GMP phosphodiesterase in rods

Question 14

G proteins family three

Answer 14

Gq (A): activates PLC-b

Question 15

G proteins family four

Answer 15

G12/13 (A): activates Rho family monomeric GTPases to regulate cytoskeleton

Question 16

3 examples of GPCR pathways

Answer 16

1. Activation of K+ channels in myocytes, B/C mediated:
   -ACh binds to GPCR
   -subunit dissociation, GDP - GTP
   -B/C subunit interaction with K+ channel to open it
   -K+ ions move out of the cell (higher to lower conc.)
   -hyperpolarization of the membrane causes reduction in frequency and power of contractions
2. Activation of adenylyl cyclase and then PKA, A mediated:
   -substrate binds to GPCR
   -subunit dissociation, GDP - GTP
   -A subunit interaction to activate adenylyl cyclase
   -ATP –> cAMP
   -cAMp activates PKA (protein kinase)
   -PKA travels to nucleus and phosphorylates CREB (a TF), activating it
   -Interaction with CRE on promotor area of specific genes
   -change in gene expression
   !!! in most cases cAMP exerts its effects by activating PKA
3. Activation of PLC to activate PKC, Gq A mediated:
   - -substrate binds to GPCR
   -subunit dissociation, GDP - GTP
   -A subunit interaction to activate PLC
   -cleaves covalent bonds of PI(4,5)P2
   -causes an increase in DAG concentration which is responsible for bringing PKC close to the membrane
   -also causes increase in IP3 concentration which binds to a receptor on the SER, opens Ca2+ channels so they flow our of the SER and into cytoplasm and activates CA2+ dependent protein kinase (CDPK)
   -CDPK activates PKC that was brought close to the membrane

Question 17

Process of camp formation

Answer 17

1. ATP changed into cAMP by adenylyl cyclase (loss of 2 phosphate groups
2. cAMP changed into AMP by cAMP phosphodiesterase (loss of 1 molecule of water)

Question 18

General structure of enzyme activated receptors (ECRs)

Answer 18

-transmembrane receptors
-N (amino) terminal faces the extracellular space, and C (carboxy) terminal faces the cytoplasm
-Extracellular domain is the ligand binding site
-Intracellular domain: long cytosolic tail containing amino acid residues (eg. tyrosine)

Question 19

2 classes of enzyme activated receptors

Answer 19

1.Tyrosine receptor kinases (TRKs)

2.Serine-theronine kinase receptors (STKRs)

!!mechanism of activation is the same for both receptor classes

Question 20

General structure of TRKs

Answer 20

same main structure as the enzyme activated receptors
EXTRA DETAILS:
-extracellular domains are very different whereas the cytosolic portions are pretty similar between all types of TRKs
-contain Tyr residues on the C terminals of the protein

Question 21

General process of activation of TRKs

Answer 21

1. binding of the receptor and the signal molecule
2. dimerisation of the receptor
3. TRK domain activation via trans and auto phosphorylation
4. anchorage of adaptor proteins needed for downstream action

Question 22

RAS cascade process

Answer 22

!! RAS is a molecular switch which becomes a kinase when activated

1. inactive TRK is activated with the binding of the signaling molecule (phosphorylation)
2. anchorage of adaptor protein brings the RAS activator protein close to the cell membrane
3. Activator proteins activates RAS (Switches GDP to GTP)
4. active RAS (now a kinase) phosphorylates MAP3k (mitogen activated kkk) and activates it
5. Causes a MAPkinase cascade which induces downstream signaling effect (Raf –> Mek –> Erk)
6. When Erk is activated it activates TFs and alters proteins already in the cell
7. causes change in cytosolic machinery AND change in gene expression due to TFs

Question 23

MAP kinases

Answer 23

Serine-threonine kinases that cause a downstream signling cascade.
The activation of membrane receptors is responsible for the activation of MAP3kinases
MAP3k –> MAP2k –> MAPk which then induces cellular responses

!!! Different initial membrane receptors trigger different MAP3ks which causes different cascades which causes different cellular responses

Question 24

LIF pathway in stem cells

Answer 24

-Leukemia inhibitory factor
-happen only in mES
LIFR= tyrosine kinase receptor!

1. LIF binds to LIFR receptor (which is heterodimeric and made up of gp130 and LIFR component)
2. Activated receptor phosphorylates JAK protein (kinase) which in turn phosphorylates STAT1/3
3. STAT1/3 passes into the nucleus and activates certain TFs
4. Increase expression in pluripotency marker genes: Nanog, Oct3/4, Myc,Klf4

Question 25

WNT cascade in hES

Answer 25

-hES is LIF dependent -WNT signaling molecule is needed instead, which binds onto the receptor 'frizzeld'. Genes that are affected due to the changing of TFs are: SOX2/OCT4

Question 26

PI3 kinase cascade mechanism

Answer 26

1. TRK dimerization upon activation (auto phosphorylated) 2. PI3 kinase enzyme is activated and anchored to the cytosolic tail of the TRK 3. PI3 activates inositol phospholipid by phosphorylating it (becoming PIP3) 4. PIP3 is used as an anchor molecule for different signaling molecules (usually kinases) 5. allows relaying of signals

Question 27

collaboration between TRKs and GPCRs

Answer 27

-in most cases the mechanisms of actions of the two receptor types run in parallel -activation of downstream signaling molecules depend on substances activated by the two receptors in diff. parts of their cascades !! activation of TRKs and GPCRs hugely increases kinase cell activity, and PLC activity. This has 2 major effects: 1. activation of TFs 2. Modulation of intracellular target proteins

Question 28

Ion channel receptors def.

Answer 28

Multimeric proteins that span across the plasma membrane. They are involved in the transmission of fast signals

Question 29

Structure of the nicotinic Ach receptor

Answer 29

-Pentamer, composed of 5 subunits: 2a, b, c, d. -Pore is formed in the center of the complex through which ions are allowed to pass -4 transmembrane domains -binding site of ACh is on the interface between a subunit and the adjacent subunit, so since there are 2a subunits, each receptor is able to bind to 2ACh

Question 30

Types of ion channel protein receptors (3)

Answer 30

1. ACH, GABA, Glycine: -pentamers (5 subunits) -N and C terminals both facing extracellular space -4 transmembrane domains -2 intracellular loops 1. Glutamate (AMPA, NMDA, Kainite): -tetramers (4 subunits) -N terminal facing extracellular space, C terminal facing cytosol -3 transmembrane domains -M2 hairpin segment which only partially goes into membrane 1. ATP receptors: -trimers (3 subunits) -N and C terminals both facing cytosol -2 transmembrane domains -1 extracellular loop which acts as ligand binding site

Question 31

Cation vs anion transport in ion channel receptors

Answer 31

1. CATION transport: Excitatory and induce depolarization 2. ANION transport: Inhibitory and induce hyperpolarization

Question 32

Control of calcium as a ubiquitous intracellular messenger

Answer 32

-Ca2+ is a secondary messenger that triggers certain cell functions (eg. secretion/ contraction of muscles/ excitability of neurons). -Ca2+ needs to be kept in tight control in cells -Different cells have different concs. of Ca2+ -Ca2+ can determine if a cell is controlled in a synchronous or asynchronous way -Kept in control using membrane ion channels (ligand and voltage gated) or exchange pumps with ATP

Question 33

Ca2+ activated mammalian proteins

Answer 33

1. Troponin C: modulation of muscle contraction 2. Calmodulin: Enzyme modulation (activates CAMkinase) 3. PLC: IP3 and DAG generation 4. IP3 receptor: Ca2+ released from intracellular stores in SER 5. Ryanodin receptor: Ca2+ released from intracellular stores in SER

Question 34

Importance of calcium signaling in neurons (2)

Answer 34

1. Ca2+ in neurons stimulates the fusion of vesicles containing neurotransmitters in the presynaptic neuron, which are then released via exocytosis in the synaptic cleft. 2. Responsible in the post synaptic neuron for events happening in the CNS (long term potentiation and synapse plasticity)

Question 35

Long term potentiation definition

Answer 35

A process involving persistent strengthening of synapses that leads to a long-lasting increase in signal transmission between neurons. Depends on the recruitment of the NMDA glutamate receptor and on intracellular Ca2+ concentration in post synaptic terminal.

Question 36

Long term potentiation process

Answer 36

1. synaptic vesicles release neurotransmitters upon arrival of action potential 2. glutamate released in synaptic cleft which binds to AMPA type glutamate receptors on post synaptic neuron 3. Na+ influx from extracellular space into post synaptic neuron to depolarise and propagate action potential 4. IN INCREASED STIMULATION: increased amounts of neurotransmitter present in cleft. This also activates the NMDA receptor on post synaptic membrane (which under normal conditions is blocked by presence of Mg2+) 5.Opening of NMDA receptor makes membrane permeable to both Na+ AND Ca2+ ions 6. Ca2+ influx into post synaptic neuron (achieved by kinases like CaMkII) RESULT: induces recruitment of new receptors to post synaptic neuron: more receptors means a higher/stronger response

Question 37

Ways in which the relation of the signaling molecule and cell response may vary depending on the signaling pathway activated (8)

Answer 37

1. TIME TAKEN for response to occur: could be a few ms for a synapse, or hours/days in the case of TRKs/GPCRs) 2. SENSITIVITY: action of hormones is triggered at low concs. (usually due to amplification of signal by secondary messenger) whereas neurotransmitters are needed at high concs. to propagate impulse 3. DYNAMIC RANGE: response in low mediator conc. kinetics (on/off simple decisions ) vs responses to large interval of signal concs. (complex metabolic responses) IN OTHER WORDS: values of conc. that trigger responses can be either binary for yes/no decisions or have a longer scale of values for diff responses with smaller increments as complexity increases. 4. PERSISTENCE: how long signals last: transient, persistent and permanent responses (in regards to how long they last) 5. SIGNAL PROCESSING: a signal molecule may either be able to trigger one sole response or a complex cascade 6. INTEGRATION: every cell response can be controlled by more than one input 7. COORDINATION: multiple responses can be triggered by a single input molecule 8. FEEDBACK: positive feedback when A stimulates the production of B which in turn stimulates A . Negative feedback when A stimulates B but B then inhibits A production

Question 38

Ways in which cells can modulate their sensitivity to an extracellular signaling molecule (5)

Answer 38

ALL WORK WITH NEGATIVE FEEDBACK FOR DESENSITISATION: 1. RECEPTOR SEQUESTRATION: upon binding of signal to the receptor, the receptor is internalized in an endosomal compartment which stores it, keeping it removed from the plasma membrane until the next event of exposure. 2. RECEPTOR DOWN REGULATION: Receptor is sent to the lysosome for degradation and is not recycled back to the plasma membrane. Less receptors present on membrane means less signals 3. RECEPTOR INACTIVATION: receptor is inactivated by a protein complex, blocking its signaling activity. (eg. b-arrestins used for the desensitisation of GPCRs) 4. INACTIVATION OF SIGNALLING PROTEINS: receptor itself is still functional but a molecule further down the signaling pathway is blocked instead 5. PRODUCTION OF INHIBITORY PROTEIN: through the signaling of the receptor, the synthesis of an inhibitory protein is triggered which blocks further signaling