Cell Communication (LE014 LE015)

Question 1

Differentiate between: Paracrine, Endocrine, and Autocrine signaling

Answer 1

Paracrine: cells in same location or tissue, does not enter the blood
Autocrine: a form of paracrine signaling in which signaling molecule affects the cell that produced it
Endocrine: cells in separate organs and tissues and requires the blood stream to transport the signaling molecule between organs.

Question 2

How do steroids act as signaling molecules?

Answer 2

Diffuse across plasma membrane - Steroids are hydrophobic molecules
Interact with receptors inside cell that will alter transcriptional activity of different genes.
Receptor will undergo conformational change upon binding
Inhibitory protein will be released (most times)
Receptor allowed to be imported to nucleus
Activate expression of early response genes – which will encode transcription factors that turn on expression of secondary response genes.
Secondary Response Genes will encode for proteins to generate changes in cellular behavior.

Question 3

What is the time scale for cellular change via Ligand Gated Ion Channels?

Answer 3

Usually very rapid

Note: this is the mechanism by which skeletal muscle is stimulated to contract.

Question 4

What does GEF stand for and what is it?

Answer 4

GEF: Guanine nucleotide Exchange Factor.
These catalyzes the process of G protein dissociating from ADP and binding ATP.

Note: G-protein, when bound to ADP, is in an inactive state. To activate it, you cannot simply phosphorylate the ADP. You must actually switch out the ADP for an ATP. This process is relatively slow, and is catalyzed by GEF. In many cases, the G-protein coupled receptor that spans the membrane 7 timesand has a GEF domain that catalyzes this process on its G-protein.

Question 5

What are two of the downstream effectors of heterotrimeric G-proteins? Which one affects Protein Kinase A? Protein Kinase C?

Answer 5

1) Adenylyl Cyclase (should think PKA)

Gs subunits activate adenylyl cyclase (on plasma membrane)
Converts ATP into cAMP 20-fold
Increased cAMP conc activates Protein Kinase A by causing its regulatory subunits to dissociate
PKA affect: metabolism by activating enzymes that break down glycogen, and alter gene expression by activating transcription factors.

2) Phospholipases (mostly phospholipase C) (should think PKC)

• Cleave a class of lipids called phosphatidylinositols (PIP)
• Generate IP3 (inositol 3-phosphate) and DAG (diacylglycerol). DAG still attached to cell membrane.
• IP3 bind to Ca channels in ER
• Ca will be released into cytoplasm
• Ca will cause many cellular events.
• Ca and CAG will activate Protein Kinase C
• DAG can also be converted to synthesize prostaglandins (mediators of inflammation and pain)

Question 6

When does phosphodiesterase hydrolyze cAMP?

Answer 6

Cells constantly express phosphodiesterase to hydrolyze cAMP and reduce the activity of Protein Kinase A. When adenylyl cyclase increases concentration of cAMP 20fold, rapidly, that’s when it overcomes phosphodiesterase’s hydrolyzing capacity.

Question 7

Which cell signaling molecule receptor spans the membrane only once? What is their mechanism?

Answer 7

Receptor Tyrosine Kinases only span the membrane once.

They contain a kinase domain on their cytoplasmic tail.
When bound to ligand, kinase domain on two receptors dimerize and phosphorylate each other. (this is why they are called RT Kinases)
Phosphorylated tails of receptors are recognized by number of different signaling proteins and their substrates.
Receptor Tyrosine Kinases will increase the signaling reactions by bringing these components together:
ie, phospholipases (cleaves to make IP3 and DAG, think about Ca2+ and PKC downstream)
ie, phosphatidylinositol kinases (which phosphorylate phosphatidylinositols to generate a patch of them on the inner leaflet of the plasma membrane

Question 8

How does cell prevent cross talk between MAP kinase pathways? Ie, not wanting MAP kinases of a given pathway from activating those in another pathway.

Answer 8

There are scaffolding proteins that MAP Kinases of the same pathway.

Question 9

What is Ras? Which cell signaling pathway commonly utilizes Ras?

Answer 9

Ras is a small GTP-binding protein.
Commonly:
Receptor Tyrosine Kinase recruits GEF for Ras
GEF catalyzes Ras to dissociate GDP and bind GTP and become activated
Activated Ras-GTP will activate MAP-KKK

Question 10

What are 3 ways in which cells terminate signaling events?

Answer 10

1) Receptor Mediated Endocytosis – utilizes clathrin-coated vesicles.
Vesicles fuse with low pH endosomes that usually dissociate the ligand from receptor.
Receptor can be recycled, or degraded in lysosome
2) Receptors can be inactivated
Modification (phosphorylation)
bounded by inhibitory proteins
3) Signaling molecule removed from outside cell

Question 11

What does the signaling molecule travel through for paracrine signaling? Endocrine signaling?

Answer 11

Paracrine – Extracellular Matrix
Note: ECM degrades the signaling molecule to contribute in controlling how far a given signal can travel
Endocrine – Blood stream

Question 12

How does paracrine signaling affect development in terms of creating a gradient?

Answer 12

During development, morphogens (signaling molecules) will spread in a gradient fashion because ECM will control
1) rate of diffusion
2) amount of signal to stay in action before ECM degrades it
so depending on distance from source of morphogen, cells will develop to different fates.

Question 13

Do all cell signaling receptor ligand – receptor binding have the same bind strength?

Answer 13

No.
Endocrine – since dilute hormones in the blood, must have high affinity interaction and bind tightly for it to work.
Cell-contact – since right there and there’s not much room for dilution, can have low affinity interaction

Question 14

Positive feedback allows for:

Answer 14

1) switch-like behavior

2) sustained response when signaling molecule has been removed

Question 15

If you see a feedback look that either has oscillations or modulation to reach an equilibrium, you should recognize that feedback as:

Answer 15

Negative Feedback Loop

Question 16

How does a steroid/ other small hydrophobic signaling molecule act on a cell? (also written up there as a question)
What is a 2-step process here and why is it important?

Answer 16

1) They will diffuse across plasma membrane
2) They will bind to a transcription factor whose Nuclear Localization Sequence is covered/inhibited by some regulatory factors
3) This bind will induce a conformational change that releases the receptor transcription factor from the regulatory factors
4) NLS will allow for the transcription factor to alter gene transcription of the PRIMARY response gene
5) PRIMARY Response genes will transcribe more transcription factors to turn on the SECONDARY response genes.
The 2 step response is important in have both a way to respond to the signal, as well as turn off the response BY:
6) the secondary transcription factors DOWN REGULATE the transcription of its own genes: the primary response genes.
*important!

Question 17

What do steroid receptors have in common?

Answer 17

They all share common domain structures because the steroids all have very similar structures: estrogen, progesterone, glucocorticoid, thyroxine, retinoic acid, etc.

Therefore, steroid receptors all have a very common ligand-binding and DNA binding structure with a variable region that accounts for the different steroids.

Question 18

Briefly: how does the ligand-gated ion channel operate in a muscle cell with Acetylcholine?
What is the response time of this signaling pathway?

Answer 18

1) Acetylcholine from neuron will bind to a sodium channel
2) Sodium channel will open up
3) Na will come flowing IN the cell, causing depolarization
4) This will trigger Ca 2+ release from SR

This signaling pathway is EXTREMELY rapid. Ie, few milliseconds or less.

Question 19

What are the 4 key steps in signal transduction through cellular signal processing?

Answer 19

1) Receptors will bind specific signaling molecules and activate cellular events via their intracellular domain
2) Several proteins relay binding state of receptor to cell machinery via their intermediary proteins.
Note: many cellular pathways can be affected with the same signaling molecule
3) Secondary messengers amplify concentration of signaling molecules
4) Cells attenuate signaling reactions to limit amount and time of cellular response. This is a way of turning off the response.

Question 20

GTP-binding proteins function as switches to indicate receptor activation. When is it considered “on” and when is it considered “off” ? What enzymes mediate switching from one to another?

Answer 20

ON: When GTP-binding protein is GTP bound
OFF: When GTP-binding protein is GDP bound
Note: NOT ATP OR ADP. ITS GTP AND GDP!

ON→OFF: GAP: GTP hydrolysis via GTPase Activating Protein

OFF→ON: GEF: GDP removal, GTP binding. (not phosphorylating!) by Guanine Nucleotide Exchange Factor.

Question 21

Structure of the Trimeric G-Protein? Where is it bound?

Answer 21

Trimeric G-Protein consists of Alpha, Beta, and Gamma subunits. The Alpha and Gamma subunits are both bound to the inner leaflet of the plasma membrane.

Question 22

How does a G-protein coupled receptor work? Start at a signal molecule binding and stop at the activation of one of the subunits.

Answer 22

1) A signal molecule will bind to the seven transmembrane receptor,
2) A conformational change will allow the receptor to act as a GEF (Guanine Nucleotide Exchange Factor) for the Alpha-protein subunit, switching out its GDP for a GTP and activating it (it = Alpha subunit)
3) Alpha subunit will dissociate from Beta-Gamma subunits (which are still bound together)
4) Alpha subunit and the Beta/Gamma subunits will have downstream effectors.

Question 23

How do Cholera Toxin and Pertussis Toxin act on adenylyl cyclase?

Answer 23

Both cholera toxin and pertussis toxin will modify G-alpha-S and G-alpha-I (respectively) by adding ADP-ribose and hyperactivating adenylyl cyclase.

Cholera Toxin- will add ADP-ribose to the activated G-alpha-S (stimulating) which will have the G-alpha-S stuck in the active state, hyperstimulate the Adenylyl Cyclase to produce way too much cAMP. This will result in increased Cl- and Na+ leak and therefore, water leaking.

Pertussis Toxin – will add ADP-ribose to the inactive G-alpha-I (inhibiting) which prevents G-alpha-I from releasing its GDP, therefore causing G-alpha-I stuck in inactive state, and unable to inhibit the adenylyl cyclase, also causing adenylyl cyclase to produce way too much cAMP.

Question 24

What is PKA and how does cAMP activate it?

Answer 24

PKA (Protein Kinase A) is a tetramer of 2 catalytic subunits and 2 regulatory subunits.
cAMP binds to the regulatory subunits and releases the catalytic subunits of PKA, which have several downstream targets such as: regulating gene transcription

Question 25

What does PI Kinase do?

Answer 25

It phosphorylates Phosphatidylinositols

Question 26

What is GAP

Answer 26

ON→OFF: GAP: GTP hydrolysis via GTPase Activating Protein

Question 27

What does Phosphlipase C cleave?

Answer 27

Phospholipase C, when activated by G-alpha-S, will cleave PIP into DAG (membrane bound) and IP3.

Question 28

Activation of MAPKKK pathway starting from the Receptor Tyrosine Kinase

Answer 28

1) Receptor Tyrosine Kinases recruit proteins, including a GEF (guanosine nucleotide exchange factor)
2) GEF activates RAS (the GTP-binding protein) by catalyzing the GDP removal, and GTP binding.
3) Activated RAS – GTP will activate MAPKKK
4) MAP KKK will activate many MAPKK
5) Each MAPKK will activate many MAPK

Note: RAS is commonly mutated in cancer.

Question 29

How do cells prevent crosstalk between various MAPKinase pathways?

Answer 29

Scaffolding proteins separate components of different MAP Kinase pathways. Cross talk could be counter productive since the same material could be used for the pathway for Cell Growth and Cell Cycle Arrest.

Question 30

Describe the process of multivesicular bodies processing receptors for degradation in lysosomes.

Answer 30

• Clathrin protein coat will cause for a vesicle to pinch off of the membrane containing the receptor.
• The problem here is that the receptors are still on the membrane outside so cannot just fuse with lysosome.
• Must pinch off again to get the receptor vesicles on the inside to ensure they get digested in the lysosome.

Question 31

True or false: Cells can detect diference in ligand concentrations.
Explain

Answer 31

True. Based on the concentration, the frequency of ion spikes (ie calcium spikes) will differ and this can determine concentration of activated downstream effectors.