Cell Signaling I

Question 1

Name the two key components required for cell-cell communication

Answer 1

Signaling or communication requires a 1) signal molecule and 2) a receptor protein, whether the receptor is membrane bound or in the cytosol doesn’t matter.

Question 2

What are the two categories or signal molecules

Answer 2

1) Hydrophilic: They bind to a receptor on the outside plasma membrane of the cell
2) Hydrophobic: These tend to be like steroids and they bind to a receptor inside the cell. They also tend to be associated with a carrier protein

Question 3

List and define the forms of intercellular signaling based on the source of the signaling molecule

Answer 3

SIGNALING IS DEFINED BASED ON THE SOURCE OF THE SIGNALING MOLECULE!!!!

1) Contact-Dependent: There is a signal on the membrane of one cell and a receptor on another
2) Paracrine: Neighboring cells release signals to act on nearby cells
3) Autocrine: When a signaling molecule is released by a cell and binds to a receptor on the same cell, or nearby cells of the same type
4) Synaptic: It is very fast and it is the release of neurotransmitters to act on an adjacent neuron
5) Endocrine: Specialized cells produce a hormone and release it into blood. This is a very slow process and the signal is usually in a very low concentration, thus the receptor must have a high affinity for the signal.

Question 4

Contact-Dependent

Answer 4

There is a signal on the membrane of one cell and a receptor on another. This is very important in development and immune response.

Question 5

Paracrine

Answer 5

Neighboring cells release signals to act on nearby cells

Question 6

Autocrine

Answer 6

When a signaling molecule is released by a cell and binds to a receptor on the same cell, or nearby cells of the same type

Question 7

Synaptic

Answer 7

It is very fast and it is the release of neurotransmitters to act on an adjacent neuron.

Carried out by neurons. It is VERY FAST signaling and takes fractions of a second. When the action potential reaches the end of the neuron the nerotransmitters are released and allowed to bind to the target cell. This is very specific because the signaling will only occur where the axon can travel to. Also, the signaling molecule doesn’t have to have a very high affinity for the receptor because the local concentration is so high

Question 8

Endocrine

Answer 8

Specialized cells produce a hormone and release it into blood. This is a very slow process and the signal is usually in a very low concentration, thus the receptor must have a high affinity for the signal.

There are specialized cells that produce a hormone and release it in the bloodstream. It then travels to distant cells in the body. This is SLOW compared to synaptic. Usually the signal is in very low concentrations so the signal receptor must have a very high affinity for the signal molecule. Dissociation constant is 10^-9-10^-12. The receptor basically pulls the signal out of solution.

Question 9

Explain how a cell can respond in a specific way to an environment that contains a multitude of different signaling molecules

Answer 9

Cells have specific receptors for specific signals. Thus, the cell may not necessarily bind to those signals. They receptors have a high affinity for their signaling molecule only.

A cell will respond to a signaling molecule only if it has a specific receptor for that molecule

They know what target to affect by the type of receptor on the target cell –> Only affect specific cells with the receptor for it.

• Often times, multiple signals are required for a cell to function properly
• For instance, you may have signal molecules A, B, and C just to keep the cell alive. There can be other molecules, D and E for example that are necessary for cell growth and division.
• Then F and G may need to be present for differentiation
• If there are no signals the cell will die

Question 10

Explain how a single signaling molecule can have different effects on different target cells

Answer 10

Different cells can have different variations of receptors that result in different signal transduction pathways within the cell to give a different result.

A hormone can have different effects in different types of cells, depending on the type of receptor present and how the cell is programmed to respond

• Signaling molecules can do different things in different tissues based on the type of receptor present and on how the cell is programmed to respond
• An example is Acetylcholine. This is a neurotransmitter released from nerve endings.
• When it makes contact with the muscle cell, it will bind to an ion channel and allow Na ions in to depolarize the membrane and cause contraction
• In heart muscle cells there is an M2 receptor that acetylcholine can bind to to cause an decreased rate and force of contraction
• In salivary glands there is a similar receptor, M3, and acetylcholine bound to it causes the cell to release its contents.
• Different receptors cause different functions and how the cell is programmed to respond to that can give different effects

Question 11

Multiple Extracellular Signals

Answer 11

Cells depend on multiple extracellular signals to stay alive. With no signal the cell would die.

Question 12

Recognize examples of hormones that have intracellular receptors

Answer 12

THESE ARE INTRACELLULAR (ACT WITHIN THE CELL) Some hydrophobic signaling molecules pass through the plasma membrane (passively or by transport) and activate nuclear receptors. Such examples are:

1) Cortisol: Regulates metabolism, particularly blood glucose concentrations.
2) Estradiol: Female sex characteristics
3) Testosterone: male sex characteristics
4) Vitamin D is another molecules with a nuclear receptor except it is cleaved at a ring. It regulated Ca concentrations in the blood.
5) Thyroxine: regulates metabolism in cells and needs a transporter to get into cells
6) Retinoic acid: involved in development and differentiation
- These are based on the cholesterol ring system and there are modifications to the cholesterol ring that allows them to bind to specific receptors.

Question 13

Explain the “early primary” and “delayed secondary” responses induced by activation of a nuclear hormone receptor

Answer 13

When these hormones bind to their receptor, the receptors dimerize in most cases and bind to specific DNA sequences to regulate gene transcription.

Early Primary Response:

• When these hormones bind to their receptor in the cell, they dimerize (not shown in figure) to form either a homo or heterodimer.
• These are basically transcription factors because they bind to specific sequence in the DNA and regulate transcription
• It shows induced synthesis of primary response proteins which are those that are directly regulated by the hormone response complex  Targets DNA to make more of this protein
• It turns out that some of these primary response proteins are actually transcription factors themselves and they can go on to regulate other genes

Delayed Secondary Response:

• It turns out that some of these primary response proteins are actually transcription factors themselves and they can go on to regulate other genes
• This is sort of a secondary response to the hormone.
• The primary response can produce proteins that actually come back and inhibit the signal from the hormone too
• The primary response proteins can come back and turn on secondary response proteins!

Question 14

Describe the general structural feature of the nuclear receptor superfamily

Answer 14

• The nuclear receptors belong to a family of proteins with similar structure and function
• They contain a DNA-binding domain, hormone-binding domain, and transcription-regulating domain(s)
• One characteristic is that they have a DNA binding domain to recognize a specific sequence.
• There is also a C-terminal part which is involved in hormone binding among other things
• The biggest difference between the receptors in the family is the length of the N-terminal region which is involved in the binding of co-activators or co-suppressors
• C-terminal is ligand-binding domain
• N-terminus is transcription-activating domain
• Typically these receptors have inhibitory protein bound to it that doesn’t allow the protein to bind to DNA
• When a hormone binds, it causes the release of that inhibitory protein, clamps down on the hormone, and part of the C and N terminal region can bind co-activator proteins that will regulate gene expression

When a hormone binds to its receptor, it causes a conformational change that results in dimerization, the release of inhibitory proteins, and the binding of co-activator proteins (or corepressors) that regulate transcription of specific genes

Question 15

Explain the basic characteristics of the three classes of cell-surface receptor proteins discussed in lecture, and indicate how the term “signal transducer” adequately describes the function of these proteins

Answer 15

1) Ion-coupled receptors: we’ve already seen how a receptor can bind an ion channel which can open or close depending on the hormone or type of channel

2) Enzyme-coupled receptors: The receptor is often just a single transmembrane protein and these have to dimerize in order for there to be an active enzyme. When a hormone binds, it brings the two molecules together, forming a dimer and activating them. Tyrosine kinases are an example.
- Another type of them don’t have any enzyme associated with it but when a signaling molecule binds, a heterodimer forms and then an enzyme from the cytosol can bind to it.

3) G-Protein-coupled Receptors:
-The biggest class are the G-Protein-Coupled Receptors
-There are three parts to it:
Receptor which is responsible for binding the hormone or ligand
G-protein which is inactive in the absence of any ligand
Enzyme: Which is inactive without the ligand

• When a signaling molecule binds the G-protein will then associate noncovalently with the receptor and you end up activating the G-protein and then the G-protein will go over to the enzyme and turn it on.
• There is a receptor, G-protein and a target protein or enzyme that is activated or sometimes turned off

The term “signal transducer” adequately describes the function of these proteins because they become active via a signal, and then they in turn, activate something else as a response, transducing the signal.

Question 16

Describe the general structural features of G-protein-linked cell surface receptor

Answer 16

• Receptor is a 7 transmembrane receptor
• There is a extracellular region involved in hormone binding (N-terminus). Then it passes through the membrane 7 times, so there are 3 extracellular and intracellular loops
• C-terminus is on the inside!
• These are called “7-transmembrane receptors” and sometimes “serpentine receptors” because they wind through the membrane like a serpent
• They only show the hormone binds to the N-terminus and this is true for a lot of types of hormones but the other extracellular loops are also involved and the hormone can bind to these as well.
• This is the amino acid sequence of the Beta-endergeric receptor
• See the N-terminus extracellular region that is often glycosylated
• There are hydrophobic residues that make a helix within the membrane
• Then see a loop and another alpha helix and so on
• It has a particularly long C-terminus tail here
• G-proteins tend to bind and get activated through the second and third intracellular loops
• It really looks more like this where the helices bundle
• Here you can see the N-terminus and 3 extracellular loops and the C-terminus and the other intracellular loops
• The hormone actually goes down and inside in between the different helices, there is not really a receptor just hanging out.
• Binding of a hormone to the receptor causes a conformational change to the receptor and activates the G-protein
• There are about 800 G-protein-coupled receptors in the human genome
• About 430 of the receptors are sensory receptors that detect odor, vision and taste. With the sensory receptors we have it is estimated that we can distinguish about 10 trillion different odors by different variations of these sensory receptors
• About 370 receptors bind hormones, neurtransmitters, and other signaling molecules
  
  • About 150 of these we don’t even know what binds to them –> Called Orphan receptors when we don’t know what binds to them. When we find out what binds to them we say they are deorphanized.
• About 50% of current drugs bind to GPCR

Question 17

Describe the general structural features of G-proteins

Answer 17

• Looking closer at the G-protein now
• It is heterotrimeric meaning it has 3 subunits that are all different, and alpha subunit that is attached to the membrane via a lipid attachment at the N-terminus
• There is a beta-subunit
• And a gamma subunit that is also attached to the membrane through a lipid
• When we activate this, the alpha subunit can dissociate from the beta and the gamma.
• The beta and the gamma will stay together
• The alpha subunit has a lot of helical structures but also some beta sheet structures too
• The alpha subunit also has the GDP-binding site
• The beta subunit is all beta sheets and it is called the seven blade propeller fold.
• The gamma subunit is small and helical
• The gamma and beta subunits attach via noncovalent interactions

Question 18

Describe how a receptor “activates” a G-protein and how, after a period of time, the G-protein inactivates itself

Answer 18

• G-protein activation by GPCRs is a multistep process
• A hormone binds to the receptor, allowing the G-protein to bind to the receptor protein noncovalently
• The binding of the hormone changes the conformation of the receptor protein, allowing it to bind to the GPCR. Then, the GPCR changes conformation, opening up the area where GDP is bound and lessening the affinity the alpha subunit has for GDP.
• Then the GDP comes off
• When GDP comes off, GTP comes on.
• When GTP comes on, the G-protein dissociated from the receptor protein and the alpha subunit dissociates from the beta-gamma.
• Now these activated pieces of the G-protein can change the activity of target proteins
• For a long time it was thought that only the alpha subunit was involved in activating the target. But now its known that the beta-gamma is involved too. Sometimes they work together and sometimes they do not

-GTP BINDS SO READILY BECAUSE IT IS VERY ABUNDANT IN THE CYTOSOL TOO

• The activated Gs (alpha subunit) protein with GTP bound binds to and activates the target protein (adenyl cyclase with Gs).
• Once the alpha subunit with GTP binds and activates it, within a few seconds the GTP is cleaved or hydrolyzed and phosphate comes off and leaves GDP behind.
• The GTPase activity of the alpha subunit is shown here.
• Once it cleaves the GTP to GDP then the alpha subunit is no longer able to bind to and activate the target so it comes off and the alpha, beta and gamma come back together in an inactive complex
• The alpha subunit can eventually hydrolyze GTP to GDP on its own but it takes several minutes for this to happen.
• When it binds to the target, the GTP is hydrolyzed within seconds so the target in a way is acting like a GAP.
• There are also RGS (Regulator of G-protein Signaling) proteins that can help in this process because they are GAPs that enhance the GTPase activity.
• Now we have an inactive G-protein and it can go back and get reactivated if the ligand is still bound to the receptor and be reactivated, continuing the process

Question 19

Recognize the structure of cAMP (cyclic AMP) and explain how it is synthesized and degraded

Answer 19

Synthesis:

• Gs activates adenylyl cyclase and creates cAMP
• cAMP is made from ATP, adenylyl cyclase cleaves the phosphodiester bond, releasing the last two phosphates from ATP. Then, the other phosphate cyclizes with the hydroxyl group at the 3 position of the ribose. This is why it is cyclic
• There are two phosphodiester bonds involved in the covalent attachment

Degradation:
-cAMP can also be degraded by the cyclic AMP phosphodiesterase and water to turn off the signal. It cleaves the phosphodiester bond to give a 5’ AMP molecule which doesn’t have the functions that cAMP does

Question 20

Gs

Answer 20

activated adenylyl cyclase

Question 21

Gi

Answer 21

inhibits adenylyl cyclase

Question 22

Gq

Answer 22

activates phospholipase-C beta

Question 23

Apply the above information about G-protein mechanism of action to describe the various steps involved in the activation of adenylyl cyclase by a Gs-coupled receptor. Also describe the return of adenylyl cyclase to the inactive state

Answer 23

There is a Gs-protein coupled receptor that will bind a signal and activate the GsPCR. Then the GsPCR will recruit the Gs-protein and activate it, which will cause the displacement of GDP for GTP on the alpha subunit which will allow the beta-gamma subunits to dissociate from it. Then, the Gs-alpha will activate the adenylyl cyclase which will cleave ATP to create cAMP. The GAP function of the andenylyl cyclase will convert the GTP to GDP on the alpha subunit and allow it to convert back to its bound state with the beta-gamma dimer.

The adenylyl cyclase will be deactivated when the alpha subunit of the G-protein dissociates from it and forms a trimer once again.

Question 24

Indicate how cyclic AMP activates protein kinase A (PKA)

Answer 24

• cAMP activates protein kinase A (PKA)
• PKA exists in an inactive state in which there are 4 subunits, 2 catalytic and 2 regulatory subunits, making a tetramer
• When the regulatory subunits are bound to the catalytic site, the catalytic subunits are inactive
• The regulatory subunits have 2 binding pockets for cAMP
• So when adenylyl cyclase activity goes up, cAMP is produced and binds to the 2 regulatory subunits (4 total cAMP can bind) which allows the catalytic subunits to detach from the regulatory subunits and makes them active
• cAMP activated PKA by binding to and associating with the regulatory elements

Question 25

Describe how PKA (protein kinase A) recognizes, and then alters the function of, specific proteins in the cell

Answer 25

PKA is a Ser/Thr kinase! Therefore, PKA phosphorylates a particular set of proteins on the Serine and Threonine residues

• Sometimes the PKA is called “Cyclic AMP-dependednt Protein Kinase A (PKA)
• The enzyme (PKA) recognizes proteins that have a particular sequence and that sequence is usually a second arginine and any amino acid (X) then Ser or Thr, then a hydrophobic amino acid.
• This phosphorylation can alter the function of the protein, either turning it on or off
• It adds a phosphate group from ATP to the Ser or Thr!

Question 26

Explain the fact that many different hormones can all activate cAMP formation and yet lead to different effects in different cells

Answer 26

The cAMP can activate Protein Kinase A (PKA) and depending on the protein makeup in the cell that was activated, the PKA can activate those proteins which can lead to a specific response needed by the cell. The protein composition is essential for a correct response. In other words, there are different proteins present within each cell type that are available to be phosphorylated by protein kinase A (PKA)

• There are a lot of hormones that increase cAMP
• These hormones have different effects
• TSH binds to receptor in thyroid gland.
• ACTH hormone binds to receptor on adrenal cortex.
• cAMP can thus do different things in different tissues
• cAMP is a signal transduction molecule that is used to take the signal from the hormone and transfer it to inside the cell
• What actually happens in the individual cell depends on what hormones are present
• In each of these tissues there are different proteins that can be activated to deactivated by PKA which leads to different effects in these different cells

Question 27

Describe how cyclic AMP, acting through PKA, can alter gene transcription

Answer 27

When the cAMP activates the PKA, the PKA can then in turn activate a protein called CREB (cAMP Response Element Binding Protein). The PKA phosphorylates the CREB, activating it and allowing it to now bind the CBP (CREB-binding protein). This can then recognize a CRE element (cAMP Response Element) on the DNA and get gene transcription.

Question 28

Discuss the role of the serine/threonine phosphoprotein phosphatases in signal transduction

Answer 28

The phosphatases remove a phosphate from the Serine or Threonine of the enzyme that was activated by PKA (protein kinase A). This deactivates the enzyme, preventing the further actions of the signal transduction pathway

Question 29

Describe a mechanism for desensitizing a G-Protein coupled receptor

Answer 29

• This is another way to stop the signal transduction
• A G-protein receptor coupled kinase called GRK (GPCR Kinase) can come in and phosphorylate the receptor, it phosphorylates in the inner loops and in the C-terminal tail. When GRK does this, the hormone receptor complex can no longer bind to a G-protein. It basically turns off the signal
• In addition, when it is phosphorylated, it attracts the protein arrestin which can bind to the phosphorylated receptor
• Arrestin can take this hormone-bound receptor and form clathrin-coated pits and endocytosis and then the receptor can be dephosphorylated and put back or be ubiquitinated and be degraded by lysosomes
• Arrestin has also been found now to stimulate other signaling pathways  It is not just turning this one off

Question 30

Describe the mechanism by which cholera toxin causes a large increase in intestinal epithelial cell cAMP levels

Answer 30

Cholera toxin contains an enzyme subunit that puts an ADP-ribose group (from NAD+) on arginine-201 of the G-alpha-s subunit. This process (called ADP-ribosylation) inactivates the GTPase activity of the G-alpha-s subunit so that it can no longer cleave the bound GTP. This thus results in continuous activations of adenylyl cyclase because the G-alpha-s is always active. This then produces large amounts of cAMP in intestinal epithelial cells. The cAMP then activates the PKA which phosphorylates the chloride ion channel, CFTR, resulting in Cl- secretion. Na+ absorption is also inhibited. Water follows the Cl- and Na+ and thus leads to voluminous diarrhea.

SUMMARY:

1) Increased cAMP
2) increased PKA
3) Activation of CFTR
4) Release of Cl- out of cell and inhibition of Na+ into cell to cause water to be more outside the cell and result in diarrhea.

Question 31

Describe the cellular signaling mechanism that occur when a signaling molecule binds to a receptor that activates the Gi protein.

Answer 31

Some signaling molecules bind to a G-protein coupled receptor that activates the Gi protein. The activation of the Gi protein is an analogous process compared to that of the Gs. The G-alpha-i subunit with bound GTP will INHIBIT the andenylyl cyclase enzyme and thus decrease cAMP. The G-beta-gamma-i dimer can activate certain potassium channels as well.