G-Protein, Ca Signaling, Stem Cells, Ser Thr Kinases

Question 1

Draw the membrane topology a G protein-coupled receptor and identify the basic structural characteristics that mediate ligand binding and coupling to G proteins.

Answer 1

There are 7 helical transmembrane proteins that circle to form an ECF facing pocket. The pocket is where a ligand can bind. There are also intracellular domains that assist in binding the ß, gamma, and Gα subunits.

Question 2

Explain how G protein coupled receptors activate hetero trimeric G proteins and diagram the GTP hydrolysis cycle of G protein signaling.

Answer 2

Agonist ligand binding favors GDP dissociation from the Gα complex which allows GTP to bind to form the active form of the G protein. This step is unfavorable without an agonist, which is why the inactive form is prevalent. When this occurs, the alpha and beta subunit which were previously closely associated to G, are able to float away to create an effect in the cell. Alpha subunit doubles as a GTPase which will eventually hydrolyze GTP to GDP and return the complex to its original inactive state. The cycle can repeat if agonist remains.

Question 3

Describe the function of IP3 and DAG in receptor signaling and how they are generated by activated G proteins?

Answer 3

When α1AR is bound by NE, Gqα dissociates and teams up withPLC to cleave PIP2. The cleavage makes IP3 and DAG. IP3 exerts effects by binding its receptor in the ER, which releases calcium. DAG binds with PKC to stimulate Ca influx into the cell. Ultimately causes vasoconstriction.

Question 4

Explain how receptor activation leads to signal termination through receptor desensitization.

Answer 4

At the same time that you activate the signaling pathway, another pathway is activated that favors desensitization of the receptor. Beta activated GRK kinase phosphorylates the receptor, which attracts ß-arrestin. (both events prevent association of another G protein). ß-arrestin interacts with clathrin and causes the receptor to be endocytosed. Internalized receptors can be re-sensitized and recycled, or they can be taken to the lysosome to be chopped up by acid.

Question 5

Give two examples of drugs that act through modulating different steps in a receptor-G protein second messenger signaling cascade.

Answer 5

Phosphodiesterase: degrades cAMP and turns it into AMP, which does not activate PKA. This prevents PKA from blocking contraction (i.e. promotes contraction). Beta blockers: Prevent activation of a pathway by being a beta receptor antagonist and decreases heart rate.

Question 6

Describe the function of cAMP in receptor signaling and how they are generated by activated G proteins?

Answer 6

cAMP is also produced by active alpha and the AC complex. It activates protein kinase A, which eventually leads to an influx of calcium, which leads to increased heart rate and contraction.

Question 7

Understand the functions of cytoplasmic Ca++ ion buffers and how these buffers affect cytoplasmic Ca++ signals.

Answer 7

When calcium enters the cell, it can either bind to an effector or bind to a buffer system.Proteins that bind calcium play a crucial role in Ca2+ signaling. The cytoplasmic buffers (e.g., parvalbumin) tend to restrict the spatial and temporal spread of Ca2+. This prevents small, specific signalling. The buffers also serve as a temporary storage site for Ca2+ while the relatively slow transport processes are operating. In the ER/SR lumen, high-capacity low affinity buffers (e.g., calsequestrin) allow large quantities of Ca2+ to be stored without the generation of a large gradient in the concentration of free Ca2+.

Question 8

Understand the routes by which extracellular Ca2+ enters the cytoplasm.

Answer 8

There is a large gradient from the ECF to the cytoplasm, which means that the onset of Ca2+ movement into the cell can be very rapid. Additionally, there is a large electrical gradient driving Ca2+ into the cell. Calcium can move into the cytoplasm from the ECF via ion channels (either passive or voltage gated). Ligand gated calcium channels and store operated calcium channels (e.g. RyR) also let Ca2+into the cell.

Question 9

Understand the routes by which Ca2+ moves out of the ER/SR into the cytoplasm.

Answer 9

This is especially critical in various types of muscle. IP3 receptors (activated by G protein receptors) and ryanodine receptors (complex, multiregulated channels). help move calcium into the cytoplasm from the ER. The nuclear envelope is closely related to the ER/SR and also contributes to calcium flowing into the cell.

Question 10

Understand the routes by which Ca2+ is extruded from the cytoplasm (a) into the extracellular space and (b) into the lumen of the ER/ SR.

Answer 10

Transporters (active). Movements are much slower than via ion channels.Ca pumps use ATP to move Ca into ECF or back into the lumen of the ER/SRNa/Ca exchangers: extrude Ca across plasma membrane or from mitochondria into cytoplasm. They derive energy from the sodium gradient.

Question 11

Learn what EF hands and C2 domains are.

Answer 11

C2 domains bind Calcium and phosphoserine. This motivates protein kinase C to bind the membrane. EF hands are parts of proteins like calmodulin that help bind Calcium. Keep in mind that there are also many Ca2+binding motifs that do not resemble C2 domains or EF hands.

Question 12

Learn the identity of the archetypical protein that contains EF hands.

Answer 12

Calcium effectors!The EF-hand motif of calmodulin is found in many other Ca2+ effectors, including parvalbumin (a cellular Ca2+ buffer), calpain (a Ca2+-activated protease) and troponin.

Question 13

Learn the identity of the archetypical protein that contains a C2 domain.

Answer 13

Proteins that need to associate with the plasma membrane.

Question 14

Learn whether these (EF C2) domains are present in other proteins.

Answer 14

They most certainly are.One example is synaptotagmin. In these, when calcium binds, it sticks to the membrane and prompts synaptic vesicles and neurotransmitters to be released.

Question 15

Understand the basics of stem cells their niches and the commitment (differentiation) of stem cells into different lineages.

Answer 15

Embryonic stem cells are pleuripotent (can develop into any different type of cell type). Adult stem cells have plasticity. Stem cell niche is a phrase loosely used in the scientific community to describe the microenvironment in which stem cells are found, which interacts with stem cells to regulate stem cell fate.

Question 16

Understand the concept of adult stem cell plasticity.

Answer 16

Traditionally, adult stem cells were through to have limited potential, only renewing the tissue from which they were derived. However, recent evidence suggests that adult stem cells from one tissue may “transdifferentiate” and be able to renew other tissue types.

Question 17

Understand the concept of reprogramming adult somatic cells into induced pluripotent stem (iPS) cells or embryonic-like stem cells.

Answer 17

Experimentally, several independent research groups have been able to genetically reprogram adult skin cells into embryonic-like stem cells [induced pluripotent stem (iPS) cells]. The therapeutic potential of iPS cells for tissue repair and regeneration is enormous; this procedure not only eliminates ethical concerns associated with generating embryonic stem cells from fertilized human embryos, but it also avoids the complication of immune rejection, since the iPS cells would be generated from the same individual in need of treatment.

Question 18

Understand the role of stem cells in the initiation and maintenance of cancer.

Answer 18

Until recently, it was generally assumed that all cancer cells were the same. However, emerging data from virtually all cancer types suggest that cancers may be maintained by a relatively small number of cancer stem cells that are resistant to traditional cancer therapies. One way to put these observations into perspective is to think of a lawn filled with dandelions. Initially after mowing the lawn, the dandelions appear to be gone, however, the roots are still intact and with time, the dandelions reappear. In the case of cancer, radiation and chemotherapy may destroy most of the tumor, but the roots, i.e. the cancer stem cells are resistant, and with time the cancer reappears. An improved understanding of cancer stem cells could result in novel therapeutic strategies that specifically target cancer stem cells for destruction and prevent tumor recurrence.

Question 19

Describe a phosphorylation reaction (including which aminoacids can be phosphorylated) and explain how it can affect a phosphorylated protein.

Answer 19

Phosphate is added to the hydroxyl group of Serine, threonine, and tyrosine. The OH group on the AA nucleophilically attacks the gamma phosphate from an ATP molecule. Phosphorylation regulates the activity of a given protein.

Question 20

List at least two other types of secondary protein modification.

Answer 20

Acetylation, glycosylation, ubiquitination.

Question 21

Describe the structure/function of a protein kinase.

Answer 21

A kinase domain consists of a small and large lobe (green and blue, respectively). ATP binds in the cleft between the lobes; interaction of the substrate is mostly with the large lobe. A “closed conformation” of the glycine rich loop in the small lobe forces the γ-Phosphate of the ATP into the right position for phosphorylation (a fast reaction). An “open conformation” of the glycine rich loop then allow exchange of the generated ADP for a new ATP (a slow reaction).

Question 22

How does kinase activity change based on its open and closed formations?

Answer 22

Thus, kinase activity is thought to require alternating open and closed conformations.

Question 23

Please describe the requirement for activation loop phosphorylation in some kinases and other regulatory mechanisms.

Answer 23

In many but not all kinases, the activation loop has to be phosphorylated for full activity. The active conformation of all kinases is highly conserved. This presents a problem for making specific inhibitors for individual kinases. However, the inactive conformations are not, since there are many ways to distort conformation to prevent activity. Generally, the ATP binding pocket is somehow distorted in inactive conformations (glycine rich loop; C-helix; activation loop). Another common regulatory theme is block of the active site by an inhibitory “pseudo-substrate” sequence.