Baranski Homework Answers
1. What are the four classes of cell-surface receptors? Give one example of each.
1. G protein-coupled receptors; epinephrine, glucagons, serotonin
2. Ion channel receptors; acetylcholine
3. Tyrosine kinase-linked receptors; erythropoietin, interferons
4. Receptors with intrinsic enzymatic activity; atrial neturetic factor, NGF, PDGF
2. a. What are the three basic modes of signal transmission from one molecule to another?
Allostery, Proximity, and Covalent Modification
b. Which of the above mechanisms regulate the following signal transduction molecules?
i. Grb2: proximity
ii. Raf: Ras induced proximity, Raf induces covalent modification on Mek
iii. p85/p110: proximity through recruitment to EGFR-P, interaction causes
allostery to activate p110
iv. PLC: proximity to bring it to the EGFR-P, covalent modification via EGFR
phosphorylation of PLC, and then proximity to bind PIP3 molecule via PLCPH
domain
3. You discover three novel proteins named after The Three Stooges: Moe, Larry, and Curly.
a. Fill in the chart. Include your calculations and equations used.
LIGAND Amt Bound
Bmax
Kd
Ligand At
(=2*Column 1)
Kd
Moe
1.3 x 105
1 x 10-8 M
2.6 x 105
Larry
8.9 x 105
3 x 10-10
1.8 x 106
Curly
9.8 x 104
8 x 10-7
2.0 x 105
K2
(=Kd*k1, k1=108
M-1sec-1)
1.0 sec-1
0.03
80
Half-life
(=0.69/k2)
0.69 sec
23
0.0086
b. Which ligand is most likely to be a neurotransmitter?
Curly, because it has the shortest half-life.
c. Which ligand had the greatest number of receptors?
Larry, because Bmax = number of receptors
4. You are measuring the specific binding of the GW ligand to its receptor using a binding assay.
In addition, a structurally similar ligand—named Jeb—has just been discovered, and you would
like to understand more about its binding characteristics. You have obtained the following data
from your binding assays:
GW:
Bound
5.8
12.3
25.0
48.1
Free
0.1
0.2
0.5
1.1
JEB:
Bound
7.1
20.3
32.4
45.1
Free
0.14
0.35
0.54
0.74
60.2
95.4
122.1
143.9
1.5
3.7
8.1
22.3
84.5
111.2
145.1
160.0
1.58
2.62
7.22
800.00
a. Create a Scatchard plot for GW. (Hint: using Excel allows you to find the equation of the
line.) What is the Kd? How many receptors are there?
In a graph of Bound vs. Free GW, bound GW (molecules/cell x 1000) would be on the yaxis, and [GW] (nM) would be on the x-axis. Make sure you understand how I got the units
for Kd that I did (below).
Bound GW / Free GW
[(molecules/cell x 1000)/nM]
Scatchard Plot
70
60
50
GW
40
Jeb
30
20
Linear
(GW)
y = -0.3863x + 62.343
10
0
0
50
100
150
200
Bound GW (molecules/cell x 1000)
Kd = -1/slope = -1/-0.4 nM-1 = 2.5 nM = 2.5 x 10-9 M
Number of receptors = x-intercept
receptors/cell
0 = -0.4x + 64  x = 160 x 1000 = 1.6 x 105
b. On the same graph, add a Scatchard plot for Jeb. What type of binding does Jeb exhibit?
Positive cooperativity
c. How can you quickly determine whether positive or negative cooperativity (or neither) exists
from a Hill plot?
Look at the slope:
Slope = 1  neither
Slope > 1  Positive cooperativity
Slope < 1  Negative cooperativity
5. Why is phosphorylation a common modification used in signaling pathways?
Phosphorylation is a reversible modification that can change the structural
characteristics of the target protein to either activate it, deactivate it, or allow/block
protein-protein interactions.
6. What are the four steps in RTK activation and signal transduction?
1. Ligand binding leads to receptor dimerization
2. Receptor dimerization causes trans-phosphorylation of catalytic domains. This
activates the catalytic domains’ kinase activity
3. Catalytic tyrosine kinase domains can further phosphorylate each other and other
protein targets (on tyrosine residues)
4. Phosphorylated proteins can recruit other proteins to form a signaling complex
7. How is substrate specificity achieved for protein phosphatases?
The substrate specificity of a phosphatase comes from the local interactions of
amino acids around the phosphorylated residue and the residues that reside in the
catalytic cleft of the phosphatase.
8. What is the target of Gs and Gi, and what effect does each have on the target?
Both target adenylyl cyclase. Gs stimulates the enzyme to produce cAMP, whereas
Gi inhibits the action of the enzyme, resulting in decreased levels of cAMP.
9. STKE: Go to http://stke.sciencemag.org/
Use the search tool to look up “Rac” and find the canonical pathway entry by G. Johnson.
a. How many canonical pathways are listed as including Rac?
7
b. For two of the pathways, what molecule(s) is/are directly upstream of Rac? (Only use
pathways for which an upstream molecule is known and find two pathways with different
upstream components.)
NK cell: PI3K Ia and Vav
EGFR: Vav
Integrin: DOCK180 and PIX/COOL
B-Cell antigen receptor: Vav
c. Click on the upstream molecules and identify the type of protein.
PI3K Ia: Lipid kinase
Vav: GEF
DOCK180: no entry
PIX/COOL: GEF
d. For the same two pathways, what molecule(s) is/are directly acted upon by Rac?
NK Cell: Pak1
EGFR: Actin
Integrin: MLK-3 and PAK
B-Cell: Raf