BIOL 201 (050)- Spring 2013 Study objectives worksheet for Exam #3

I. Be able to describe the key components of the ECM and their organization within a cellular context.

(1.) Below are four key proteins that comprise the ECM. For each, describe their localization (intracellular, integral membrane protein, or extracellular) and with which of the other component(s) each directly interacts. Integrin:

Actin:

Collagen:

Fibronectin:

IIa. Define the terms affinity, potency, and efficacy. Understand the relationship among them and what each can tell us about a given hormone (ligand). Be able to apply these concepts to appropriate graphs.

IIb. Understand the mechanistic difference between competitive and noncompetitive receptor antagonists. Be able to describe how each affects the parameters listed in (IIa) above.

(2.) The graph below depicts results of an experiment in which a biological response (effect) was measured following an increasing concentration of an agonist for a receptor responsible for this effect (black line; open squares). The same agonist was then

applied following application of either antagonist #1 (red line; open circles) or antagonist #2 (blue line; open triangles). The number of receptors did not change in any condition, and the concentration of antagonists #1 and #2 were identical and did not change.

(a) What type of antagonists are #1 and #2 (competitive or noncompetitive)? Explain your reasoning using terms listed in Objective IIa. (b) Based on the graph, and that the concentration of #1 and #2 were the same in the experiment, which antagonist has a higher affinity for the receptor? Explain your reasoning. (c) In the space below, first redraw the graph with just the No antagonist curve as a starting point. Now, keeping everything else the same as above, assume #1 and #2 are noncompetitive antagonists. Draw curves for how you would predict they would look relative to the No antagonist line (i.e. the shape and position of the curve is important; the exact numbers are not).

III. Understand the concepts of enzyme kinetics (e.g. substrates, products, transition state), especially as they relate to free energy. Be able to identify and describe Vmax and Km both conceptually and graphically.

(3.) The Michaelis-Menten graph below represents the reaction rate of an enzyme versus an increasing concentration of substrate. (a) Identify where the (approximate) Vmax and Km would be found on this graph. (b) Draw curves that would reflect the kinetic changes expected upon addition of either a competitive inhibitor or a noncompetitive (allosteric) inhibitor. (c) Describe in words the mechanistic difference between competitive and noncompetitive inhibitors in terms of binding sites (relative to the substrate) and the basic way inhibition occurs in both situations.

IV. Understand the fundamental differences between lipid-soluble and watersoluble hormone action at a target cell. (4a.) Draw and label the key components of the signal transduction pathway for cortisol (a lipid-soluble hormone). Make sure to label the proper cellular compartments in which each component localizes and where each step takes place in the cell.

(4b.) Draw and label the key components of the signal transduction pathway for epinephrine (a water-soluble hormone) that activates a GPCR. Again, label the cellular location for each key step and contrast this scenario with that for cortisol above.

V. Be able to describe the nucleotide cycle for both heterotrimeric and monomeric G-proteins, including comparing and contrasting the key steps for each. Understand the role for the GDP- and GTP-bound conformations and the mechanisms for the conversion between them. (5.) As discussed in class, the G-protein, Transducin, transmits signals from a GPCR in the retina in response to light photons. Laboratory mice harboring a mutation in one of

the genes involved in this signaling pathway, which prevents activated Transducin from returning to the off state, have impaired visual recovery from bright flashes of light. (a.) In which nucleotide-bound state is Transducin trapped in this situation?

(b.) What type of chemical reaction is being impaired?

(c.) Which of the following is least likely to be the mutated gene in these mice: the GPCR (rhodopsin), Transducin itself, or an RGS (GAP) protein?

(d.) Activated Transducin increases the activity of an enzyme (PDE) that degrades (decreases the amount of) a small molecule called cGMP. Which of these components serves as the effector and which is the second messenger? As such, how would you expect the overall levels of cGMP to compare between these mutant mice and normal control mice?

VI. Know the primary functions for each of the major cell-cell adhesion (interaction) mechanisms discussed. Be able to identify the key proteins involved in each of these and what (if any) cytoskeleton components are involved.

VII. Know and understand the key steps involved in Receptor Tyrosine Kinase signaling pathways. Be able to compare and contrast this pathway with that for

GPCRs. Understand how these receptors lead to activation of G-proteins and be able to describe their guanine nucleotide cycle. (7.) Cancer cells often have increased activity of the VEGF receptor, a receptor tyrosine kinase (RTK) that allows for increased blood vessel formation to feed the growing tumor. (a) What type of G-proteins function with RTKs such as the VEGF receptor?

(b) When RTKs are activated by hormone (ligand) they first form dimers. What chemical modification occurs to the receptors upon dimer formation?

(c) Suppose the cancer cells contain a mutated VEGF receptor that increases its activity. How would the level of G-protein activation be predicted to change as a result?

(d) New treatment strategies include antagonists to the VEGF receptor itself (e.g. the drug Avastin). Other potential treatment strategies include antagonists for additional kinases in the cell that are not VEGF (nor other RTKs). Describe where these kinases fit into the VEGF signaling pathway and why they might also be useful against the cancer cells.

VIII. Understand the relationship between Gibbs free energy and reaction rates. Be able to identify the key components of a free energy graph and how it describes a reaction. Understand the role of enzymes (catalysts) in this process. (8.) Below are two Gibbs free energy graphs each representing a hypothetical reaction. (a) Which of the two reactions (left or right) will proceed more favorably? Using the principles of free energy, make an argument to support you answer.

(b) What is the term used to describe the hump in the middle of each curve? Why is this parameter an important consideration in the overall reaction?

(c) On the left graph, draw a curve that would represent this reaction in the presence of an enzyme (catalyst) and explain your reasoning.