CELL BIOLOGY - Essay Example

1. Norepinephrine acts at a G-protein-linked receptor on heart muscle to make the heart beat faster by the effect of the G protein on a protein kinase. Would the following enhance or inhibit this effect of norepinephrine? Answer each part.A. A high concentration of a non-hydrolyzable analog of GTP. ⇒Inhibit Binding of norepinephrine activates the receptor that in turn activates the G protein. However, for the next step to occur, the G protein must hydrolyze GTP to GDP. In the presence of a saturating amount of non-hydrolyzable GTP, the G protein will be stalled in the inactive state and the result would be inhibition.B. Modification of the norepinephrine-receptor-linked G protein  subunit by cholera toxin.

⇒Enhance Cholera toxin alters the α subunit such that the G protein becomes constitutively activated. The result is an increase in cAMP and associated kinases that would amplify the cascade effect and therefore enhance the effect of norepinephrine.C. Mutations in the norepinephrine receptor that weaken the interaction between the receptor and norepinephrine. The answer depends on whether the receptor is still activated under the conditions of the weaker interactions. If the weaker interaction is still sufficient to activate the receptor but also allows for a faster exchange rate for norepinephrine molecules, then the result would be to enhance the effect of norepinephrine.

If, instead, the interaction was not sufficient to activate the receptor, then the effects of norepinephrine would be inhibited because the inactive receptor would not respond to the norepinephrine signal. D. Mutations in the norepinephrine receptor that weaken the interaction between the receptor and the G protein. This will likely inhibit the effects of norepinephrine, because although binding of norepinephrine will still activate the receptor at the same rate, the receptor would no longer be stably associated with the G protein, and the signal will not be transduced from activated receptor to inactive G protein as efficiently.E. Mutations in the G protein  subunit that weaken the interaction between  and the protein kinase.

If the α subunit is responsible for activating adenylate cyclase and signaling an increase in cAMP and activation of resulting cAMP-dependent kinases, then altering the interaction between the  and the protein kinase will have no effect. If instead, the  - protein kinase interaction is necessary for amplification of the norepinephrine signal, then weakening the interaction will have the effect described above (i.e. it will depend on whether or not the weaker interaction is still sufficient to activate the kinase.)F. Inhibition of protein phosphatases.

⇒By inhibiting protein phosphatases in general, protein kinases—activated by phosphorylation— and their downstream targets will have longer half lives because phosphatases won’t be available to hydrolyze their phosphate groups. If these kinases and downstream targets are required to potentiate the norepinephrine signal, then the effects of norepinephrine would be enhanced. 2. Receipt of extracellular signals can cause changes in cellular physiology. Some changes occur on a very fast time scale (i.e., in less than a second) whereas other changes occur long after the receipt of a signal (i.e., hours).A. Name an intracellular change that could result in a quick change in cell physiology.

⇒phosphorylation/dephosphorylationB. Name an intracellular change that could lead to a slow change in cellular physiology.⇒Gene expression/protein synthesis C. Explain why the response you named in A results in a quick change while the response you named in B results in a slow change.Phosphorylation and dephosphorylation of proteins occurs rapidly with materials constantly available in the cell. Thus, nothing needs to be synthesized de novo for these changes to take place, and they are quickly reversible.

Moreover, the process of phosphorylation and dephosphorylation are redundant “switch reactions” that are very common and of low complexity. Gene expression and protein synthesis consist of numerous steps, require active transport of materials to different compartments in the cell, and are inherently complex, because specific genes need to be targeted for transcription, and from transcribed genes, specific levels of protein need to be translated. These steps are highly regulated and they require a great deal of energy.

For these reasons, the changes come about slowly.3. The rod photoreceptors in the eye are extremely sensitive to light. Rod cells sense light through a signal transduction cascade involving light-activation of a G-protein coupled receptor that activates a G-protein, which activates cyclic GMP phosphodiesterase. Describe how you would expect the addition of the following drugs to affect the light-sensing ability of the rod cells. Explain your answers.A. A drug that is an inhibitor of cyclic GMP phosphodiesterase?

A cyclic GMP phosphodiesterase inhibitor would decrease the phosphodiesterase activity, which normally acts to convert cGMP to 5’GMP. The result would be a greater amount of cGMP, which would act to keep sodium channels open, potentiating depolarization. Since light sensing is associated with hyperpolarization, this would inhibit light sensing ability. B. A drug that is a nonhydrolyzable analog of GTP?If GTP cannot be hydrolyzed, then the signal will not be transduced from the photoreceptor to the G protein, and the cascade would end prematurely.

The result would be an inhibition of light sensing ability.4. The growth factor Superchick stimulates proliferation of cultured chicken cells. The receptor that binds Superchick is a receptor tyrosine kinase, and many tumor cell lines have mutations in the gene that encodes this receptor. Which one of the following types of mutations would be expected to induce uncontrolled cell proliferation?(a) A mutation that prevents localization of the receptor to the plasma membrane.(b) A mutation that prevents dimerization of the receptor.(c) A mutation that destroys the kinase activity of the receptor.(d) A mutation that prevents recognition of the receptor by phosphatases.(e) A mutation that prevents ligand binding.

In general, Superchick would be assumed to act as a stimulant for the receptor tyrosine kinases, promoting their dimerization, auto-phosphorylation, and activation of downstream targets. Furthermore, mutations in the receptor tyrosine kinase that are associated with tumor cells probably causes these receptors to be constitutively “on” such that they behave as though growth factors are always present, and constantly signal the cell to divide. Such a mutation might, for instance, not recognize phosphatases, and for that reason, would not be easily “turned off.”5. When activated by the signal, the platelet-derived growth factor (PDGF) receptor phosphorylates itself on multiple tyrosines (as indicated below by the circled Ps; the numbers next to these Ps indicate the amino acid number of the tyrosine).

These phosphorylated tyrosines serve as docking sites for proteins that interact with the activated PDGF-receptor. These proteins are indicated in the figure below, and include the proteins A, B, C, and D. The PDGF-receptor is activated when it binds to the signal, PDGF. One of the cellular responses to PDGF is an increase in DNA synthesis, which can be measured by incorporation of radioactive thymidine into the DNA. To determine whether protein A, B, C, and/or D are responsible for activation of DNA synthesis, you construct mutant versions of the PDGF-receptor that retain one or more tyrosine phosphorylation sites.

You express these mutant versions in cells that do not make a PDGF-receptor. In these cells, the various versions of the PDGF-receptor are expressed normally and in response to PDGF binding, become phosphorylated on whichever tyrosines remain. You measure the level of DNA synthesis in cells that express the various mutant receptors and obtain the data shown below.A. From these data, which, if any, of these proteins A, B, C, and D are involved in the stimulation of DNA synthesis by PDGF? Why?

Proteins A and D appear to additively stimulate DNA synthesis by PDGF. Even when proteins B and C cannot bind the receptor (trial 6), DNA synthesis is unaffected. However, when both A and D are missing (trials 3 and 4), there is no detectable synthesis. Likewise, when either A or D is missing, there is a partial decrease in protein synthesis.B. Which, if any, of these proteins inhibit DNA synthesis? Why?Protein B inhibits DNA synthesis. From these data, it is possible that B inhibits DNA synthesis indirectly by inhibiting D.

Comparison of trials 5 and 7 shows that B decreases the effect of D alone, suggesting that it acts as an inhibitor. C. Which, if any, of these proteins appear to play no detectable role in DNA synthesis? Why?C appears to have no effect. Comparing lanes 4 and 9 suggests that the interaction of the receptor with protein C alone is no different from the absence of all protein interactions. In addition, comparison of lanes 5 and 8 show that the activation effect of protein D is unchanged by the presence or absence of protein C.

6 Two protein kinases, PK1 and PK2, work sequentially in a signal transduction pathway. You create cells that contain inactivating mutations in either PK1 or PK2 and find that these cells no longer respond to an extracellular signal. You also create cells containing a version of PK1 that is permanently active and find that the cells behave as if they are receiving the signal even when the signal is not present. When you introduce the permanently active version of PK1 into cells that have an inactivating mutation in PK2, you find that these cells also behave as though they are receiving the signal even when no signal is present.

From these results, does PK1 activate PK2 or does PK2 activate PK1? Explain your answer.PK2 activates PK1; PK1 follows PK2 in the transduction cascade. This is evident because permanently active PK1 is necessary and sufficient for signal transduction, so we know that it must interact directly with downstream targets. Moreover, since PK1 activates these targets even when PK2 is inactive, we know that PK1 must come after PK2 in the sequence, because active PK2 is neither necessary nor sufficient for signal transduction.

ReferencesBerne, Robert M. et. al. Physiology. Missouri: Mosby. 2004. Devlin, Thomas M. Textbook of Biochemistry with Clinical Correlations. New York: Wiley-Liss, Inc. 1997.