Active mutants. Some protein kinases are inactive unless they are phosphorylated on key serine or threonine residues. In some cases, active enzymes can be generated by mutating these serine or threonine residues to aspartate. Explain.

In the pocket. SH2 domains bind phosphotyrosine residues in deep pockets on their surfaces. Would you expect SH2 domains to bind phosphoserine and phosphothreonine with high affinity? Why or why not?

On–off. Why is the GTPase activity of G proteins crucial to the proper functioning of a cell? Why have G proteins not evolved to catalyze GTP hydrolysis more efficiently?

Viva la différence. Why is the fact that a monomeric hormone binds simultaneously to two identical receptor molecules, thus promoting the formation of a dimer of the receptor, considered remarkable?

Antibodies mimicking hormones. Antibodies have two identical antigen-binding sites. Remarkably, antibodies to the extracellular parts of growth-factor receptors often lead to the same cellular effects as does exposure to growth factors. Explain this observation.

Facile exchange. A mutated form of the α subunit of the heterotrimeric G protein has been identified; this form readily exchanges nucleotides even in the absence of an activated receptor. What would be the effect on a signaling pathway containing the mutated α subunit?

Making connections. Suppose that you were investigating a newly discovered growth-factor signal-transduction pathway. You found that, if you added GTPγS, a nonhydrolyzable analog of GTP, the duration of the hormonal response increased. What can you conclude?

Diffusion rates. Usually, rates of diffusion vary inversely with molecular weights; so smaller molecules diffuse faster than do larger ones. In cells, however, calcium ion diffuses more slowly than does cAMP. Propose a possible explanation.

Negativity abounds. Fura-2 is not effective for the study of calcium levels in intact, living cells. On the basis of how Fura-2 is depicted in Section 14.1, why is it ineffective?

Awash with glucose. Glucose is mobilized for ATP generation in muscle in response to epinephrine, which activates G_αs. Cyclic AMP phosphodiesterase is an enzyme that converts cAMP into AMP. How would inhibitors of cAMP phosphodiesterase affect glucose mobilization in muscle?

Getting it started. The insulin receptor, on dimerization, cross-phosphorylates the activation loop of the other receptor molecule, leading to activation of the kinase. Propose how this phosphorylation event can take place if the kinase starts in an inactive conformation.

Many defects. Considerable effort has been directed toward determining the genes in which sequence variation contributes to the development of type 2 diabetes. Approximately 800 genes have been implicated. Propose an explanation for this observation.

Growth-factor signaling. Human growth hormone binds to a cell-surface membrane protein that is not a receptor tyrosine kinase. The intracellular domain of the receptor can bind other proteins inside the cell. Furthermore, studies indicate that the receptor is monomeric in the absence of hormone but dimerizes on hormone binding. Propose a possible mechanism for growth-hormone signaling.

Receptor truncation. You prepare a cell line that overexpresses a mutant form of EGFR in which the entire intracellular region of the receptor has been deleted. Predict the effect of overexpression of this construct on EGF signaling in this cell line.

Hybrid. Suppose that, through genetic manipulations, a chimeric receptor is produced that consists of the extracellular domain of the insulin receptor and the transmembrane and intracellular domains of the EGF receptor. Cells expressing this receptor are exposed to insulin, and the level of phosphorylation of the chimeric receptor is examined. What would you expect to observe and why? What would you expect to observe if these cells were exposed to EGF?

Total amplification. Suppose that each β-adrenergic receptor bound to epinephrine converts 100 molecules of G_αs into their GTP forms and that each molecule of activated adenylate cyclase produces 1000 molecules of cAMP per second. With the assumption of a full response, how many molecules of cAMP will be produced in 1 s after the formation of a single complex between epinephrine and the β-adrenergic receptor?

Nerve-growth-factor pathway. Nerve-growth factor (NGF) binds to a protein tyrosine kinase receptor. The amount of diacylglycerol in the plasma membrane increases in cells expressing this receptor when treated with NGF. Propose a simple signaling pathway and identify the isoform of any participating enzymes. Would you expect the concentrations of any other common second messengers to increase on NGF treatment?

Redundancy. Because of the high degree of genetic variability in tumors, typically no single anticancer therapy is universally effective for all patients, even within a given tumor type. Hence, it is often desirable to inhibit a particular pathway at more than one point in the signaling cascade. In addition to the EGFR-directed monoclonal antibody cetuximab, propose alternative strategies for targeting the EGF signaling pathway for antitumor drug development.

Distant relatives. The structure of adenylate cyclase is similar to the structures of some types of DNA polymerases, suggesting that these enzymes derived from a common ancestor. Compare the reactions catalyzed by these two enzymes. In what ways are they similar?

Kinase inhibitors as drugs. Functional and structural analysis indicates that Gleevec is an ATP-competitive inhibitor of the Bcr-Abl kinase. In fact, many kinase inhibitors under investigation or currently marketed as drugs are ATP competitive. Can you suggest a potential drawback of drugs that utilize this particular mechanism of action?

Establishing specificity. You wish to determine the hormone-binding specificity of a newly identified membrane receptor. Three different hormones, X, Y, and Z, were mixed with the receptor in separate experiments, and the percentage of binding capacity of the receptor was determined as a function of hormone concentration, as shown in graph A.

Binding issues. A scientist wishes to determine the number of receptors specific for a ligand X, which he has in both radioactive and nonradioactive form. In one experiment, he adds increasing amounts of radioactive X and measures how much of it is bound to the cells. The result is shown as total activity in the following graph. Next, he performs the same experiment, except that he includes a several hundredfold excess of nonradioactive X. This result is shown as nonspecific binding. The difference between the two curves is the specific binding.

Counting receptors. With the use of experiments such as those described in Problems 21 and 22, the number of receptors in the cell membrane can be calculated. Suppose that the specific activity of the ligand is 10¹² cpm per millimole and that the maximal specific binding is 10⁴ cpm per milligram of membrane protein. There are 10¹⁰ cells per milligram of membrane protein. Assume that one ligand binds per receptor. Calculate the number of receptor molecules present per cell.