Question

Research in cell biology and metabolism has progressed due to the discovery of molecules that artificially stimulate or inhibit glucagon/epinephrine and insulin signaling pathways. Let’s say you are working in a lab cataloging the effects of a library of small molecules on these pathways and have a “hit” on molecule 1stAVNGR. Preliminary data on molecule 1stAVNGR indicates that the cardiac isoform of PFK2/FBPase2 is doubly phosphorylated when this molecule is present at micromolar concentrations in cell cultures. Given this context answer the following questions.

a. Under these conditions what is the predicted degree of association between the regulatory subunits and the catalytic subunits of PKA?

b. Further investigation of molecule 1stAVNGR indicates elevated levels of cAMP within the cell despite the absence of epinephrine or glucagon. Hypothesize two possible explanations for this data.

c. When cell cultures are given both molecule 1stAVNGR and molecule RedSKLL (a G-protein inhibitor) cAMP levels remain high (again despite the absence of epinephrine or glucagon). Given this new information hypothesize a possible explanation for the data.

Answer 1

Hi,
The signal cascade events during activation of G proteins by glucagon or epinephrine are as follows:
- activation of G-proteins
- increase in cAMP
- activation of PKA
- phosphorylation of PFK2
- activates enzymes to either convert glycogen to glucose or visa versa.

Now, that the inhibitor in present, the PFK remains doubly phosphorylated ( at ser and thr). The PKA enzyme has subunits , 2 catalytic and 2 regulatory. Regulatory units always block the catalytic units. When cAMP binds to the regulatory subunits, it dissociates from the catalytic subunit. The fee catalytic subunits now phosphorylate the Ser and Thr of PFK2. In the given case, the PFK is phosphotylated, so the catalytic and regulatory subunits of PKA are not interacting with each other.

The elevated cAMP can arise due to 1. Constitutive activation of G-protein receptors by the Drug. This produces continues cAMP even though downstream channels are blocked. Another hypothesis, the drug is inhibiting the enzyme cAMP phosphodiesterase, which degrades cAMP. In the absence of stimuli, the cAMP levels remain high.

When the drug and a G-protein inhibitor is added to the cell, the cAMP remains high. That means the drug is not constitutively expressing the G-protein receptors. Rather it is acting at the cAMP phosphodiesterase level.