Please give an explanation to the answer given Questions 20 through 22 refer to
ID: 214746 • Letter: P
Question
Please give an explanation to the answer given
Questions 20 through 22 refer to GPCR signaling as depicted. Congratulations! You survived your first month working at no has a notoriously high employee attrition rate/body count. You have a new projectSydlc AMP characterizing a dopamine- activated GPCR (typically expressed in neurons) whose signaling probably follows the same basic outline as the generic GPCR scheme shown (right Your project manager wants you to formulate hypotheses before you run tests for signaling kinase activities in a mouse cell culture line that expresses only the dopamine receptor you want to study. Akt kinase 20. You plan to pre-treat your mouse cells with a chemical analog of GTP called GTP-+S. GTP-y-S substitutes a sulfur atom in place of an oxygen on the third (gamma) phosphate of GTP resulting in a GTP mimic that cannot be hydrolyzed by most GTPases. In other words, GTP-y-S is like a version of GTP than can never be converted to GDP, which leaves most GTPases locked in a GTP-bound conformation. Your pre-treatment of cells with GTP-r-S results in lots of GTP-y-S entering the cytosol, so what might you expect to see when you treat cells with dopamine and measure kinase activities? Compared to cells receiving no GTP-y-S pre-treatment, GTP A) Higher activation of PKA, PKC and CaM-kinase B) Higher activation of PKA, but normal activation of both PKC and CaM-kinase C) Normal activation of PKA, but lower activation of both PKC and CaM-kinase D) Normal activation of PKA and PKC, but lower activation of CaM-kinase E) Normal activation of PKA, lower activation of CaM-kinase, & higher activation of PKC 21. Next your boss wants you to pre-treat your cells with a chelator chemical called EGTA that is highly selective for binding calcium. When in the cytosol, EGTA should sequester any available free calcium ions, keeping the cytosolic [Ca2] very low-even when the cytosol is challenged by a burst of calcium released by a calcium channel. What might you expect to see when you treat cells with dopamine and measure kinase activities? A) Higher activation of PKA, PKC and CaM-kinase B) Higher activation of PKA, but normal activation of both PKC and CaM-kinase C) Normal activation of PKA, but lower activation of both PKC and CaM-kinase D) Normal activation of PKA and PKC, but lower activation of CaM-kinase E) Normal activation of PKA, lower activation of CaM-kinase, & higher activation of PKCExplanation / Answer
G proteins are composed of three subunits- , , and . During inactivated state, the subunit has a bound GDP which on receiving an appropriate signal from the corresponding receptor gets replaced by a molecule of GTP. The exchange of GTP in place of GDP makes the protein active and dissociates it into two functional subunits- a GTP bound subunit and a complex. The subunit has an intrinsic GTPase activity and it must hydrolyse its bound GTP into GDP back in order to reassociate with complex and becomes inactive. But the GTPase activity is very weak to promote the hydrolysis single-handedly. The GTPase activity of the subunit is greatly enhanced by the binding of a second protein, which can be either its target protein or a specific protein known as a regulator of G protein signaling (RGS). RGS proteins act as -subunit-specific GTPase activating proteins (GAPs), and catalyzes the GTP hydrolysis and thus shutts off G-protein-mediated responses.
20. As the chemical analog of GTP, GTP--S blocks the conversion of GTP into GDP, it competitively prevents the subunit of the G protein to activate its hydrolytic activity and thus the G proteins will remain active for longer time. As the diagram indicates that G protein signaling activates PKA via cyclic AMP and both PKC and CaM-kinase via Ca2+ therefore, the correct answer is a) or GTP--S pre-treatment leads to higher activation of PKA, PKC and CAM-kinase.
21. G protein activates the plasma-membrane-bound enzyme phospholipase C- which cleaves phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] into inositol 1,4,5-trisphosphate [IP3] and diacylglycerol. IP3 is a water soluble molecule which then leaves plasma membrane and reaches endoplasmic reticulum (ER), binds to and opens IP3-gated Ca2+-channels in the ER membrane. Thus Ca2+ stored in the ER gets released in the cytosol. Ca2+ is a secondary messenger molecule which participates both in the activation of PKC (becomes activated at the cytoplasmic face of plasma membrane by a combination of Ca2+, diacylglycerol, and the negatively charged membrane phospholipid phosphatidylserine) and CaM kinase (becomes activated on binding Ca2+). When the cell receives a pre-treatment of a calcium chelator, EGTA it conjugates with the whole calcium pool available inside the cytosol. If G protein signaling remains unaffected then PKA signaling will remain active. Thus when only EGTA pre-treament is given then the correct answer is C) or EGTA pre-treatment leads to normal activation of PKA but lower activation of both Cam-kinase and PKC.
22. RNA intereference (RNAi) is an gene silencing method where the expression of a particular gene is knocked down at the post-transcriptional level, via using a short (~20-22 bp ) length of nucleotides complementary to the 3' UTR region of the cognate mRNA and thus preventing it from being translated and degrading it. When calmodulin expression is made low by RNAi pre-treatment then normal activation of both PKA and PKC takes place but lower activation of CaM-kinase will occur.