Following their activation upon binding toligand, G protein–coupled receptors,GP
ID: 3453 • Letter: F
Question
Following their activation upon binding toligand, G protein–coupled receptors,GPCRs,
in turn activate G proteins and therebyinitiate a signaling cascade. Followingligand
binding, GPCRs become phosphorylated by Gprotein receptor kinases (GRKs) which, in
turn, leads to desensitization of thereceptor such that continued stimulation byligand
results in a waning responsiveness of thecell. Arrestins are proteins that bind toGPCRs
and are involved in this desensitization.In order to understand GPCR-arrestin
interactions, the122adrenergic receptor (122AR), a GPCR, and its interaction with
arrestin are subjected tostudy.
artificial membranepreparation. Included in this preparation are either purifiedGs
protein or purifiedGRK2 or purified arrestin, as indicated by + symbols onthe
figure below.Epinephrine is added as indicated and the GTPase activity ofthe
preparation is thenmeasured. Why is GTPase activity measured in this assay?Under
what conditions isGs activated? How does the presence of the kinaseGRK2 affect
Gs activation? Howdoes the presence of arrestin affect Gs activation? Howdo
the results withGRK2 relate to the findings in part (a) above? What can youdeduce
Explanation / Answer
Here are my thoughts:
1. GTPase activity is measured because their activity levelsdirectly relate to the rate at which the GTP protein is beingphosphorylated and activated, since GTPase is the protein thatphosphorylates the GTP protein.
2. GTPase is activated when BOTH Gs and Epinepherine arepresent. If either is missing, Gs will not activate (proven byresults in tests 1 and 6 when either is missing, and confirmed intests 2-5 when both are present, allowing the activity topeak).
3. GRK2 decreases the activity levels of GTPase (proven bytest 3 vs. test 2) by desensitizing the receptor protein,decreasing response rates over time to an extended stimulation by aligand.
4. Arrestins work with the GRK2 proteins in desensitizing thereceptor protein, further decreasing the rate of GTPase activity(proved in test 4 vs. test 3)
5. You can say that the experiment confirms part A'sstatements about GRK's desensitizing role in the cell's G protein,since the presence of the molecule reduces GTPase's activitylevels.
6. B arrestin must be a sort of cofactor or coenzyme to theGRK protein, because it increases GRK's effectiveness since GTPaseactivity levels decreased much further when B arrestin was addedwith GRK2, but had no effect when B arrestin was added withoutGRK2, proving that B arrestin needs GRK2 to have any effect on thecell.
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