Following theiractivation upon binding to ligand, G protein–coupledreceptors, GP
ID: 3454 • Letter: F
Question
Following theiractivation upon binding to ligand, G protein–coupledreceptors, GPCRs,
in turn activate Gproteins and thereby initiate a signaling cascade. Followingligand
binding, GPCRsbecome phosphorylated by G protein receptor kinases (GRKs) which,in
turn, leads todesensitization of the receptor such that continued stimulation byligand
results in a waningresponsiveness of the cell. Arrestins are proteins that bind toGPCRs
and are involved inthis desensitization. In order to understandGPCR-arrestin
interactions,the 122adrenergic receptor (122AR), a GPCR, and its interaction with
c. When the 122AR is activated, a signaling cascade isinitiated that leads to cAMP
dependentphosphorylation of protein X. Cells were transfected withcontrol
silencing RNA (siRNA) or arrestin siRNA (see Chapter 8 of thetext) to prevent
expression of arrestin. The cells were then incubated inepinephrine and, at various
times, aliquots ofthe preparation were prepared for Western blotting usingan
antibody directedagainst phosphorylated protein X. The data are shownbelow.
What is a likelyexplanation for the differing pattern of protein X phosphorylationin
Explanation / Answer
If you understand some of the terminology, then this question isnot as difficult as it might seem at first. First understand GPCR's, which are receptors that upon binding ofligand initiate a cascade of events intracellularly, leading tocellular events. In this case, the GPCR is the122AR and the ligand is epinephrine, the cellular eventbeing analyzed is the Phosphorylation of protein X. Now, to prevent over excitation of the GPCR's, there are mechanismsthat desensitize (make less sensitive) the receptor so that even ifepinephrine binds, it won't lead to phosphorylation of proteinX. The mechanism for desensitization is carried out by thearrestin, so normally if arrestin is functioning, youwill have decreased sensitivity and hence decreased phosphorylationof Protein X. So the experiment is basically looking at the role ofarrestin. In one condition, arrestin is expressedand present (control siRNA-no effect on arrestin) while inthe 2nd condition, the arrestin gene is inhibited using siRNAtechnology. siRNA uses short RNA strands that basicallyinhibit protein synthesis. So when control siRNA is being used, you wouldn't expect to see adecreased function of arrestin, hence you would havedesensitization of the 122AR and hence have lessProtein X. Therefore, in the first row of the Western blot,you see that Protein X phosphorylation levels decrease over time(normal at 2 and 5 min, less at 10 min, none afterwards). By comparison, if you block arrestin using the siRNA, youwouldn't have a desensitization of 122AR so you willcontinue to have high levels of Protein X in the phosphorylatedform. This is exactly what the 2nd row of the western blotshows. Phosphorylated Protein X concentration remains highand unabated until the max exposure time (30 minutes). Hope this helps.