1. You\'re studying cellular signaling through G-Protein Coupled Receptors (GPCR
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Question
1. You're studying cellular signaling through G-Protein Coupled Receptors (GPCRs) Specifically you're working on a pair of newly identified GPCRs, GPCR-X and GPCR-Y. Each binds the same small ligand, but activates different heterotrimeric G-proteins that act on the effector protein, adenylyl cyclase. You have access to cells that express GPCR-X and NOT GPCR-Y, and other cells that express GPCR-Y and NOT GPCR-X. You and your grad student mentor find that the binding of ligand has opposite effects on adenylyl cyclase activity for each cell type. GPCR-X activation causes an increase in adenyly cyclase activity, while GPCR-Y activation causes a decrease in adenylyl cyclase activity. A. (3pts) What can you and your grad student mentor measure experimentally to examine adenylyl cyclase activity upon treatment with ligand? B. (3pts) Based on the above stated effects on adenylyl cyclase activity, what type of G-protein is activated by each receptor?Explanation / Answer
A. When the ligand binds to the GPCR receptor, it activates or represses adenylase cyclase activity. Adenylyl cyclase converts ATP to cyclic AMP (cAMP). cAMP activates several kinases such as protein kinase A. These kinases will regulate several functions such as gene transcription, cellular metabolism etc. In order to analyze adenylase cyclase activity. cAMP levels can be monitored in the two cell lines when treated with [?-32P]ATP over time. If the signaling results in adenylase cyclase activation, cyclic [32P]AMP will be released, which can be measured after lysis of cells by radioimmunoassay specific for cAMP. cAMP levels will reduce if the enzyme is not activated after binding of the ligand. If adenylyl cyclase is activated, the levels of cAMP will increase over time.
B. Adenylyl cyclase is protein that is regulated by different proteins that include the G-proteins (guanine nucleotide binding regulatory proteins) Gs and G1, and Ca2+-calmodulin. Binding of ligand to the receptor causes activation of adenylyl cyclase, which will then cleave ATP to cAMP. cAMP is a ubiquitous intracellular signaling molecule that activates cAMP-dependent protein kinase A (PKA).
GPCRs associate with heterotrimeric G-proteins (green, that have three different subunits: alpha, beta, and gamma.
Binding of a ligand to the activating receptor couples the GDP bound heterotrimeric Gs protein to the receptor. GDP is replaced by GTP on the Gs protein. The Gs protein dissociates into the Gs?-GTP subunit and the ?? complex. The Gs?-GTP subunit activates the adenylyl cyclase enzyme. When GTP is hydrolysed by the GTPase activity of alpha subunit of G-protein, the adenylase cyclase activity will be inhibited.
Binding of ligand to inhibitory receptor activates the Gi class of proteins, through similar events. Most probably, the Gl?-GTP directly inhibits adenylyl cyclase. It is also possible ?? complexes liberated from Gl in combination with inactivating free Gs?-GTP could also cause inhibition of adenylyl cyclase.
GPCR-X causes activation of adenylyl cyclase. Epinephrine is a G-protein that activates beta adrenergic receptors in the heart. The binding will activated Gsa type of G protein.
GPCR-Y causes inhibition of adenylyl cyclase. GABAB receptors will activate the Gia subunit of G protein. Hence, Gia type of G protein is activated.
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