In the absence of ligand both subunits are close

Info iconThis preview shows page 1. Sign up to view the full content.

View Full Document Right Arrow Icon
This is the end of the preview. Sign up to access the rest of the document.

Unformatted text preview: You would tether a YFP tag to the Gb subunit and a CFP tag to the Ga subunit. In the absence of ligand both subunits are close together and so when CFP is excited at 440nm FRET occurs between CFP and YFP and so light of 527nm is emitted. When ligand is bound to wildtype receptor the Ga subunit binds GTP and dissociates from the Gbg subunits. Now when CFP is excited, light emits at 490nm since no FRET can occur. (iii) While trying to understand the basis for mutant B you look at receptor localization in the presence and absence of ligand using the GFP tagged versions of the receptors from part (i). What do you expect to see in the wildtype cells after extended exposure (30mins) to ligand? You would expect to see the receptor localized at the plasma membrane when ligand is not present. After 30 mins of ligand exposure you would expect to see some of the GFP 7.06 Spring 2004 PS 2 KEY 7 of 7 fluorescence in the cytoplasm in a punctate pattern, since upon extended interaction with the ligand the receptors are internalized into endosomes using b-arrestin. In the cells expressing mutant B the receptor is found to localize predominantly to the cytoplasm in the presence or absence of ligand. How could you explain this? How could you demonstrate this experimentally? b–arrestin could be constitutively binding to the mutant RKR receptor in the presence and absence of ligand, resulting in the receptor being internalized and hence there is insufficient receptor on the surface to activate the required signaling pathways. To demonstrate this you could express a version of b-arrestin that is tagged with YFP and look for colocalization with mutant RKR-GFP in the endosomes in the absence of the MC ligand. (iv) The RKR receptor has a conserv...
View Full Document

Ask a homework question - tutors are online