1. Construct one plot including the data for the WT and the MC4R mutants presenting the differences in membrane localisation. 2.Obtain approximate Emax and EC50 values from the graphs. 3.Why the ratio of PE/GFP fluorescence is measured in the FACS experiment, instead of measuring only total PE fluorescence? 4.Explain the effect that each mutation causes in the function of MC4R, and how they can be linked to disease. This question relates to the effect of different mutations on the Melanocortin-4 Receptor, a GPCRthat upon hormone stimulation activates adenylyl cyclase via stimulatory G proteins to elevateintracellular CAMP levels. Pharmacological studies established MC4R as a critical factor inregulating energy homeostasis. The endogenous ligands for MC4R include the agonist -MSH andthe antagonist agouti-related protein (AgRP).Several MCR4 mutations have been reported in various patient cohorts with eating disorders. Inorder to investigate the effects of each individual mutation, HEK293 cells were transfected withWild-Type MC4R or with the following MC4R mutants: P78L, 1102T and N240S. Three differentparameters were measured for each variant: Cell surface expression, -MSH binding andintracellular cAMP production.Cell surface expression was assayed used HEK293 cells transfected with N-terminal Flag and C-terminal GFP tagged wild-type and mutated MC4Rs. Cells were harvested and treated withmonoclonal anti-Flag antibody in conditions in which the primary antibody binds only toreceptors located at the cell surface. A subsequent incubation with phycoerythrin (PE)-conjugatedanti-mouse antibody was used for fluorescence-based sorting using FACS. The ratio of PE/GFPfor WT and MC4R mutants was measured in triplicates and is represented as a percentage of theWT PE/GFP ratio.WTpmol CAMP/106 cellsP78L1102TN240S7006005004003002001001000-le-12359598Plot showing the different levels of cAMP produced upon stimulation of cells transfected with the different mutants and WT• 1102T■ WTN240S▲ P78LRatio PE/GFP (%WT)le-11 le-101003882101.…………l .…………lle-09 le-08[NDP-MSH], Mle-07T1001e-06336999700-600-500400-300-200-1000le-05

A G protein-coupled receptor (GPCR) helps control how much energy we have. The hormone called MC4R…

1. Construct one plot including the data for the WT and the MC4R mutants presenting the differences in membrane localisation. 2.Obtain approximate Emax and EC50 values from the graphs. 3.Why the ratio of PE/GFP fluorescence is measured in the FACS experiment, instead of measuring only total PE fluorescence? 4.Explain the effect that each mutation causes in the function of MC4R, and how they can be linked to disease.

1. Construct one plot including the data for the WT and the MC4R mutants presenting the differences in membrane localisation. 2.Obtain approximate Emax and EC50 values from the graphs. 3.Why the ratio of PE/GFP fluorescence is measured in the FACS experiment, instead of measuring only total PE fluorescence? 4.Explain the effect that each mutation causes in the function of MC4R, and how they can be linked to disease.

Human Physiology: From Cells to Systems (MindTap Course List)

9th Edition

ISBN:9781285866932

Author:Lauralee Sherwood

Publisher:Lauralee Sherwood

Chapter19: The Peripheral Endocrine Glands

Section: Chapter Questions

Problem 10RE

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