Interaction of HA-MC4R-mCerulean3 with Gs-YFP

Graduate #18
Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology

Ansley Scott - Jackson State University
Co-Author(s): Eugene Nyamugenda and Giulia Baldini, University of Arkansas for Medical Sciences, Little Rock, AR

---

Melanocortin-4 receptor (MC4R) is a G-protein coupled receptor expressed primarily in the brain, where it functions to regulate food intake and energy expenditure. The mechanism behind MC4R is not completely understood, thus the objective is to study the signaling properties and interaction between MC4R and G-alpha stimulatory subunit (Gs). Fluorescent proteins, fluorophores that are able to absorb fluorescence at one wavelength and emit at a longer wavelength, have been found to aid in study of protein localization in live cells. Fluorescent proteins vary in ability to fold and in brightness. Previous studies have been performed using HA-MC4R-GFP, MC4R tagged with HA and cloned with Green Fluorescent Protein (GFP) and HA-MC4R-CFP, MC4R tagged with HA and cloned with Cyan Fluorescent Protein (CFP). In Neuro2A (N2A) cells stably transfected with HA-MC4R-GFP, it was observed that the protein was present at the endosomal compartment and in the plasma membrane. Yet, in N2A cells transiently transfected with HA-MC4R-CFP, few cells expressed the protein of interest at cell surface, suggesting that HA-MC4R-CFP may be retained in the endoplasmic reticulum as an unfolded protein. In this study, the fluorophore mCerulean3, a more robust and brilliant variant of CFP, was subcloned downstream of HA-MC4R in a mammalian gene expressing vector. With the aforementioned knowledge, the goal of this project is to determine whether HA-MC4R-mCerulean3 will be expressed at the plasma membrane and endosomal compartment more efficiently than HA-MC4R-CFP, making it a better tool for studying the interaction of MC4R with Gs. Measurement of cellular distribution of HA-MC4R-GFP, HA-MC4R-CFP and HA-MC4R-mCerulean3 transfected into N2A cells indicated that HA-MC4R-mCerulean 3 localized at the plasma membrane and endosomal compartment in a manner better than HA-MC4R-CFP as expected. This indicates that HA-MC4R-mCerulean3 may be a better tool for FRET experiments, leading to a better understanding of the interaction of MC4R and Gs and their link to obesity.

Not Submitted

Funder Acknowledgement(s): NIH Grant 5R25HL108825-05 (SURP-UAMS); NIH Grants R01-DK102206 (Giulia Baldini); Intramural Funding Support from UAMS College of Medicine Research Council; LSAMP Bridges to Doctorate - Jackson State University

Faculty Advisor: Giulia Baldini, gbaldini@uams.edu