This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We plan to study the kinetics of the expression of the M1 muscarinic receptor on the plasma membrane of Chinese hamster ovary (CHO) cells using a fusion protein consisting of a conditional aggregation domain (CAD) fused to the N-terminus of the human M1 receptor and green fluorescent protein (GFP) fused to the C-terminus. Following transient expression in CHO cells, the fusion protein (GFP-M1-CAD) is trapped in the ER, but can be induced to exit following application of a ligand (AP21998) that binds to the CAD and causes dissociation of fusion protein aggregates in the ER. After exiting the ER, the CAD domain is cleaved by furin, and the M1-GFP receptor travels to the plasma membrane. We plan to explore the factors that influence the plasma membrane expression of the M1 receptor using this fusion protein and TIRF.
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