Expression, Purification And Biophysical Characterization Of Large Fragments Of The Apelin Receptor Enabling Delineation Of A Juxtamembrane Helix With Amphipathicity Necessary For Plasma Membrane Localization.

Abstract

The apelin receptor (AR or APJ) is a class-A G-protein coupled receptor (GPCR) activated by various isoforms of the peptide hormones apelin and, during embryonic development, apela. The apelin-AR system has been proposed as a therapeutic target for cardiovascular disease, central nervous system disorders, and various cancers. Because GPCRs are both inherently dynamic and function in a heterogeneous lipid bilayer environment, nuclear magnetic resonance (NMR) spectroscopy is well-suited for their characterization. NMR spectroscopy, however, requires multi-milligram quantities of proteins enriched with 15N, 13C, and/or 2H isotopes. Full-length isotope-enriched GPCRs are very challenging to produce; therefore, I have employed the “divide and conquer” approach to study the AR in two pieces. Here, for the first time, I have demonstrated the use of small adenine- and thymine-rich (AT-rich) gene tags to enhance the expression of the 137 amino acid N-terminal portion of AR including the first three transmembrane segments (TM1-3) in Escherichia coli. Condition optimization in pursuit of an ideal membrane-mimetic environment has also been carried out. In vitro translation of full-length AR divided into two pieces, each fused with a split-intein fragment, is also demonstrated using an E. coli-based cell-free expression system. I also expressed, isotope-enriched, and purified the 71 amino acid intracellular C-terminal tail of the AR to allow correlation of its structure, membrane-interactions, and function. Biophysical characterization indicate the presence of a juxtamembrane 8th helix upon exposure to membranous environments. This helix is, putatively, common to class-A GPCRs but poorly characterized from a structure-function perspective. Directly correlating to biophysical data showing differences in helicity and micelle binding, I used immunofluorescence experiments to unambiguously show the importance of amphipathy of this helix in plasma membrane localization of the receptor. Overall, this work details both the challenges faced and successes achieved in characterization of the AR using NMR spectroscopy.