Structure, Dynamics and Topology of the N-terminus and First Transmembrane Segment of APJ

Abstract

APJ is a G-protein coupled receptor expressed in the cardiovascular system, central nervous system and several other tissues. Activation of APJ by the peptide ligand apelin has defined roles in cardiovascular regulation, in glucose metabolism and in tumour growth. Transmembrane proteins such as APJ are difficult to study due to expression, solubility and refolding difficulties. For this reason we have produced a fragment of APJ containing the functionally essential N-terminal region and first transmembrane helix of the receptor (APJ55). Through a combination of circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy we have characterized APJ55. CD spectroscopy indicates that APJ55 only properly refolds in specific detergents, with the anionic detergents sodium dodecylsulphate and 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG) being the best. NMR spectroscopy has provided an initial structure of APJ55. As a complement to this structure, relaxation studies and paramagnetic spin label titration demonstrated the dynamics and topology of APJ55 in the LPPG micelle. Finally the structure of APJ55 has been placed into the context of full length APJ using a homology model. APJ55 provides a new system to probe apelin-APJ interactions and is a basis for study of additional regions of APJ.