Structural features of the apelin receptor N-terminal tail and first transmembrane segment implicated in ligand binding and receptor trafficking

Abstract

G-protein coupled receptors (GPCRs) comprise a large family of membrane proteins with rich functional diversity. Signaling through the apelin receptor (AR or APJ) influences the cardiovascular system, central nervous system and glucose regulation. Pathophysiological involvement of apelin has been shown in atherosclerosis, cancer, human immunodeficiency virus-1 (HIV-1) infection and obesity. Here, we present the high-resolution nuclear magnetic resonance (NMR) spectroscopy-based structure of the N-terminus and first transmembrane (TM) segment of AR (residues 1-55, AR55) in dodecylphosphocholine micelles. AR55 consists of two disrupted helices, spanning residues D14-K25 and A29-R55(1.59). Molecular dynamics (MD) simulations of AR built from a hybrid of experimental NMR and homology model-based restraints allowed validation of the AR55 structure in the context of the full-length receptor in a hydrated bilayer. AR55 structural features were functionally probed using mutagenesis in full-length AR through monitoring of apelin-induced extracellular signal-regulated kinase (ERK) phosphorylation in transiently transfected human embryonic kidney (HEK) 293A cells. Residues E20 and D23 form an extracellular anionic face and interact with lipid headgroups during MD simulations in the absence of ligand, producing an ideal binding site for a cationic apelin ligand proximal to the membrane-water interface, lending credence to membrane-catalyzed apelin-AR binding. In the TM region of AR55, N46(1.50) is central to a disruption in helical character. G42(1.46), G45(1.49) and N46(1.50), which are all involved in the TM helical disruption, are essential for proper trafficking of AR. In summary, we introduce a new correlative NMR spectroscopy and computational biochemistry methodology and demonstrate its utility in providing some of the first high-resolution structural information for a peptide-activated GPCR TM domain.

Small expression tags enhance bacterial expression of the first three transmembrane segments of the apelin receptor

“…It is also evident that the secondary structure composition of the protein in SDS micelles is essentially the same at both pH 6.00±0.05 and 7.00±0.05 ( Figure 6 and Table 3). Despite the suitability of DPC for solubilization of AR55 in our previous structural study (Langelaan et al 2013), the CD spectrum for the DPC sample of AR_TM1-3 resembles that of a protein rich in β-strand secondary structure, as upheld by deconvolution which estimates a proportion of only ~3% helical structure. This is indicative of improper folding and/or aggregation; hence, DPC micelle conditions were not pursued further.…”

“…The apelin/AR system has been shown to have roles in regulation of fluid homeostasis (De Mota et al 2004), angiogenesis during tumour formation (Sorli et al 2007), adipoinsular axis function , regulation of the cardiovascular (Kagiyama et al 2005) and central nervous systems (Kralisch et al 2007), and as a co-receptor in human immunodeficiency virus type-I infection (Cayabyab et al 2000). To date, the only high-resolution structural information available for AR is for the AR55 fragment (the 55 N-terminal amino acids, including the first TM segment (Langelaan et al 2013)), shedding light on the positioning of N-terminal amino acids previously reported to be involved in apelin binding and activation.…”

“…However, obtaining sufficient quantities of protein is typically a bottleneck in structural characterization of GPCRs, or any other class of membrane proteins, using NMR spectroscopy (Gautier et al 2010). As a result of the difficulties in expression of isotope-labeled full-length GPCRs, fragments of receptors ranging in size from 20-120 amino acids have been studied (Langelaan et al 2013;Tiburu et al 2011;Tikhonova and Costanzi 2009;Tyukhtenko et al 2009;Zhao et al 2006;Zou et al 2008). Ste2p, a yeast GPCR, is the only example where larger fragments (4 and 5 transmembrane (TM) segments) have been reported for structural characterization by NMR spectroscopy (Cohen et al 2014;Potetinova et al 2012).…”

Pyrene–Apelin Conjugation Modulates Fluorophore– and Peptide–Micelle Interactions

“…The AR55 region comprising the N-terminal tail and first transmembrane segment has been structurally characterized in DPC, 26 SDS (PDB entry 2LOT), and LPPG micelles (PDB entry 2LOV). A pair of acidic residues (E20 and D23; colored red, Figure 1C) in the AR Nterminal tail has been implicated in apelin-AR binding through mutagenesis, 50 with this region exhibiting structural convergence to provide an anionic patch.…”

“…Mutations were confirmed through sequencing (GENEWIZ). Expression and purification for wildtype and mutant AR55 protein constructs followed our previously detailed inclusion body preparation-based method, 26 with each product mass confirmed using positive mode electrospray ionization mass spectrometry (Dalhousie Mass Spectrometry Laboratory).…”