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

https://doi.org/10.1016/j.bbamem.2013.02.005Get rights and content
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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-R551.59. 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, N461.50 is central to a disruption in helical character. G421.46, G451.49 and N461.50, 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.

Graphical abstract

Highlights

► We present the structure of the N-terminal portion of the human apelin receptor (AR). ► Combining NMR data and MD simulation, we place this in the full-length AR context. ► The first transmembrane helix of AR is a kinked helix and is required for trafficking. ► The AR N-terminal tail has an anionic surface ideal for binding its ligand, apelin. ► This is one of the first structural characterizations of a peptide-activated GPCR.

Abbreviations

AR
apelin receptor
AR55
residues 1–55 of AR
BSA
bovine serum albumin
CSI
chemical shift index
DMEM
Dulbecco's modified eagle medium
DPC
dodecylphosphocholine
DPPC
dipalmitoylphosphatidylcholine
DSS
sodium 2,2-dimethyl-2-silapentane-5-sulfonate
DTT
dithitothreitol
ERK
extracellular signal-regulated kinase
FBS
fetal bovine serum
GPCR
G-protein coupled receptor
HA
hemagglutinin
HEK
human embryonic kidney
HSQC
heteronuclear single quantum coherence
IPTG
isopropyl β-D-1-thiogalactopyranoside
LB
Luria broth
MD
molecular dynamics
NMR
nuclear magnetic resonance
NOE
nuclear Overhauser enhancement
NOESY
NOE spectroscopy
PCR
polymerase chain reaction
PEI
polyethylenimine
PDB
Protein Data Bank
RMSD
root mean square deviation
TFA
trifluoracetic acid
TM
transmembrane
TOCSY
total correlation spectroscopy

Keywords

Apelin receptor
Membrane protein structure
Divide and conquer
Biomolecular NMR spectroscopy
Homology model
Molecular dynamics simulations

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