Membrane Phospholipid Analogues as Molecular Rulers
to Probe the Position of the Hydrophobic Contact Point of Lysophospholipid
Ligands on the Surface of G‑Protein-Coupled Receptor during
Membrane Approach
When lipid mediators
bind to G-protein-coupled receptors (GPCRs),
the ligand first enters the lipid bilayer, then diffuses laterally
in the cell membrane to make hydrophobic contact with the receptor
protein, and finally enters the receptor’s binding pocket.
In this process, the location of the hydrophobic contact point on
the surface of the receptor has been little discussed even in cases
in which the crystal structure has been determined, because the ligand
binding pocket is buried inside the transmembrane (TM) domains. Here,
we coupled an activator ligand to a series of membrane phospholipid
surrogates, which constrain the depth of entry of the ligand into
the lipid bilayer. Consequently, via measurement of the receptor-activating
activity as a function of the depth of entry into the membrane, these
surrogates can be used as molecular rulers to estimate the location
of the hydrophobic contact point on the surface of GPCR. We focused
on lysophosphatidylserine (LysoPS) receptor GPR34 and prepared a series
of simplified membrane-lipid-surrogate-conjugated lysophospholipid
analogues by attaching alkoxy amine chains of varying lengths to the
hydrophobic tail of a potent GPR34 agonist. As expected, the activity
of these lipid-conjugated LysoPS analogues was dependent on chain
length. The predicted contact position matches the position of the
terminal benzene ring of a nonlipidic ligand that protrudes between
TMs 4 and 5 of the receptor. We further found that the nature of the
terminal hydrophilic functional group of the conjugated membrane lipid
surrogate strongly influences the activity, suggesting that lateral
hydrophilic contact of LysoPS analogues with the receptor’s
surface is also crucial for ligand–GPCR binding.