Ligand Modulation of Lateral Segregation of a G-Protein-Coupled Receptor into Lipid Microdomains in Sphingomyelin/Phosphatidylcholine Solid-Supported Bilayers

Abstract

Increasing evidence supports the idea that the plasma membrane bilayer is characterized by a laterally inhomogeneous mixture of lipids, having an organized structure in which lipid molecules segregate into small domains or patches. Such microdomains are characterized by high contents of sphingolipids that form thicker liquid-ordered regions having resistance to extraction with nonionic detergents. The existence of lipid lateral segregation has been demonstrated in both model and biological membranes, although its role in protein sorting and membrane function still remains unclear. In the present studies, plasmon-waveguide resonance (PWR) spectroscopy was employed to investigate the properties of microdomains in a model system consisting of a solid-supported lipid bilayer composed of a 1:1 mixture of palmitoyloleoylphosphatidylcholine (POPC) and brain sphingomyelin (SM), and their influence on the partitioning and functioning of the human delta opioid receptor (hDOR; a G-protein coupled receptor, GPCR). Resonance signals corresponding to two microdomains (POPC-rich and SM-rich) were observed in such bilayers, and the sorting of the receptor into each domain was highly dependent on the type of ligand that was bound. When no ligand was bound, the receptor incorporated preferentially into the POPC-rich domain; when an agonist or an antagonist was bound, the receptor incorporated preferentially into the SM-rich component, although with a two-fold greater propensity for this microdomain in the case of the agonist. G-protein binding to the agonist-bound receptor in the SM-rich domain occurred with a 30-fold higher affinity than to the receptor in the PC-rich domain. The binding of agonist to an unliganded receptor in the bilayer produced receptor trafficking from the PC-rich into the SM-rich component. Since the SM-rich domain is thicker than the PC-rich domain, and previous studies with the hDOR have shown that the receptor elongates upon agonist-activation, we propose that hydrophobic matching between the receptor and the lipid is a driving force for receptor trafficking to the SM-rich component.Keywords microenvironmental effects; G-protein binding and activation; phospholipids; sphingolipids; protein sorting For the last three decades the fluid mosaic model of Singer and Nicholson (1) has provided the foundation for the understanding of membrane structure. According to this model, membranes

FRET imaging reveals that functional neurokinin-1 receptors are monomeric and reside in membrane microdomains of live cells

“…A reason for this finding might be that the microdomains are able to recruit heterotrimeric G proteins as was observed in recent experiments (11,18). This recruitment will lead to an effective increase by a factor of up to 80 of the respective concentrations of the various signaling partners inside the domains.…”

“…Several reports based on biochemical (10), plasmon-resonance spectroscopy (11), single-molecule microscopy (12), and fluorescence recovery after photobleaching experiments (13) propose that GPCRs are localized in microdomains, but a clear demonstration of the existence and nature of such microdomains in living cells remains elusive, in particular because the interpretation of biochemical data can be rather equivocal (14)(15)(16)(17). Compartmentalization in form of microdomains was proposed to explain the efficiency of signal transduction at the low physiological surface concentrations of the signaling partners by their enrichment inside specialized signaling platforms (10,18).…”

The Two NK-1 Binding Sites Correspond to Distinct, Independent, and Non-Interconvertible Receptor Conformational States As Confirmed by Plasmon-Waveguide Resonance Spectroscopy

“…It should be noted that not all receptor molecules that are added to the PWR cell sample are inserted into the lipid bilayer. Indeed, previous studies performed with the hDOR, where the amount of receptor in the lipid bilayer was determined, have shown that only ∼5% of the receptor that is added to the cell sample is incorporated into the bilayer (24). It has also to be mentioned that the receptor preparation is quite pure and does not contain detectable receptor-interacting proteins, such as G-proteins as observed by SDS gel electrophoresis (see the Supporting Information).…”

The Significance of G Protein-Coupled Receptor Crystallography for Drug Discovery

“…Alternatively, realtime plasmon waveguide resonance spectroscopy can be useful as both a kinetic tool and a means of predicting the pharmacological action of ligands. The PWR method offers increased sensitivity over traditional SPR by detecting plasmon shifts in two dimensions; it can report mass rearrangements associated with the conformational plasticity of the GPCR system that correlate with pharmacological activity (Devanathan et al, 2004;Alves et al, 2005;Hruby and Tollin, 2007;Georgieva et al, 2008).…”