Ligand modulation of lateral segregation of a G-protein-coupled receptor into lipid microdomains in sphingomyelin/phosphatidylcholine solid-supported bilayers

Abstract

A growing body of 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 that are resistant 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 these 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 was incorporated preferentially into the POPC-rich domain; when an agonist or antagonist was bound, the receptor was incorporated preferentially into the SM-rich component, although with a 2-fold greater propensity for this microdomain in the case of the agonist. Binding of G-protein to the agonist-bound receptor in the SM-rich domain occurred with a 30-fold higher affinity than binding to the receptor in the PC-rich domain. The binding of the agonist to an unliganded receptor in the bilayer produced receptor trafficking from the PC-rich to 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 is elongated 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.

Figure 1

PWR spectra obtained for a solid-supported lipid bilayer containing a 1:1 mixture of POPC and SM (panels A,D, curves 1) and for the incorporation of agonist (DPDPE)-bound hDOR (∼10 nM, final concentration in the sample cell) into the lipid bilayer (panels A,D, curves 2). Spectra were obtained with 543.5 nm (green) exciting light using either p- (panels A,B,C) or s-polarized light (panel D,E,F). Experimental PWR spectra and simulation of the POPC:SM (1:1) bilayer (panels B,E), and upon hDOR incorporation into the bilayer (panels C,F), are shown. In panels B,C,E,F, the solid curves containing the open circles show the experimental spectra and the simulated spectra, respectively; the solid curves with the closed symbols represent the deconvoluted single lipid component spectra for POPC (closed circles) and SM (closed triangles), respectively, obtained from the simulated fits. The simulated fit to the experimental spectra is the result of an appropriately weighted sum of the component curves.

Figure 2

PWR spectra obtained for a solid-supported lipid bilayer containing a 1:1 mixture of POPC and SM (panels A,B, curves 1) and for the incorporation of unliganded hDOR into the lipid bilayer (∼10 nM, final concentration in the sample cell) (panels A,B, curves 2), obtained with 543.5 nm (green) exciting light using either p- (panels A,C) or s- polarized light (panels B,D). Panels C and D represent the simulation results for the incorporated receptor, as described in the legend to Figure 1, with the difference that the receptor is now unliganded.

Figure 3

PWR spectra obtained for the supported lipid bilayer containing a 1:1 mixture of POPC and SM (panels A,B, curves 1) and for the incorporation of antagonist (NTI)-bound hDOR into the lipid bilayer (panels A,B, curves 2) obtained with 543.5 nm (green) exciting light using either p- (panels A,C) or s-polarized light (panels B,D). Panels C and D represent the simulation results for the incorporated receptor, as described in the legend to Figure 1, with the difference that the receptor is now bound to the antagonist, naltrindole.

Figure 4

Time evolution of PWR spectra, obtained with s-polarized light, upon agonist (DPDPE) addition to the receptor when incorporated into a POPC:SM (1:1) lipid bilayer (panel A). Looking at the right shoulder, going from left to right the spectra represent increasing time intervals: (solid line) 0 min; (···) 2 min; (---) 6 min; (–––) 8 min; (– – –) 16 min; and (— — —) 20 min. Panel B represents a plot of the spectral shifts obtained in panel A as a function of time. The solid curve is a fit of the data to a single exponential using the following equation: Y=Y_max × (1−exp(−k × X)), where Y_max is the maximal rate obtained, k is the first-order rate constant and the half time is 0.693/k. The fit was obtained with GraphPad Prism (San Diego, CA).

Figure 5

Binding curves obtained for G-protein interaction (G_1α2-βγ) with the DPDPEliganded hDOR when reconstituted into a POPC:SM (1:1) bilayer obtained using p- (■) and s-polarized light (▲). Solid curves correspond to hyperbolic fits to the data performed using the following equation that describes the 1:1 binding of a ligand to a receptor: Y = (B_max × X)/ (K_D+ X). B_max represents the maximum concentration bound and K_D is the concentration of ligand required to reach half-maximal binding. Dissociation constant (K_D) values are given in Table 2.