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 are viewed as laterally homogeneous lipid assemblies where the bilayer functions as a non-interacting two-dimensional solvent, having little influence on membrane protein function. During the last decade, evidence has accumulated indicating that lateral segregation into lipid microdomains exists in membranes (see 2
for a review). Microdomain formation occurs by spontaneous aggregation of certain naturally occurring lipids that spontaneously aggregate in the plane of the membrane driven solely by distinctive intermolecular interactions, including van der Waals forces between the long, largely saturated chains of sphingomyelin and glycosphingolipids, as well as hydrogen bonding between adjacent glycosyl moieties (3
). Operationally, lipid laterally segregated microdomains have been defined by their insolubility in nonionic detergents at 4°C, and by a light buoyant density on sucrose gradients (3
). Microdomains are also characterized by a higher degree of molecular order (induced by the presence of saturated fatty acyl chains and sometimes cholesterol), and by being thicker than the surrounding liquid-disordered lipids in the membrane.
Different methodologies have been used to investigate lipid microdomains, both in vivo
and employing model membranes, such as non-ionic detergent extraction at low temperatures, cholesterol depletion, and direct observation by different microscopic and spectroscopic methods (AFM, FRET, FRAP, FCS, SPT, etc.; see 3
for a review). Depending on the method employed and whether model membranes or cells were studied, results on the size, lifetime and composition of microdomains are diverse and controversial. Apart from these controversies, there is common agreement on the existence of microdomains in model membranes that form spontaneously in mixtures of SM and unsaturated phospholipids such as DOPC and POPC, in both the presence and absence of cholesterol (2
One of the most important roles of lipid microdomains at the cell surface may be their function in signal transduction. Given the overall low abundance of key signaling molecules in cells, one way to account for the rapid response characteristic of signal transduction is that cells concentrate signaling molecules in membrane microdomains. Such domains could be viewed as platforms that serve to localize the requisite components, facilitating their interaction and supporting signaling. In this view, a given receptor and its effector molecules would be localized in a single microdomain. Activation by the hormone signal would lead to a rapid and efficient signal transduction due to the close proximity of the interacting partners. Specificity of the signaling could be enhanced by receptor restriction to a particular domain containing a different subset of signaling partners. In another view, complementary components of a signaling pathway would be segregated into different domains under basal conditions, and following activation those components could be joined by domain fusion. Alternatively, domains could contain nearly complete signaling pathways that would be activated when a receptor or another molecule is recruited into the microdomain, promoting interaction and leading to signaling. Microdomains could provide more specificity and diversity in the signaling cascade by providing compartmentalization to the different components involved or by modulating the intrinsic activities of the proteins located therein. The relative biological importance of these various mechanisms remains to be elucidated.
Under basal conditions, some GPCRs are almost exclusively located in microdomains (e.g. more than 90% of the gonadotrophin-releasing hormone receptor (6
)), whereas others are present in a small amount (e.g. <10% of the oxytocin receptor (7
)). In other cases, the localization of GPCRs to lipid microdomains is modulated by ligand binding (for reviews see 9
). For the β2
-adrenergic receptor (11
) and the adenosine A1
), treatment with agonist causes translocation of the cognate receptor out of lipid microdomains. By contrast, the angiotensin II type receptor (13
), the muscarinic receptor (14
) and the bradykinin B1
) are targeted to microdomains upon activation by agonist. Others, such as the endothelin receptor are apparently unaffected by agonist binding (18
). However, the functional significance of agonist-induced receptor localization has not been demonstrated. It is possible that ligand-induced movement of receptor into lipid microdomains may promote receptor association with G-proteins or effector enzymes that are enriched in this compartment. Since laterally segregated membrane regions are involved in endocytosis, the sequestration of receptor into those domains may be involved in desensitization via removal from the cell surface.
Another issue is hydrophobic matching involving the length of the transmembrane domains of the protein, because a liquid-ordered bilayer is thicker than a liquid-disordered one. These parameters play a role in protein sorting to the cell surface (19
). However, how precisely the transmembrane domains need to be matched with the thickness of the bilayer is an open issue. So far, no detailed analysis has been carried out to find out how transmembrane proteins having different transmembrane domain lengths partition into the different domains.
In the present work we have investigated the partitioning of a GPCR (i.e. the human delta-opioid receptor, hDOR) between PC-rich and SM-rich domains in a model system consisting of solid-supported lipid bilayers, and the resulting effects of the microenvironmental differences on G-protein binding affinity and activation. This receptor belongs to family A of GPCRs and is involved in a plethora of biological events such as analgesia, locomotive activity, blood pressure, gastrointestinal motility, learning and memory (21
). It should be mentioned that the hDOR is mainly located in the spinal cord and in the brain, areas whose membranes are very rich in unsaturated lipids and sphingolipids, and thus have the potential for microdomain formation. This receptor has been the subject of interest in our laboratories over the last several years, and we have investigated ligand-induced conformational changes and receptor-G protein interactions using plasmon-waveguide resonance (PWR) spectroscopy (22
We have also used PWR spectroscopy to examine solid-supported lipid bilayers consisting of pure lipids (DOPC, POPC and SM), and PC:SM binary mixtures (24
). We have shown that a single lipid bilayer formed from the PC:SM mixture consists of microdomains enriched in the SM component, coexisting with PC-rich domains, whose different optical properties allow observation of separate resonances in a PWR experiment. These are formed spontaneously over time as a consequence of lateral phase separation. The uniqueness of the PWR technique is that it allows the mass and structural characteristics of a lipid membrane to be determined for a single bilayer in both steady-state and kinetic modes. Unlike other techniques for domain visualization, PWR can also provide insights into microenvironmental effects on protein functional properties (23
) in model systems that simulate a natural lipid bilayer. Simulation of the PWR spectra allowed the deconvolution of the complex spectra of the binary mixtures into single resonance spectra corresponding to the two microdomains. Comparison of the spectra of the mixtures with those of the pure lipids has shown that the resonance occurring at smaller angles is due to a microdomain containing mainly PC molecules, with a small admixture of SM (PC-rich component), whereas the higher angle resonance consists predominantly of a microdomain composed of SM molecules, with a small admixture of PC (SM-rich component). The ability to resolve resonances corresponding to these bilayer regions is due to their differing microstructure and optical properties, with the PC areas less well ordered, less tightly packed and thinner than the SM areas as a consequence of the presence of unsaturated fatty acyl chains.
In the present study, the partitioning of the hDOR between the two domains (PCand SM-rich domains) that form spontaneously in a POPC:SM (1:1) bilayer was directly determined by comparing the spectral shifts obtained for each resonance upon receptor insertion, and the amount of receptor in each microdomain was quantified by spectral simulation. The partitioning of the receptor between the two domains was found to be highly dependent on the type of ligand that was bound to the receptor. Binding of agonist to the unliganded receptor in the POPC:SM lipid bilayer resulted in receptor trafficking from the PC-rich to the SM-rich domain. We have also measured the binding affinities of G-protein to each of these receptor populations and have shown that the affinity for the agonist-bound hDOR in the SM-rich domain was 30-fold higher than for receptor in the PC-rich domain.