Lipids, lipid rafts and caveolae: Their importance for GPCR signaling and their centrality to the endocannabinoid system

Abstract

Scientific views of cell membrane organization are presently changing. Rather than serving only as the medium through which membrane proteins diffuse, lipid bilayers have now been shown to form compartmentalized domains with different biophysical properties (rafts/caveolae). For membrane proteins such as the G protein coupled receptors (GPCRs), a raft domain provides a platform for the assembly of signaling complexes and prevents cross-talk between pathways. Lipid composition also has a strong influence on the conformational activity of GPCRs. For certain GPCRs, such as the cannabinoid receptors, the lipid bilayer has additional significance. Endocannabinoids such as anandamide (AEA) are created in a lipid bilayer from lipid and act at the membrane embedded CB₁ receptor. Endocannabinoids exiting the CB₁ receptor are transported either by a carrier-mediated or a simple diffusion process to the membrane of the postsynaptic cell. Following cellular uptake, perhaps via caveolae/lipid raft-related endocytosis, AEA is rapidly metabolized by a membrane-associated enzyme, fatty acid amide hydrolase (FAAH) located in the endoplasmic reticulum. The entry point for AEA into FAAH appears to be from the lipid bilayer. This review explores the importance of lipid composition and lipid rafts to GPCR signaling and then focuses on the intimate relationship that exists between the lipid environment and the endocannabinoid system.

Introduction

Views of how cell membranes are organized are presently changing. The lipid bilayer that constitutes these membranes is no longer understood to be a homogeneous fluid in which membrane proteins diffuse. Cell biological and biophysical studies have indicated that there is selective confinement of proteins and lipids in discrete regions of the membrane, called lipid rafts. These raft regions are composed mainly of sphingolipids and cholesterol in the outer leaflet, somehow connected to domains of unknown composition in the inner leaflet (Simons and Vaz, 2004). Specific classes of proteins are associated with the rafts. Caveolae, a special type of lipid raft, are plasma membrane invaginations that have been implicated in a variety of cellular processes, including signal transduction, endocytosis, transcytosis and cholesterol trafficking. It has been proposed that the function of the lipid raft is the spatial concentration of specific sets of proteins in order to increase the efficiency and specificity of signal transduction by facilitating interactions between proteins and by preventing cross-talk between pathways (Moffett et al., 2000). The topic of lipid rafts has been extensively reviewed [see (Harder and Engelhardt, 2004, Helms and Zurzolo, 2004, Mayor and Rao, 2004, Salaun et al., 2004, Simons and Vaz, 2004) for recent reviews].

In model membranes, raft lipids have been proposed to exist in a separate phase similar to the liquid ordered (l_o) phase (Brown and London, 1997, Schroeder et al., 1994). Acyl chains of lipids in the l_o phase are tightly packed and highly ordered and extended. In mixtures of phospholipids, sphingolipids, and cholesterol at concentrations similar to those in the plasma membrane at 37 °C (Ahmed et al., 1997), the presence of unusually long saturated acyl chains on sphingolipids promotes phase separation and formation of the l_o phase. This model membrane raft formation is thought likely to mimic raft formation in biological membranes as well (Ahmed et al., 1997).

Cell membranes that are in the disordered phase have been found to be fully solubilized by non-ionic detergents. Detergent resistant membranes (DRMs) have been found to be rich in sphingolipids and cholesterol. The long saturated acyl chains and high acyl chain melting temperature of sphingolipids mediate their association in detergent resistant domains. These sphingolipid and cholesterol-rich domains have the properties of the liquid-ordered (l_o) phase previously described in model membranes. Detergent-resistant membranes (DRMs) isolated from cells are thought to be derived from rafts in living cells because there is a good correlation between the onset of the ordered phase and the acquisition of detergent insolubility in model membranes (Ahmed et al., 1997, Schroeder et al., 1998). Several lines of investigation support the idea that DRMs are not detergent-induced artifacts, but exist as domains in cell membranes (Brown and London, 1997).

Using Electrospray Ionization/Mass Spectrometric Analysis, Pike et al. reported that lipid rafts isolated in the absence of detergent not only contain, at their core, the high cholesterol DRM material discussed above, but also contain loosely associated lipids that are enriched in anionic phospholipids, plasmalogens, and arachidonic acid-containing molecular species. The enrichment in lipid rafts of arachidonic acid-containing phospholipids implies that these domains may represent a localized pool of substrate for the generation of arachidonic acid in response to cell activation. Further, phosphatidylserine is substantially enriched in this compartment, suggesting it may be important for the function of these domains, etc. (Pike et al., 2002).

It has been postulated that proteins with a high affinity for an ordered lipid environment are selectively recruited to rafts (Schroeder et al., 1994). Proteins modified with saturated fatty acyl chains that could partition favorably into l_o phase domains would be expected to partition into l_o domains. The best characterized acyl modifications of proteins which target proteins to DRMs are structures that include dual saturated acyl chains (Moffett et al., 2000). These are glycosylphosphatidylinositol membrane anchors (Arreaza and Brown, 1995, Rodgers et al., 1994) [which contain predominantly saturated fatty acids; McConville and Ferguson, 1993], modification with tandem amide-linked myristate and thioester-linked palmitate (Milligan et al., 1995, Shenoy-Scaria et al., 1994), and modification with tandem thioester-linked palmitate chains (Arni et al., 1998).