Lipid raft and regulation of G-protein coupled receptors signaling

G-protein coupled receptors are the most abundant class of receptors in the human body. These receptors are transmembrane proteins showing the ligand-binding site on the extracellular surface whereas possess on the intracellular region a unique class of signaling molecules called G protein which regulate the intracellular messengers (G proteins are so named since they bind the guanine nucleotides GTP and GDP). Each transmembrane region consists of a single α helix, and the α helices are arranged in a characteristic structural motif that is similar in all membranes of this receptor class. In the resting state (non-stimulated), the cytoplasmic domain of the receptor is non-covalently linked to a G protein complex formed of α and βγ subunits. Upon activation, the α subunit exchanges GDP for GTP. The α-GTP subunit then dissociates from βγ subunit, and they both cross the inner leaflet of the plasma membrane to interact with a number of different effectors. These effectors include adenylate cyclase, phospholipase C, various ion channels, and other classes of proteins.

Signal mediated by G proteins are usually terminated by the hydrolysis of GTP to GDP, which is catalyzed by the inherent GTPase activity of the α subunit.

The intracellular molecular pathways recruited by the GPCRs are strictly dependent on the typology of the α subunit, indeed a large number of Gα

protein isoforms have been identified, with specific effect on their target.

The most common are distinguished in Gα_s, Gα_i ,Gα_q and Gα_12/13):

-­‐ Gαs induces the activation of adenilate cyclase;

-­‐ Gαi orGαo have an inhibitory effect on the adenilate cyclase activity;

-­‐ Gαq is responsible of the a Ca²⁺ intracellular levels increase;

-­‐ Gα12/13 is involved in the cytoskeleton proteins regulation by a Rho-GTPase.

Moreover, the βγ complex of the G proteins can also act as second messenger molecules, although their actions are not as thoroughly characterized.

Synthesis of GPCRs starts in the ER where they form either homo- or heterodimeric structures. Following ER exit, GPCRs transit through the Golgi apparatus and they undergo through additional modifications, such as oligosaccharide processing, fatty acylation and/or myristoylation in order to allow their final segregation in the lipid rafts. Indeed, at the end of these chemical reactions, GPCRs are packaged in exocytic transport vesicles and enter the endosomal system where they are subsequently targeted to the plasma membrane [162].

A large number of G-protein-coupled receptors are enriched in the lipid rafts or caveolae. This includes β1- and β2-adrenergic receptors, adenosine 1 (A1) receptor, angiotensin II type 1 (AT1) receptor, bradykinin B₁ and B₂ receptors, S1P1 receptor (S1PR1) and also muscarinic cholinergic receptor M2 [163-171]. Switching from inactive and active state, these receptors differently move between raft and non-raft region and mediate their intracellular signaling. Indeed, A1 and β2-adrenergic

receptor initially localize in the lipid raft and subsequently translocate into non-raft membranes after activation, whereas M₂, AT1, S1PR1 as well as B₁ receptors are targeted to the rafts upon activation by the agonist. Thus, ligand interaction induces changes in the receptor localization between raft and non-raft compartments. Moreover, ligands can also induce the translocation of the receptors into the lipid rafts, where G proteins and effector enzymes are localized to promote the development of the signaling. However, for some GPCRs, such as muscarinic acetylcholine receptor or AT1, interaction with the substrate leads to desensitization via a mechanism which involves the sequestration of the receptor from the cell surface [172] since both caveolar and non-caveolar lipid rafts are known to be involved in the endocytosis [137, 147, 150, 154]. Thus, for receptors that are recruited into the lipid rafts following agonist activation, it is possible that recruitment of rafts not only initiates signaling but also represents the first stage of the desensitization of the signal.

Caveolins have been shown to interact with several GPCRs regulating their signaling, as A1 receptor, M₂ receptor, and also B₂ receptor. The interaction with caveolin not only serve for the localization of the receptor, but also profoundly influence signaling from certain neurotransmitter receptors, indeed Cav-1 is known to be a lipid raft regulatory protein [173].

A particular experiment has used the “caveolin scaffolding domain“ as a bait to screen a library of synthetic peptides containing a motif able to bind Cav-1. GPCRs as α-adrenergic receptor, muscarinic receptor, endothelin receptor have been found to contain this peptidic motif but it is still undefined if it is responsible for association with the caveolar-domain [174].

After their segregation in the lipid rafts, localization of G-protein coupled receptor depend on their distribution between planar lipid raft and caveolae microdomains. However, a paper of Phil Oh in 2001 showed that after isolation of caveolae from other non-caveolar lipid raft microdomains in endothelial cells, it has been observed that Gα_q-protein interacts directly with caveolin, targeting the receptor in the caveolae, while Gαs, Gαi are targeted to lipid raft that does not contain caveolins [175] [176] (Figure 1.4). It has been reported that AT1 receptor, as a Gα_q protein-coupled receptor, even if contains the “caveolin scaffolding domain”, it is highly expressed in non-caveolar lipid raft. However, mutations of the “caveolin scaffolding domain” causes a reduction of AT1 expression on cell surface, meaning that Cav-1 bound has an important role in mediating the receptor sorting in plasma membrane even if it will be not localize in caveolae domain [177].

Figure.1.4 Phil O. et al. Molecular Biology of the Cell. 2001. Receptors localization in caveolae (V) and lipid rafts (LR), isolated from endothelial cell plasma membrane. Gi and Gs appear to be concentrated primarily in the lipid raft, while Gq is preferentially associated to caveolae

Thus, different G proteins may segregate into different subtype of lipid rafts depending on the presence of other components of the cells. Beside their localization it has been reported that an intact lipid rafts is necessary for the G protein-mediated signaling, indeed alteration of the lipid composition could affect the shift of the receptor from basal to active state and vice-versa. This has been confirmed by in-vitro experiments in cells treated with methyl-β-cyclodextrin, which is known to cause extraction of cholesterol from plasma membrane. Depletion of cholesterol destroys the tight lipid-order of the lipid rafts. This negatively affects the signaling of G protein targeted in the lipid rafts upon their activation, whereas facilitating the pathways mediated by G protein, which translocate in non-raft domains after activation. Indeed, depletion of cholesterol has been associated to impairment of particular G-protein mediated signaling, such as bradykinin-stimulated phosphatydil-inositol turnover [178], as well as thrombin–stimulated phosphatidic acid generation and phosphatidyl-inositol 3,4,5 triphosphate production is inhibited by cholesterol depletion [179]. On the contrary, both myocyte contraction and adenilate cyclase activation mediated by β2-adrenergic and adenosine-1 receptors, respectively, are enhanced by cholesterol depletion [165, 166], whereas supplementation of cholesterol and their fixation in the lipid raft environment inhibit the activity.

These findings suggest the important role that cholesterol and lipid rafts composition play in the regulation of G protein signaling, which is also dependent not only on the localization of the receptors in the lipid rafts or caveolae, but also on the partitioning of these protein between the rafts and non-rafts domains changing from a basal to an active state and vice-versa [136].