Signal transduction via G-protein-coupled receptors (GPCRs) is a fundamental pathway through which the functions of an individual cell can be integrated within the demands of a multicellular organism. Since this family of receptors first discovered, the proteins that constitute this signaling cascade and their interactions with one another have been studied intensely. In parallel, the pivotal role of lipids in the correct and efficient propagation of extracellular signals has attracted ever increasing attention. This is not surprising given that most of the signal transduction machinery is membrane-associated and therefore lipid-related. Hence, lipid-protein interactions exert a considerable influence on the activity of these proteins. This review focuses on the post-translational lipid modifications of GPCRs and G proteins (palmitoylation, myristoylation, and isoprenylation) and their significance for membrane binding, trafficking and signaling. Moreover, we address how the particular biophysical properties of different membrane structures may regulate the localization of these proteins and the potential functional consequences of this phenomenon in signal transduction. Finally, the interactions that occur between membrane lipids and GPCR effector enzymes such as PLC and PKC are also considered.

Most drugs currently used for human therapy interact with proteins, altering their activity to modulate the pathological cell physiology. In contrast, 2-hydroxy-9-cis-octadecenoic acid (Minerval) was designed to modify the lipid organization of the membrane. Its structure was deduced following the guidelines of the mechanism of action previously proposed by us for certain antitumor drugs. The antiproliferative activity of Minerval supports the above-mentioned hypothesis. This molecule augments the propensity of membrane lipids to organize into nonlamellar (hexagonal H II ) phases, promoting the subsequent recruitment of protein kinase C (PKC) to the cell membrane. The binding of the enzyme to membranes was marked and significantly elevated by Minerval in model (liposomes) and cell (A549) membranes and in heart membranes from animals treated with this drug. In addition, Minerval induced increased PKC␣ expression (mRNA and protein levels) in A549 cells. This drug also induced PKC activation, which led to a p53-independent increase in p21 CIP expression, followed by a decrease in the cellular concentrations of cyclins A, B, and D3 and cdk2. These molecular changes impaired the cell cycle progression of A549 cells. At the cellular and physiological level, administration of Minerval inhibited the growth of cancer cells and exerted antitumor effects in animal models of cancer without apparent histological toxicity. The present results support the potential use of Minerval and related compounds in the treatment of tumor pathologies.

After activation, agonist-occupied G protein-coupled receptors are phosphorylated by G protein-coupled receptor kinases and bind cytosolic ␤-arrestins, which uncouple the receptors from their cognate G proteins. Recent studies on the ␤ 2 -adrenergic receptor have demonstrated that ␤-arrestin also targets the receptors to clathrin-coated pits for subsequent internalization and activation of mitogen-activated protein kinases. We and others have previously shown that muscarinic acetylcholine receptors (mAChRs) of the m1, m3, and m4 subtype require functional dynamin to sequester into HEK-293 tsA201 cells, whereas m2 mAChRs sequester in a dynamin-independent manner. To investigate the role of ␤-arrestin in mAChR sequestration, we determined the effect of overexpressing ␤-arrestin-1 and the dominant-negative inhibitor of ␤-arrestin-mediated receptor sequestration, ␤-arrestin-1 V53D, on mAChR sequestration and function. Sequestration of m1, m3, and m4 mAChRs was suppressed by 60 -75% in cells overexpressing ␤-arrestin-1 V53D, whereas m2 mAChR sequestration was affected by less than 10%. In addition, overexpression of ␤-arrestin-1 V53D as well as dynamin K44A significantly suppressed m1 mAChR-mediated activation of mitogen-activated protein kinases. Finally, we investigated whether mAChRs sequester into clathrincoated vesicles by overexpressing Hub, a dominant-negative clathrin mutant. Although sequestration of m1, m3, and m4 mAChRs was inhibited by 50 -70%, m2 mAChR sequestration was suppressed by less than 10%. We conclude that m1, m3, and m4 mAChRs expressed in HEK-293 tsA201 cells sequester into clathrin-coated vesicles in a ␤-arrestin-and dynamin-dependent manner, whereas sequestration of m2 mAChRs in these cells is largely independent of these proteins.Exposure of many G protein-coupled receptors (GPCRs) 1 to their agonists results within seconds to minutes in attenuation of receptor responsiveness. An important step in this process of receptor desensitization is the rapid phosphorylation of agonist-bound receptors by G protein-coupled receptor kinases (1). These kinases phosphorylate serine and threonine residues located in the third cytoplasmic loop (i.e. m1 and m2 muscarinic acetylcholine receptors (mAChRs)) (2, 3) or in the cytoplasmic carboxyl-terminal tail of the receptors (for example, ␤ 2 -adrenergic receptors) (4). After phosphorylation, cytosolic ␤-arrestins bind with increased affinity to the receptors and sterically inhibit further coupling of the receptors with G proteins (1). To date, two ␤-arrestin isoforms, ␤-arrestin-1 (arrestin 2) and ␤-arrestin-2 (arrestin 3) have been identified, each undergoing alternative splicing (1). Both isoforms are ubiquitously expressed, with ␤-arrestin-1 being the major ␤-arrestin expressed in many tissues (1). Recent evidence indicates that ␤-arrestins do not only bind to GPCRs but also associate with nanomolar affinity with clathrin heavy chains and target ␤-arrestin-bound GPCRs to the clathrin-coated pits, leading to receptor internalization (5, 6). This process ...

Sustained stimulation of muscarinic acetylcholine receptors (mAChRs) and other G protein-coupled receptors usually leads to a loss of receptor binding sites from the plasma membrane, referred to as receptor sequestration. Receptor sequestration can occur via endocytosis of clathrin-coated vesicles that bud from the plasma membrane into the cell but may also be accomplished by other, as yet ill-defined, mechanisms. Previous work has indicated that the monomeric GTPase dynamin controls the endocytosis of plasma membrane receptors via clathrin-coated vesicles. To investigate whether mAChRs sequester in a receptor subtype-specific manner via dynamin-dependent clathrin-coated vesicles, we tested the effect of overexpressing the dominant-negative dynamin mutant K44A on m1, m2, m3, and m4 mAChR sequestration in HEK-293 cells. The m1, m2, m3, and m4 mAChRs sequestered rapidly in HEK-293 cells following agonist exposure but displayed dissimilar sequestration pathways. Overexpression of dynamin K44A mutant fully blocked m1 and m3 mAChR sequestration, whereas m2 mAChR sequestration was not affected. Also, m4 mAChRs, which like m2 mAChRs preferentially couple to pertussis toxin-sensitive G proteins, sequestered in a completely dynamin-dependent manner. Following agonist removal, sequestered m1 mAChRs fully reappeared on the cell surface, whereas sequestered m2 mAChRs did not. The distinct sequestration of m2 mAChRs was also apparent in COS-7 and Chinese hamster ovary cells. We conclude that the m2 mAChR displays unique subtype-specific sequestration that distinguishes this receptor from the m1, m3, and m4 subtypes. These results are the first to demonstrate that receptor sequestration represents a new type of receptor subtype-specific regulation within the family of mAChRs.Muscarinic acetylcholine receptors (mAChRs) 1 belong to the superfamily of plasma membrane receptors that regulate a large number of signal transduction pathways via activation of heterotrimeric GTP-binding proteins (G proteins). The mAChR family consists of five subtypes of cloned mAChRs and can be subdivided into two functional groups: the m1, m3, and m5 subtypes, which preferentially couple to the G q family of G proteins, and the m2 and m4 subtypes, which effectively activate the G i family of G proteins. The m1, m2, m3, and m4 receptors are widely expressed in the central nervous system and peripheral tissues, whereas m5 receptors are present in only minute amounts in hippocampus and other areas of the brain (1). Prolonged exposure of mAChRs and other G proteincoupled receptors to agonists usually results in the attenuation of the cellular response. One molecular mechanism of attenuation involves the phosphorylation of the receptors by G protein-coupled receptor kinases (GRKs) and increased binding of the inhibitory protein ␤-arrestin to the phosphorylated receptors, thereby inhibiting the coupling with G proteins (2). Another regulatory mechanism is the sequestration of receptors by which G protein-coupled receptors become inaccessible for hydrophil...

G protein-coupled receptors (GPCRs) 1 constitute the main class of membrane receptors and form the widest gene family known in the mammalian genome. Therefore, signal transduction via G proteins represents one of the most important ways of cell signaling (for a review, see Ref. 1). Upon activation by agonists, GPCRs undergo conformational changes that induce the activation of heterotrimeric G proteins, promoting the exchange of the GDP bound to the G␣ subunit for GTP. This exchange provokes the dissociation of the G␣ subunit from the G␤␥ dimer and enables both molecular entities to modulate the activity of specific effectors or other signaling proteins (e.g. G protein receptor kinases).The association of both GPCRs and G proteins to the plasma membrane makes them susceptible to their lipid environment so that lipid-protein interactions are crucial to their function. In recent years, evidence has accumulated showing that the plasma membrane organization is more complex than a simple "chaotic sea" of bipolar lipid molecules in a liquid-crystalline state that only serves to support membrane proteins (2). In fact, differentiated membrane domains with specific protein and lipid compositions can exist in a cell such as the basal and apical membranes of epithelial/endothelial cells, the pre-and postsynaptic membranes of neuronal synapses, or lipid rafts and caveolae. Moreover, the extracellular and cytosolic leaflets of the plasma membrane differ in their lipid composition (3), further demonstrating the relevance of lipids in the organization and function of the membrane. Natural membranes are composed of different amphitropic molecules that differ in their propensity to form secondary lipid structures that influence the structural properties of the membrane (for a review, see Ref. 4). In this context, hexagonal (H II ) structures regulate the localization and activity of some key membrane signaling proteins (5, 6). Indeed, we have recently been able to show that changes in the lipid composition of erythrocyte cell membranes can influence the membrane association of G proteins and protein kinase C in vivo (7). Moreover, the neural membranes of cold-adapted fishes contain higher levels of phosphatidylethanolamine (PE) species in winter than in summer (8). These lipid changes probably lower the solidto-liquid and lamellar-to-hexagonal phase transition temperatures of membranes to maintain protein function and cell signaling in neurons and other cells. These data suggest that hexagonal phase propensity plays a major role in regulating physiological processes and might also participate in the control of other pivotal cellular events influenced by membrane proteins, such as cell growth or energy metabolism.Our main goal was to elucidate the role of the membrane lipid structure in the association to membranes of heterotrimeric G proteins and the molecular entities formed after their receptor-mediated activation. For this purpose we used synthetic membranes (liposomes) and purified G proteins as model systems because their li...

Guanine nucleotide-binding proteins, G proteins, propagate incoming messages from receptors to effector proteins. They switch from an inactive to active state by exchanging a GDP molecule for GTP, and they return to the inactive form by hydrolyzing GTP to GDP. Small monomeric G proteins, such as Ras, are involved in controlling cell proliferation, differentiation and apoptosis, and they interact with membranes through isoprenyl moieties, fatty acyl moieties, and electrostatic interactions. This protein-lipid binding facilitates productive encounters of Ras and Raf proteins in defined membrane regions, so that signals can subsequently proceed through MEK and ERK kinases, which constitute the canonical MAP kinase signaling cassette. On the other hand, heterotrimeric G proteins undergo co/post-translational modifications in the alpha (myristic and/or palmitic acid) and the gamma (farnesol or geranylgeraniol) subunits. These modifications not only assist the G protein to localize to the membrane but they also help distribute the heterotrimer (Galphabetagamma) and the subunits generated upon activation (Galpha and Gbetagamma) to appropriate membrane microdomains. These proteins transduce messages from ubiquitous serpentine receptors, which control important functions such as taste, vision, blood pressure, body weight, cell proliferation, mood, etc. Moreover, the exchange of GDP by GTP is triggered by nucleotide exchange factors. Membrane receptors that activate G proteins can be considered as such, but other cytosolic, membranal or amphitropic proteins can accelerate the rate of G protein exchange or even activate this process in the absence of receptor-mediated activation. These and other protein-protein interactions of G proteins with other signaling proteins are regulated by their lipid preferences. Thus, G protein-lipid interactions control the features of messages and cell physiology.

Free triterpenic acids (TTPs) present in plants are bioactive compounds exhibiting multiple nutriceutical activities. The underlying molecular mechanisms have only been examined in part and mainly focused on anti-inflammatory properties, cancer and cardiovascular diseases, in all of which TTPs frequently affect membrane-related proteins. Based on the structural characteristics of TTPs, we assume that their effect on biophysical properties of cell membranes could play a role for their biological activity. In this context, our study is focused on the compounds, oleanolic (3β-hydroxy-12-oleanen-28-oic acid, OLA), maslinic (2α,3β-dihydroxy-12-oleanen-28-oic acid, MSL) and ursolic ((3β)-3-hydroxyurs-12-en-28-oic acid, URL) as the most important TTPs present in orujo olive oil. X-ray diffraction, differential scanning calorimetry, (31)P nuclear magnetic resonance and Laurdan fluorescence data provide experimental evidence that OLA, MSL and URL altered the structural properties of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and DPPC-Cholesterol (Cho) rich membranes, being located into the polar-hydrophobic interphase. Specifically, in DPPC membranes, TTPs altered the structural order of the L(β'), phase without destabilizing the lipid bilayer. The existence of a nonbilayer isotropic phase in coexistence with the liquid crystalline L(α) phase, as observed in DPPC:URL samples, indicated the presence of lipid structures with high curvature (probably inverted micelles). In DPPC:Cho membranes, TTPs affected the membrane phase properties increasing the Laurdan GP values above 40°C. MSL and URL induced segregation of Cho within the bilayer, in contrast to OLA, that reduced the structural organization of the membrane. These results strengthen the relevance of TTP interactions with cell membranes as a molecular mechanism underlying their broad spectrum of biological effects.

Olive oil consumption leads to high monounsaturated fatty acid intake, especially oleic acid, and has been associated with a reduced risk of hypertension. However, the molecular mechanisms and contribution of its different components to lower blood pressure (BP) require further evaluation. Here, we examined whether a synthetic, non-boxidation-metabolizable derivative of oleic acid, 2-hydroxyoleic acid (2-OHOA), can normalize BP in adult spontaneously hypertensive rats (SHRs) and whether its antihypertensive action involves cAMP-dependent protein kinase A (PKA) and Rho kinase, two major regulators of vascular smooth muscle contraction. Oral administration of 2-OHOA to SHRs induced sustained systolic BP decreases in a time-dependent