Platelet P2Y1 receptor exhibits constitutive G protein signaling and ß-arrestin 2 recruitment.

BACKGROUND: Purinergic P2Y1 and P2Y12 receptors (P2Y1-R and P2Y12-R) are G protein-coupled receptors (GPCR) activated by adenosine diphosphate (ADP) to mediate platelet activation, thereby playing a pivotal role in hemostasis and thrombosis. While P2Y12-R is the major target of antiplatelet drugs, no P2Y1-R antagonist has yet been developed for clinical use. However, accumulating data suggest that P2Y1-R inhibition would ensure efficient platelet inhibition with minimal effects on bleeding. In this context, an accurate characterization of P2Y1-R antagonists constitutes an important preliminary step. RESULTS: Here, we investigated the pharmacology of P2Y1-R signaling through Gq and ß-arrestin pathways in HEK293T cells and in mouse and human platelets using highly sensitive resonance energy transfer-based technologies (BRET/HTRF). We demonstrated that at basal state, in the absence of agonist ligand, P2Y1-R activates Gq protein signaling in HEK293T cells and in mouse and human platelets, indicating that P2Y1-R is constitutively active in physiological conditions. We showed that P2Y1-R also promotes constitutive recruitment of ß-arrestin 2 in HEK293T cells. Moreover, the P2Y1-R antagonists MRS2179, MRS2279 and MRS2500 abolished the receptor dependent-constitutive activation, thus behaving as inverse agonists. CONCLUSIONS: This study sheds new light on P2Y1-R pharmacology, highlighting for the first time the existence of a constitutively active P2Y1-R population in human platelets. Given the recent interest of P2Y12-R constitutive activity in patients with diabetes, this study suggests that modification of constitutive P2Y1-R signaling might be involved in pathological conditions, including bleeding syndrome or high susceptibility to thrombotic risk. Thus, targeting platelet P2Y1-R constitutive activation might be a promising and powerful strategy for future antiplatelet therapy.

Glycerophosphodiesterase 3 (GDE3) is a lysophosphatidylinositol-specific ectophospholipase C acting as an endocannabinoid signaling switch.

Endocannabinoid signaling plays a regulatory role in various (neuro)biological functions. 2-arachidonoylglycerol (2-AG) is the most abundant endocannabinoid, and although its canonical biosynthetic pathway involving phosphoinositide-specific phospholipase C and diacylglycerol lipase α is known, alternative pathways remain unsettled. Here, we characterize a noncanonical pathway implicating glycerophosphodiesterase 3 (GDE3, from GDPD2 gene). Human GDE3 expressed in HEK293T cell membranes catalyzed the conversion of lysophosphatidylinositol (LPI) into monoacylglycerol and inositol-1-phosphate. The enzyme was equally active against 1-acyl and 2-acyl LPI. When using 2-acyl LPI, where arachidonic acid is the predominant fatty acid, LC-MS analysis identified 2-AG as the main product of LPI hydrolysis by GDE3. Furthermore, inositol-1-phosphate release into the medium occurred upon addition of LPI to intact cells, suggesting that GDE3 is actually an ecto-lysophospholipase C. In cells expressing G-protein-coupled receptor GPR55, GDE3 abolished 1-acyl LPI-induced signaling. In contrast, upon simultaneous ex-pression of GDE3 and cannabinoid receptor CB2, 2-acyl LPI evoked the same signal as that induced by 2-AG. These data strongly suggest that, in addition to degrading the GPR55 LPI ligand, GDE3 can act as a switch between GPR55 and CB2 signaling. Coincident with a major expression of both GDE3 and CB2 in the spleen, spleens from transgenic mice lacking GDE3 displayed doubling of LPI content compared with WT mice. Decreased production of 2-AG in whole spleen was also observed, supporting the in vivo relevance of our findings. These data thus open a new research avenue in the field of endocannabinoid generation and reinforce the view of GPR55 and LPI being genuine actors of the endocannabinoid system.

GHSR-D2R heteromerization modulates dopamine signaling through an effect on G protein conformation.

The growth hormone secretagogue receptor (GHSR) and dopamine receptor (D2R) have been shown to oligomerize in hypothalamic neurons with a significant effect on dopamine signaling, but the molecular processes underlying this effect are still obscure. We used here the purified GHSR and D2R to establish that these two receptors assemble in a lipid environment as a tetrameric complex composed of two each of the receptors. This complex further recruits G proteins to give rise to an assembly with only two G protein trimers bound to a receptor tetramer. We further demonstrate that receptor heteromerization directly impacts on dopamine-mediated Gi protein activation by modulating the conformation of its α-subunit. Indeed, association to the purified GHSR:D2R heteromer triggers a different active conformation of Gαi that is linked to a higher rate of GTP binding and a faster dissociation from the heteromeric receptor. This is an additional mechanism to expand the repertoire of GPCR signaling modulation that could have implications for the control of dopamine signaling in normal and physiopathological conditions.

Deciphering biased inverse agonism of cangrelor and ticagrelor at P2Y12 receptor.

P2Y12 receptor (P2Y12-R) is one of the major targets for drug inhibiting platelet aggregation in the treatment/prevention of arterial thrombosis. However, the clinical use of P2Y12-R antagonists faces some limitations, such as a delayed onset of action (clopidogrel) or adverse effect profile (ticagrelor, cangrelor), justifying the development of a new generation of P2Y12-R antagonists with a better clinical benefit-risk balance. Although the recent concept of biased agonism offers the possibility to alleviate undesirable adverse effects while preserving therapeutic outcomes, it has never been explored at P2Y12-R. For the first time, using highly sensitive BRET2-based probes, we accurately delineated biased ligand efficacy at P2Y12-R in living HEK293T cells on G protein activation and downstream effectors. We demonstrated that P2Y12-R displayed constitutive Gi/o-dependent signaling that is impaired by the R122C mutation, previously associated with a bleeding disorder. More importantly, we reported the biased inverse agonist efficacy of cangrelor and ticagrelor that could underlie their clinical efficacy. Our study points out that constitutive P2Y12-R signaling is a normal feature of the receptor that might be essential for platelets to respond faster to a vessel injury. From a therapeutic standpoint, our data suggest that the beneficial advantages of antiplatelet drugs might be more related to inverse agonism at P2Y12-R than to antagonism of ADP-mediated signaling. In the future, deciphering P2Y12-R constitutive activity should allow the discovery of more selective biased P2Y12-R blockers demonstrating therapeutic advantages over classical antiplatelet drugs by improving therapeutic outcomes and concomitantly relieving undesirable adverse effects.

Ligand-specific conformational transitions and intracellular transport are required for atypical chemokine receptor 3-mediated chemokine scavenging.

The atypical chemokine receptor ACKR3 contributes to chemotaxis by binding, internalizing, and degrading the chemokines CXCL11 and CXCL12 to shape and terminate chemotactic gradients during development and immune responses. Although unable to trigger G protein activation, both ligands activate G protein-independent ACKR3 responses and prompt arrestin recruitment. This offers a model to specifically study ligand-specific receptor conformations leading to G protein-independent signaling and to functional parameters such as receptor transport and chemokine degradation. We here show chemokine specificity in arrestin recruitment, by different effects of single amino acid substitutions in ACKR3 on arrestin in response to CXCL12 or CXCL11. Chemokine specificity in receptor transport was also observed, as CXCL11 induced faster receptor internalization, slower recycling, and longer intracellular sojourn of ACKR3 than CXCL12. Internalization and recycling rates of the ACKR3 R1423.50A substitution in response to each chemokine were similar; however, ACKR3 R1423.50A degraded only CXCL12 and not CXCL11. This suggests that ligand-specific intracellular receptor transport is required for chemokine degradation. Remarkably, the failure of ACKR3 R1423.50A to degrade CXCL11 was not caused by the lack of arrestin recruitment; rather, arrestin was entirely dispensable for scavenging of either chemokine. This suggests the involvement of another, yet unidentified, ACKR3 effector in scavenging. In summary, our study correlates ACKR3 ligand-specific conformational transitions with chemokine-dependent receptor transport dynamics and points toward unexpected ligand specificity in the mechanisms of chemokine degradation.

Dual agonist occupancy of AT1-R-α2C-AR heterodimers results in atypical Gs-PKA signaling.

Hypersecretion of norepinephrine (NE) and angiotensin II (AngII) is a hallmark of major prevalent cardiovascular diseases that contribute to cardiac pathophysiology and morbidity. Herein, we explore whether heterodimerization of presynaptic AngII AT1 receptor (AT1-R) and NE α2C-adrenergic receptor (α2C-AR) could underlie their functional cross-talk to control NE secretion. Multiple bioluminescence resonance energy transfer and protein complementation assays allowed us to accurately probe the structures and functions of the α2C-AR-AT1-R dimer promoted by ligand binding to individual protomers. We found that dual agonist occupancy resulted in a conformation of the heterodimer different from that induced by active individual protomers and triggered atypical Gs-cAMP-PKA signaling. This specific pharmacological signaling unit was identified in vivo to promote not only NE hypersecretion in sympathetic neurons but also sympathetic hyperactivity in mice. Thus, we uncovered a new process by which GPCR heterodimerization creates an original functional pharmacological entity and that could constitute a promising new target in cardiovascular therapeutics.

Apolipoprotein A-I enhances proliferation of human endothelial progenitor cells and promotes angiogenesis through the cell surface ATP synthase.

BACKGROUND: Human endothelial progenitor cells (hEPC) correspond to a subtype of stem cells which, in the presence of angiogenic stimuli, can be mobilized from bone marrow to circulation and then recruited to the damaged endothelium, where they differentiate into mature endothelial cells. High-density lipoproteins (HDL) increase the level and functionality (proliferation, migration, differentiation, angiogenesis capacity) of circulating hEPC; however, the contribution of receptors for HDL and/or apolipoprotein A-I (apoA-I), the main HDL apolipoprotein, in these effects is still unclear. On mature endothelial cells, the cell surface F1-ATP synthase has been previously characterized as a high affinity receptor of apoA-I, whereas the scavenger receptor SR-BI mainly binds with fully lipidated HDL and displays a poor affinity for lipid-free apoA-I. Furthermore, it was shown that apoA-I binding to surface ATP synthase on mature endothelial cells promotes cell proliferation, whereas inhibits apoptosis. In this work, we aimed to determine the effect of apoA-I in the proliferation and the angiogenic capacity of early hEPC, and the contribution of the cell surface ATP synthase in these events. RESULTS: We first evidenced that early hEPC express the ATP synthase at the surface of nonpermeabilized cells, where it is not colocalized with MitoTracker, a mitochondria marker. ApoA-I (50 µg/mL) increases hEPC proliferation (+14.5%, p<0.001) and potentiates the effect of hEPC on a cellular model of angiogenesis, with an increase of +31% (p<0.01) in branch point counting and in tubule length. These effects of apoA-I were totally reversed in the presence of ATP synthase inhibitors, such as IF1 or oligomycin, whereas the inhibition of the HDL receptor, SR-BI, partially inhibits these events. CONCLUSIONS: These results provide the first evidence that surface ATP synthase is expressed on early hEPC, where it mediates apoA-I effects in hEPC proliferation and in angiogenesis. This knowledge could be helpful for future investigations focused on the regulation of the number and functionality of these cells and in the development of new therapies for the treatment of diseases, such as cardiovascular disease.

Role of the ubiquitin-proteasome system in the regulation of P2Y13 receptor expression: impact on hepatic HDL uptake.

The protective effect of high density lipoproteins (HDL) against atherosclerosis is mainly attributed to their capacity to transport excess cholesterol from peripheral tissues back to the liver for further elimination into the bile, a process called reverse cholesterol transport (RCT). Recently, the importance of the P2Y13 receptor (P2Y13-R) was highlighted in HDL metabolism since HDL uptake by the liver was decreased in P2Y13-R deficient mice, which translated into impaired RCT. Here, we investigated for the first time the molecular mechanisms regulating cell surface expression of P2Y13-R. When transiently expressed, P2Y13-R was mainly detected in the endoplasmic reticulum (ER) and strongly subjected to proteasome degradation while its homologous P2Y12 receptor (P2Y12-R) was efficiently targeted to the plasma membrane. We observed an inverse correlation between cell surface expression and ubiquitination level of P2Y13-R in the ER, suggesting a close link between ubiquitination of P2Y13-R and its efficient targeting to the plasma membrane. The C-terminus tail exchange between P2Y13-R and P2Y12-R strongly restored plasma membrane expression of P2Y13-R, suggesting the involvement of the intra-cytoplasmic tail of P2Y13-R in expression defect. Accordingly, proteasomal inhibition increased plasma membrane expression of functionally active P2Y13-R in hepatocytes, and consequently stimulated P2Y13-R-mediated HDL endocytosis. Importantly, proteasomal inhibition strongly potentiated HDL hepatic uptake (>200 %) in wild-type but not in P2Y13-R-deficient mice, thus reinforcing the role of P2Y13-R expression in regulating HDL metabolism. Therefore, specific inhibition of the ubiquitin-proteasome system might be a novel powerful HDL therapy to enhance P2Y13-R expression and consequently promote the overall RCT.

Serum IF1 concentration is independently associated to HDL levels and to coronary heart disease: the GENES study.

HDL is strongly inversely related to cardiovascular risk. Hepatic HDL uptake is controlled by ecto-F1-ATPase activity, and potentially inhibited by mitochondrial inhibitor factor 1 (IF1). We recently found that IF1 is present in serum and correlates with HDL-cholesterol (HDL-C). Here, we have evaluated the relationship between circulating IF1 and plasma lipoproteins, and we determined whether IF1 concentration is associated with the risk of coronary heart disease (CHD). Serum IF1 was measured in 648 coronary patients ages 45-74 and in 669 matched male controls, in the context of a cross-sectional study on CHD. Cardiovascular risk factors were documented for each participant, including life-style habits and biological and clinical markers. In controls, multivariate analysis demonstrated that IF1 was independently positively associated with HDL-C and apoA-I (r = 0.27 and 0.28, respectively, P < 0.001) and negatively with triglycerides (r = -0.23, P < 0.001). Mean IF1 concentration was lower in CHD patients than in controls (0.43 mg/l and 0.53 mg/l, respectively, P < 0.001). In multivariate analyses, following adjustments on cardiovascular risk factors or markers, IF1 was negatively related to CHD (P < 0.001). This relationship was maintained after adjustment for HDL-C or apoA-I. This study identifies IF1 as a new determinant of HDL-C that is inversely associated with CHD.

Endosome-to-cytosol transport of viral nucleocapsids.

During viral infection, fusion of the viral envelope with endosomal membranes and nucleocapsid release were thought to be concomitant events. We show here that for the vesicular stomatitis virus they occur sequentially, at two successive steps of the endocytic pathway. Fusion already occurs in transport intermediates between early and late endosomes, presumably releasing the nucleocapsid within the lumen of intra-endosomal vesicles, where it remains hidden. Transport to late endosomes is then required for the nucleocapsid to be delivered to the cytoplasm. This last step, which initiates infection, depends on the late endosomal lipid lysobisphosphatidic acid (LBPA) and its putative effector Alix/AIP1, and is regulated by phosphatidylinositol-3-phosphate (PtdIns3P) signalling via the PtdIns3P-binding protein Snx16. We conclude that the nucleocapsid is exported into the cytoplasm after the back-fusion of internal vesicles with the limiting membrane of late endosomes, and that this process is controlled by the phospholipids LBPA and PtdIns3P and their effectors.

Inverse agonist efficacy of selatogrel blunts constitutive P2Y12 receptor signaling by inducing the inactive receptor conformation.

Selatogrel is a potent inhibitor of adenosine diphosphate (ADP) binding to the P2Y12 receptor, preventing platelet activation. We have previously shown that the P2Y12 receptor constitutively activates Gi- and Go-protein-mediated signaling in human platelets. Here, we report that selatogrel acts as an inverse agonist of the P2Y12 receptor. Specifically, using bioluminescence resonance energy transfer2 (BRET2) probes, selatogrel, ticagrelor, and elinogrel were shown to stabilize the inactive form of the Gαi/o-Gßγ complex in cells with recombinant expression of the P2Y12 receptor. In dose-response experiments, while selatogrel exhibited a maximal efficacy similar to ticagrelor, selatogrel was approximately 100-fold more potent than ticagrelor. Quantification of relative cyclic adenosine monophosphate (cAMP) levels in cells expressing the cAMP BRET1 sensor (CAMYEL probe) confirmed that selatogrel completely abolished the constitutive activity of the P2Y12 receptor. In agreement, selatogrel increased basal cAMP levels in human platelets, confirming inverse agonism on the endogenous human platelet P2Y12 receptor. In agreement with the biochemical phenotype of inverse agonism efficacy of selatogrel, the 2.8 Angstrom resolution cocrystal structure of selatogrel bound to the P2Y12 receptor confirmed that selatogrel stabilizes the inactive, basal state of the receptor. Selatogrel bound to pocket 1, spanning helix III to VII. Furthermore, the binding mode of selatogrel, suggesting steric overlap with the proposed binding site of ADP and the ADP analog 2-methylthioadenosine diphosphate (2MeSADP), agrees with the functional characterization of selatogrel preventing platelet activation by blocking ADP binding to the P2Y12 receptor.

Atomic force microscopy-single-molecule force spectroscopy unveils GPCR cell surface architecture.

G protein-coupled receptors (GPCRs) form the largest family of cell surface receptors. Despite considerable insights into their pharmacology, the GPCR architecture at the cell surface still remains largely unexplored. Herein, we present the specific unfolding of different GPCRs at the surface of living mammalian cells by atomic force microscopy-based single molecule force spectroscopy (AFM-SMFS). Mathematical analysis of the GPCR unfolding distances at resting state revealed the presence of different receptor populations relying on distinct oligomeric states which are receptor-specific and receptor expression-dependent. Moreover, we show that the oligomer size dictates the receptor spatial organization with nanoclusters of high-order oligomers while lower-order complexes spread over the whole cell surface. Finally, the receptor activity reshapes both the oligomeric populations and their spatial arrangement. These results add an additional level of complexity to the GPCR pharmacology until now considered to arise from a single receptor population at the cell surface.

A severe form of abetalipoproteinemia caused by new splicing mutations of microsomal triglyceride transfer protein (MTTP).

Abetalipoproteinemia is a rare autosomal recessive disease characterized by low lipid levels and by the absence of apoB-containing lipoproteins. It is the consequence of microsomal triglyceride transfer protein (MTTP) deficiency. We report two patients with new MTTP mutations. We studied their functional consequences on the triglyceride transfer function using duodenal biopsies. We transfected MTTP mutants in HepG2 and HeLa cells to investigate their association with protein disulfide isomerase (PDI) and their localization at the endoplasmic reticulum. These children have a severe abetalipoproteinemia. Both of them had also a mild hypogammaglobulinemia. They are compound heterozygotes with c.619G>T and c.1237-28A>G mutations within the MTTP gene. mRNA analysis revealed abnormal splicing with deletion of exon 6 and 10, respectively. Deletion of exon 6 (Δ6-MTTP) introduced a frame shift in the reading frame and a premature stop codon at position 234. Despite the fact that Δ6-MTTP and Δ10-MTTP mutants were not capable of binding PDI, both MTTP mutant proteins normally localize at the endoplasmic reticulum. However, these two mutations induce a loss of MTTP triglyceride transfer activity. These two mutations lead to abnormal truncated MTTP proteins, incapable of binding PDI and responsible for the loss of function of MTTP, thereby explaining the severe abetalipoproteinemia phenotype of these children.

P2Y13 receptor is critical for reverse cholesterol transport.

UNLABELLED: A major atheroprotective functionality of high-density lipoproteins (HDLs) is to promote "reverse cholesterol transport" (RCT). In this process, HDLs mediate the efflux and transport of cholesterol from peripheral cells and its subsequent transport to the liver for further metabolism and biliary excretion. We have previously demonstrated in cultured hepatocytes that P2Y(13) (purinergic receptor P2Y, G protein-coupled, 13) activation is essential for HDL uptake but the potential of P2Y(13) as a target to promote RCT has not been documented. Here, we show that P2Y(13)-deficient mice exhibited a decrease in hepatic HDL cholesterol uptake, hepatic cholesterol content, and biliary cholesterol output, although their plasma HDL and other lipid levels were normal. These changes translated into a substantial decrease in the rate of macrophage-to-feces RCT. Therefore, hallmark features of RCT are impaired in P2Y(13)-deficient mice. Furthermore, cangrelor, a partial agonist of P2Y(13), stimulated hepatic HDL uptake and biliary lipid secretions in normal mice and in mice with a targeted deletion of scavenger receptor class B type I (SR-BI) in liver (hypomSR-BI-knockout(liver)) but had no effect in P2Y(13) knockout mice, which indicate that P2Y(13)-mediated HDL uptake pathway is independent of SR-BI-mediated HDL selective cholesteryl ester uptake. CONCLUSION: These results establish P2Y(13) as an attractive novel target for modulating RCT and support the emerging view that steady-state plasma HDL levels do not necessarily reflect the capacity of HDL to promote RCT.

Hrs and SNX3 functions in sorting and membrane invagination within multivesicular bodies.

After internalization, ubiquitinated signaling receptors are delivered to early endosomes. There, they are sorted and incorporated into the intralumenal invaginations of nascent multivesicular bodies, which function as transport intermediates to late endosomes. Receptor sorting is achieved by Hrs--an adaptor--like protein that binds membrane PtdIns3P via a FYVE motif-and then by ESCRT complexes, which presumably also mediate the invagination process. Eventually, intralumenal vesicles are delivered to lysosomes, leading to the notion that EGF receptor sorting into multivesicular bodies mediates lysosomal targeting. Here, we report that Hrs is essential for lysosomal targeting but dispensable for multivesicular body biogenesis and transport to late endosomes. By contrast, we find that the PtdIns3P-binding protein SNX3 is required for multivesicular body formation, but not for EGF receptor degradation. PtdIns3P thus controls the complementary functions of Hrs and SNX3 in sorting and multivesicular body biogenesis.

The ESCRT-I subunit TSG101 controls endosome-to-cytosol release of viral RNA.

Like other enveloped viruses, vesicular stomatitis virus infects cells through endosomes. There, the viral envelope undergoes fusion with endosomal membranes, thereby releasing the nucleocapsid into the cytoplasm and allowing infection to proceed. Previously, we reported that the viral envelope fuses preferentially with the membrane of vesicles present within multivesicular endosomes. Then, these intra-endosomal vesicles (containing nucleocapsids) are transported to late endosomes, where back-fusion with the endosome limiting membrane delivers the nucleocapsid into the cytoplasm. In this study, we show that the tumor susceptibility gene 101 (Tsg101) subunit of the endosomal sorting complexes required for transport (ESCRT)-I complex, which mediates receptor sorting into multivesicular endosomes, is dispensable for viral envelope fusion with endosomal membranes and viral RNA transport to late endosomes but is necessary for infection. Our data indicate that Tsg101, in contrast to the ESCRT-0 component Hrs, plays a direct role in nucleocapsid release from within multivesicular endosomes to the cytoplasm, presumably by controlling the back-fusion process. We conclude that Tsg101, through selective interactions with its partners including Hrs and Alix, may link receptor sorting and lysosome targeting to the back-fusion process involved in viral capsid release.

Stimulation of cell surface F1-ATPase activity by apolipoprotein A-I inhibits endothelial cell apoptosis and promotes proliferation.

OBJECTIVE: Several findings argue for a protective effect of high-density lipoproteins (HDLs) against endothelial dysfunction. The molecular mechanisms underlying this protective effect are not fully understood, although recent works suggest that the actions of HDL on the endothelium are initiated by multiple interactions between HDLs (lipid or protein moiety) and cell surface receptors. We previously showed that the mitochondrial related F(1)-ATPase is a cell surface receptor for HDLs and their main atheroprotective apolipoprotein (apoA-I). Herein we test the hypothesis that the cell surface F(1)-ATPase may contribute to the ability of apoA-I and HDLs to maintain endothelial cell survival. METHODS AND RESULTS: Cell imaging and binding assays confirmed the presence of the F(1)-ATPase at the surface of human umbilical vein endothelial cells (HUVECs) and its ability to bind apoA-I. Cell surface F(1)-ATPase activity (ATP hydrolysis into ADP) was stimulated by apoA-I and was inhibited by its specific inhibitor IF(1)-H49K. Furthermore the antiapoptotic and proliferative effects of apoA-I on HUVECs were totally blocked by the F(1)-ATPase ligands IF(1)-H49K, angiostatin and anti-betaF(1)-ATPase antibody, independently of the scavenger receptor SR-BI and ABCA1. CONCLUSIONS: This study suggests an important contribution of cell surface F(1)-ATPase to apoA-I-mediated endothelial cell survival, which may contribute to the atheroprotective functions of apoA-I.

PLIF: A rapid, accurate method to detect and quantitatively assess protein-lipid interactions.

Phosphoinositides are a type of cellular phospholipid that regulate signaling in a wide range of cellular and physiological processes through the interaction between their phosphorylated inositol head group and specific domains in various cytosolic proteins. These lipids also influence the activity of transmembrane proteins. Aberrant phosphoinositide signaling is associated with numerous diseases, including cancer, obesity, and diabetes. Thus, identifying phosphoinositide-binding partners and the aspects that define their specificity can direct drug development. However, current methods are costly, time-consuming, or technically challenging and inaccessible to many laboratories. We developed a method called PLIF (for "protein-lipid interaction by fluorescence") that uses fluorescently labeled liposomes and tethered, tagged proteins or peptides to enable fast and reliable determination of protein domain specificity for given phosphoinositides in a membrane environment. We validated PLIF against previously known phosphoinositide-binding partners for various proteins and obtained relative affinity profiles. Moreover, PLIF analysis of the sorting nexin (SNX) family revealed not only that SNXs bound most strongly to phosphatidylinositol 3-phosphate (PtdIns3P or PI3P), which is known from analysis with other methods, but also that they interacted with other phosphoinositides, which had not previously been detected using other techniques. Different phosphoinositide partners, even those with relatively weak binding affinity, could account for the diverse functions of SNXs in vesicular trafficking and protein sorting. Because PLIF is sensitive, semiquantitative, and performed in a high-throughput manner, it may be used to screen for highly specific protein-lipid interaction inhibitors.

SNX12 role in endosome membrane transport.

In this paper, we investigated the role of sorting nexin 12 (SNX12) in the endocytic pathway. SNX12 is a member of the PX domain-containing sorting nexin family and shares high homology with SNX3, which plays a central role in the formation of intralumenal vesicles within multivesicular endosomes. We found that SNX12 is expressed at very low levels compared to SNX3. SNX12 is primarily associated with early endosomes and this endosomal localization depends on the binding to 3-phosphoinositides. We find that overexpression of SNX12 prevents the detachment (or maturation) of multivesicular endosomes from early endosomes. This in turn inhibits the degradative pathway from early to late endosomes/lysosomes, much like SNX3 overexpression, without affecting endocytosis, recycling and retrograde transport. In addition, while previous studies showed that Hrs knockdown prevents EGF receptor sorting into multivesicular endosomes, we find that overexpression of SNX12 restores the sorting process in an Hrs knockdown background. Altogether, our data show that despite lower expression level, SNX12 shares redundant functions with SNX3 in the biogenesis of multivesicular endosomes.

Role of SNX16 in the dynamics of tubulo-cisternal membrane domains of late endosomes.

In this paper, we report that the PX domain-containing protein SNX16, a member of the sorting nexin family, is associated with late endosome membranes. We find that SNX16 is selectively enriched on tubulo-cisternal elements of this membrane system, whose highly dynamic properties and formation depend on intact microtubules. By contrast, SNX16 was not found on vacuolar elements that typically contain LBPA, and thus presumably correspond to multivesicular endosomes. We conclude that SNX16, together with its partner phosphoinositide, define a highly dynamic subset of late endosomal membranes, supporting the notion that late endosomes are organized in distinct morphological and functional regions. Our data also indicate that SNX16 is involved in tubule formation and cholesterol transport as well as trafficking of the tetraspanin CD81, suggesting that the protein plays a role in the regulation of late endosome membrane dynamics.