Our laboratory was interested in characterizing the molecular composition of non-caveolar lipid rafts. Thus, we generated monoclonal antibodies to lipid raft proteins of human myelomonocytic cells. Two of these proteins, KE04p and C8orf2, were found to be highly enriched in the detergent-insoluble, buoyant fraction of sucrose gradients in a cholesterol-dependent manner. They contain an evolutionarily conserved domain placing them in the prohibitin family of proteins. In contrast to other family members, these two proteins localized to the ER. Furthermore, the extreme N-termini of KE04p and C8orf2 were found to be sufficient for heterologous targeting of GFP to the ER in the absence of classical ER retrieval motifs. We also demonstrate that all prohibitin family members rely on sequences in their extreme N-termini for their distinctive subcellular distributions including the mitochondria, plasma membrane and Golgi vesicles. Owing to their subcellular localization and their presence in lipid rafts, we have named KE04p and C8orf2, ER lipid raft protein (erlin)-1 and erlin-2, respectively. Interestingly, the ER contains relatively low levels of cholesterol and sphingolipids compared with other organelles. Thus, our data support the existence of lipid-raft-like domains within the membranes of the ER.

The pathogenicity of Plasmodium falciparum is due to the unique ability of infected erythrocytes (IRBCs) to adhere to vascular endothelium. We investigated whether adhesion of IRBCs to CD36, the major cytoadherence receptor on human dermal microvascular endothelial cells (HDMECs), induces intracellular signaling and regulates adhesion. A recombinant peptide corresponding to the minimal CD36-binding domain from P falciparum erythrocyte membrane protein 1 (PfEMP1), as well as an anti-CD36 monoclonal antibody (mAb) that inhibits IRBC binding, activated the mitogen-activated protein (MAP) kinase pathway that was dependent on Src-family kinase activity. Treatment of HDMECs with a Src-family kinase-selective inhibitor (PP1) inhibited adhesion of IRBCs in a flow-chamber assay by 72% (P < .001). More importantly, Src-family kinase activity was also required for cytoadherence to intact human microvessels in a human/severe combined immunodeficient (SCID) mouse model in vivo. The effect of PP1 could be mimicked by levamisole, a specific alkaline-phosphatase inhibitor. Firm adhesion to PP1-treated endothelium was restored by exogenous alkaline phosphatase. In contrast, inhibition of the extracellular signal-regulated kinase 1/2 (ERK 1/2) and p38 MAP kinase pathways had no immediate effect on IRBC adhesion. These results suggest a novel mechanism for the modulation of cytoadherence under flow conditions through a signaling pathway involving CD36, Src-family kinases, and an ectoalkaline phosphatase. Targeting IntroductionThe adhesion of infected erythrocytes (IRBCs) containing mature stages of the parasite Plasmodium falciparum to host endothelium is a key pathogenic process of the infection. 1 The resulting sequestration of IRBCs in the microvasculature can lead to tissue hypoxia, metabolic disturbances, multiorgan dysfunction, and ultimately the death of 1 to 2 million patients worldwide annually. 2 Cytoadherence is mediated by the parasite protein P falciparum erythrocyte membrane protein 1 (PfEMP1) 3 and endothelial receptor molecules, including CD36, 4,5 intercellular adhesion molecule 1 (ICAM-1), 6 E-selectin, and vascular cell adhesion molecule 1 (VCAM-1). 7 Under physiologic flow conditions, IRBCs have been shown to interact synergistically with the different adhesion molecules on microvascular endothelium in a shear-dependent manner, 8,9 mimicking the adhesive events of the leukocyte recruitment cascade. 10 IRBCs tether and roll on ICAM-1, 8,9 VCAM-1, 8 and P-selectin. 8 These low-affinity interactions by themselves are not sufficient for the arrest of the rolling cells, but they enhance the subsequent adhesion of almost all clinical parasite isolates tested to CD36. 8,9 Peptide-mapping studies on CD36 reveal that residues 145 to 171, 11 and to a lesser extent 97 to 110, 12 are important for IRBC adhesion, while the critical region of PfEMP1 involved in binding to CD36 is localized to a 179-amino acid sequence within the cysteine-rich interdomain region 1 (CIDR1). 13 The above investigations focused on the extracell...

The group of stomatin/prohibitin/flotillin/HflK/C (SPFH) domain-containing proteins comprise members of diverse subcellular localization and function. Association with detergent-resistant membranes (DRMs) and the propensity to form oligomers are two common properties of SPFH domain proteins and likely important for the function of these proteins. Our laboratory recently discovered two novel members of this protein group, which, based on their endoplasmic reticulum (ER) localization and association with DRMs, were named ER lipid raft-associated protein (erlin)-1 and -2. Here we characterized erlin oligomerization and identified domains within the erlins responsible for oligomerization and DRM association. Using co-immunoprecipitation and sucrose density gradient centrifugation approaches on endogenous and ectopically expressed erlin proteins, we found that they formed homo-and hetero-oligomers and were part of large multimeric complexes. These properties were independent of their DRM association. By analyzing truncation and point mutants of erlin-2 we discovered that interaction between erlin monomers (oligomerization) and association with high molecular weight complexes require distinct regions within the protein. Although oligomerization and DRM association were mediated by a region immediately downstream of the SPFH domain (residues 228 -300), integration into high molecular weight complexes was absolutely dependent on a phenylalanine residue C-terminal of this region (Phe-305), which lies within a short stretch of hydrophobic residues. Our data demonstrate that lower order oligomerization and incorporation into multimeric complexes are two separate biochemical properties of the erlins, because they are mediated by distinct regions.