Expression of flotillins in the human placenta: potential implications for placental transcytosis

A proteomics survey of human placental syncytiotrophoblast (ST) apical plasma membranes revealed peptides corresponding to flotillin-1 (FLOT1) and flotillin-2 (FLOT2). The flotillins belong to a class of lipid microdomain-associated integral membrane proteins that have been implicated in clathrin- and caveolar-independent endocytosis. In the present study, we characterized the expression of the flotillin proteins within the human placenta. FLOT1 and FLOT2 were coexpressed in placental lysates and BeWo human trophoblast cells. Immunofluorescence microscopy of first-trimester and term placentas revealed that both proteins were more prominent in villous endothelial cells and cytotrophoblasts (CTs) than the ST. Correspondingly, forskolin-induced fusion in BeWo cells resulted in a decrease in FLOT1 and FLOT2, suggesting that flotillin protein expression is reduced following trophoblast syncytialization. The flotillin proteins co-localized with a marker of fluid-phase pinocytosis, and knockdown of FLOT1 and/or FLOT2 expression resulted in decreased endocytosis of cholera toxin B subunit. We conclude that FLOT1 and FLOT2 are abundantly coexpressed in term villous placental CTs and endothelial cells, and in comparison, expression of these proteins in the ST is reduced. These findings suggest that flotillin-dependent endocytosis is unlikely to be a major pathway in the ST, but may be important in the CT and endothelium.     

Androgen receptor phosphorylation. Regulation and identification of the phosphorylation sites

Activation of signal transduction kinase cascades has been shown to alter androgen receptor (AR) activity. Although it has been suggested that changes in AR phosphorylation might be directly responsible, the basal and regulated phosphorylations of the AR have not been fully determined. We have identified the major sites of AR phosphorylation on ARs expressed in COS-1 cells using a combination of peptide mapping, Edman degradation, and mass spectrometry. We describe the identification of seven AR phosphorylation sites, show that the phosphopeptides seen with exogenously expressed ARs are highly similar to those seen with endogenous ARs in LNCaP cells and show that specific agonists differentially regulate the phosphorylation state of endogenous ARs in LNCaP prostate cancer cells. Treatment of LNCaP cells with the synthetic androgen, R1881, elevates phosphorylation of serines 16, 81, 256, 308, 424, and 650. Ser-94 appears constitutively phosphorylated. Forskolin, epidermal growth factor, and phorbol 12-myristate 13-acetate increase the phosphorylation of Ser-650. The kinetics of phosphorylation of most sites in response to hormone or forskolin is temporally delayed, reaching a maximum at 2 h post-stimulation. The exception is Ser-81, which continues to display increasing phosphorylation at 6 h. These data provide a basis for analyzing mechanisms of cross-talk between growth factor signaling and androgen in prostate development, physiology, and cancer.     

Kinetic mechanism of quinone oxidoreductase 2 and its inhibition by the antimalarial quinolines

Quinone oxidoreductase 2 (QR2) purified from human red blood cells was recently shown to be a potential target of the quinoline antimalarial compounds [Graves et al., (2002) Mol. Pharmacol. 62, 1364]. QR2 catalyzes the two-electron reduction of menadione via the oxidation of N-alkylated or N-ribosylated nicotinamides. To investigate the mechanism and consequences of inhibition of QR2 by the quinolines further, we have used steady-state and transient-state kinetics to define the mechanism of QR2. Importantly, we have shown that QR2 when isolated from an overproducing strain of E. coli is kinetically equivalent to the enzyme from the native human red blood cell source. We observe ping-pong kinetics consistent with one substrate/inhibitor binding site that shows selectivity for the oxidation state of the FAD cofactor, suggesting that selective inhibition of the liver versus red blood cell forms of malaria may be possible. The reductant N-methyldihydronicotinamide and the inhibitor primaquine bind exclusively to the oxidized enzyme. In contrast, the inhibitors quinacrine and chloroquine bind exclusively to the reduced enzyme. The quinone substrate menadione, on the other hand, binds nonspecifically to both forms of the enzyme. Single-turnover kinetics of the reductive half-reaction are chemically and kinetically competent and confirm the inhibitor selectivity seen in the steady-state experiments. Our studies shed light on the possible in vivo potency of the quinolines and provide a foundation for future studies aimed at creating more potent QR2 inhibitors and at understanding the physiological significance of QR2.