The leptin receptor system of human platelets.

Obesity is associated with elevated levels of leptin in the blood. Elevated leptin is a risk factor for thrombosis in humans, and leptin administration promotes platelet activation and thrombosis in the mouse. The current study examines the effect of leptin on human platelets, and provides initial insights into the nature of the leptin receptor on these platelets. Leptin potentiated the aggregation of human platelets induced by low concentrations of ADP, collagen and epinephrine. However, the response varied significantly between donors, with platelets from some donors (approximately 40%) consistently responding to leptin (responders) and those from other donors (approximately 60%) never responding (non-responders). Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) experiments showed that platelets from both groups only express the signaling form of the leptin receptor, and that responder platelets express higher levels of this receptor than non-responders. Ligand-binding assays demonstrate specific, saturable binding of leptin to platelets from both groups with apparent K(d) values of 76 +/- 20 nM for responders and 158 +/- 46 nM for non-responders. Thus, the decreased sensitivity of non-responder platelets to leptin does not result from the absence of the signaling form of this receptor, but may reflect differences in its level of expression and/or affinity for leptin. These preliminary studies demonstrate that platelets are a major source of leptin receptor in the circulation, and suggest that leptin-responsive individuals may have a higher risk for obesity-associated thrombosis than non-responsive individuals.

Plasminogen activator inhibitor-1 regulates cell adhesion by binding to the somatomedin B domain of vitronectin.

Plasminogen activator inhibitor-1 (PAI-1) binds to the somatomedin B (SMB) domain of vitronectin. It inhibits the adhesion of U937 cells to vitronectin by competing with the urokinase receptor (uPAR; CD87) on these cells for binding to the same domain. Although the inhibitor also blocks integrin-mediated cell adhesion, the molecular basis of this effect is unclear. In this study, the effect of the inhibitor on the adhesion of a variety of cells (e.g., U937, MCF7, HT-1080, and HeLa) to vitronectin was assessed, and the importance of the SMB domain in these interactions was determined. Although PAI-1 blocked the adhesion of all of these cells to vitronectin-coated wells, it did not block adhesion to a variant of vitronectin which lacked the SMB domain. Interestingly, HT-1080 and U937 cells attached avidly to microtiter wells coated with purified recombinant SMB (which does not contain the RGD sequence), and this adhesion was again blocked by the inhibitor. These results affirm that PAI-1 can inhibit both uPAR- and integrin-mediated cell adhesion, and demonstrate that the SMB domain of vitronectin is required for these effects. They also show that multiple cell types can employ uPAR as an adhesion receptor. The use of purified recombinant SMB should help to further define this novel adhesive pathway, and to delineate its relationship with integrin-mediated adhesive events.

Direct binding of occupied urokinase receptor (uPAR) to LDL receptor-related protein is required for endocytosis of uPAR and regulation of cell surface urokinase activity.

Low-density lipoprotein receptor-related protein (LRP) mediates internalization of urokinase:plasminogen activator inhibitor complexes (uPA:PAI-1) and the urokinase receptor (uPAR). Here we investigated whether direct interaction between uPAR, a glycosyl-phosphatidylinositol-anchored protein, and LRP, a transmembrane receptor, is required for clearance of uPA:PAI-1, regeneration of unoccupied uPAR, activation of plasminogen, and the ability of HT1080 cells to invade extracellular matrix. We found that in the absence of uPA:PAI-1, uPAR is randomly distributed along the plasma membrane, whereas uPA:PAI-1 promotes formation of uPAR-LRP complexes and initiates redistribution of occupied uPAR to clathrin-coated pits. uPAR-LRP complexes are endocytosed via clathrin-coated vesicles and traffic together to early endosomes (EE) because they can be coimmunoprecipitated from immunoisolated EE, and internalization is blocked by depletion of intracellular K(+). Direct binding of domain 3 (D3) of uPAR to LRP is required for clearance of uPA-PAI-1-occupied uPAR because internalization is blocked by incubation with recombinant D3. Moreover, uPA-dependent plasmin generation and the ability of HT1080 cells to migrate through Matrigel-coated invasion chambers are also inhibited in the presence of D3. These results demonstrate that GPI-anchored uPAR is endocytosed by piggybacking on LRP and that direct binding of occupied uPAR to LRP is essential for internalization of occupied uPAR, regeneration of unoccupied uPAR, plasmin generation, and invasion and migration through extracellular matrix.

Platelet activating factor receptor (PAF-R) is found in a large endosomal compartment in human umbilical vein endothelial cells.

In previous studies, we have localized the platelet activating factor receptor (PAF-R) in situ on the surface of the endothelium in a number of microvascular beds without providing information on its intracellular location. In the present study, we used human umbilical vein cells (HUVECs) as a model to immunolocalize PAF-R by light and electron microscopic procedures. We raised two different polyclonal antibodies against synthetic peptides of the C- and N-terminal of PAF-R and used them for immunolocalization studies. By immunofluorescence, we found that the anti-C-terminal antibody (CPAF-R) stains an extensive intracellular tubular network. By electron microscopy, using a preembedding staining procedure, we detected PAF-R on the surface of the plasmalemma in a staining pattern similar to that described on microvascular endothelia in situ, but at a considerably lower density. Immunogold labeling of thin frozen sections revealed the presence of PAF-R on the plasmalemma, and especially in an extensive network of tubular-vesicular elements and vesicles associated with it. No detectable amounts of PAF-R were found in the endoplasmic reticulum (ER) or in Golgi cisternae. Double immunofluorescence labeling with antibodies for compartment marker proteins and PAF-R revealed that PAF-R localizes in an endosomal compartment. Confocal microscopy showed that PAF-R colocalizes in this compartment together with the transferrin receptor (Tf-R) and the thrombin receptor (TH-R), but it also showed that the colocalization was partial rather than complete. These findings suggest that the endosomal network is either discontinuous or, conversely, that the proteins in its membrane do not have a fully randomized distribution.

Endocytic trafficking of megalin/RAP complexes: dissociation of the complexes in late endosomes.

Megalin (gp330) is a member of the low-density lipoprotein receptor gene family. Like other members of the family, it is an endocytic receptor that binds a number of specific ligands. Megalin also binds the receptor-associated protein (RAP) that serves as an exocytic traffic chaperone and inhibits ligand binding to the receptor. To investigate the fate of megalin/RAP complexes, we bound RAP glutathione-S-transferase fusion protein (RAP-GST) to megalin at the surface of L2 yolk sac carcinoma cells and followed the trafficking of the complexes by immunofluorescence and immunogold labeling and by their distribution on Percoll gradients. We show that megalin/RAP-GST complexes, which are internalized via clathrin-coated pits, are delivered to early endosomes where they accumulate during an 18 degrees C temperature block and colocalize with transferrin and transferrin receptor. Upon release from the temperature block, the complexes travel to late endosomes where they colocalize with rab7 and can be coprecipitated with anti-RAP-GST antibodies. Dissociation of the complex occurs in late endosomes and is most likely triggered by the low pH (approximately 5.5) of this compartment. RAP is then rapidly delivered to lysosomes and degraded whereas megalin is recycled to the cell surface. When the ligand, lipoprotein lipase, was bound to megalin, the receptor was found to recycle through early endosomes. We conclude that in contrast to receptor/ligand complexes, megalin/RAP complexes traffic through late endosomes, which is a novelty for members of the low-density lipoprotein receptor gene family.

Identification of the second cluster of ligand-binding repeats in megalin as a site for receptor-ligand interactions.

Megalin is a large cell surface receptor that mediates the binding and internalization of a number of structurally and functionally distinct ligands from the lipoprotein and protease:protease inhibitor families. To begin to address how megalin is able to bind ligands with unique structurally properties, we have mapped a binding site for apolipoprotein E (apoE)-beta very low density lipoprotein (beta VLDL), lipoprotein lipase, aprotinin, lactoferrin, and the receptor-associated protein (RAP) within the primary sequence of the receptor. RAP is known to inhibit the binding of all ligands to megalin. We identified a ligand-binding site on megalin by raising mAb against purified megalin, selected for a mAb whose binding to megalin is inhibited by RAP, and mapped the epitope for this mAb. mAb AC10 inhibited the binding of apoE-beta VLDL, lipoprotein lipase, aprotinin, and lactoferrin to megalin in a concentration-dependent manner. When cDNA fragments encoding the four cysteine-rich ligand-binding repeats in megalin were expressed in a baculovirus system and immunoblotted with AC10, it recognized only the second cluster of ligand-binding repeats. The location of the epitope recognized by mAb AC10 within this domain was pinpointed to amino acids 1111-1210. From these studies we conclude that the binding of apoE-beta VLDL, lactoferrin, aprotinin, lipoprotein lipase, and RAP to megalin is either competitively or sterically inhibited by mAb AC10 suggesting that these ligands bind to the same or closely overlapping sites within the second cluster of ligand-binding repeats.

The expression of megalin (gp330) and LRP diverges during F9 cell differentiation.

The receptor-associated protein, RAP, is a chaperonin-like molecule that binds to two members of the low density lipoprotein receptor (LDLR) superfamily-megalin (gp330) and the LDL receptor-related protein (LRP). In F9 embryonal carcinoma cells, expression of RAP mRNA increases when differentiation is induced with retinoic acid and dibutyryl-cyclic AMP. We have investigated the expression of megalin and LRP and their interaction with RAP in F9 cells using biochemical and immunocytochemical methods. Both receptors are expressed in uninduced F9 cells, but only megalin co-precipitates with RAP. When F9 cells were induced to differentiate into parietal endoderm, the expression of megalin was dramatically increased. The expression of megalin exceeded that of LRP and RAP by an order of magnitude and both receptors co-precipitated with RAP. By immunoelectron microscopy, megalin and LRP were localized to clathrin-coated pits at the cell surface in both undifferentiated and differentiated F9 cells, whereas RAP was found mainly in the ER. A sizeable pool of LRP was also detected in the ER. When F9 cells were grown in suspension in the presence of RA and induced to develop into embryoid bodies, the expression of megalin and LRP segregated into different cell types: megalin was found in the outer epithelial layer and LRP in the stem cells of the inner core. Our results demonstrate that F9 cells induced to differentiate in monolayer culture express megalin, LRP and RAP, and RAP is capable of interacting simultaneously with both receptors. In embryoid bodies the expression of megalin and LRP diverges, and only megalin is expressed in the outer epithelial layer.(ABSTRACT TRUNCATED AT 250 WORDS)

Membrane traffic and sorbitol release during osmo- and volume regulation in isolated rat renal inner medullary collecting duct cells.

In response to hypotonic stress, cells of the inner medullary collecting duct (IMCD) undergo swelling followed by a regulatory volume decrease (RVD) and a transient release of organic osmolytes such as sorbitol. In this study, we tested the hypothesis whether membrane recycling is involved in the latter process. Therefore, the state of submembranal actin and the cellular uptake or release of the fluid-phase marker fluorescein isothiocyanate (FITC)-dextran (FD) were investigated as related to changes in membrane permeability for sorbitol. After exposure to hypotonic medium the submembranal actin web rapidly disaggregated but it started to reorganize after 5 min of incubation. The basal-lateral pole of IMCD cells showed a significant uptake of extracellular FD after 100 sec. After 5 min, part of this fluorescence intensity had moved towards the cell center but the main part remained submembranal. Disintegration of the actin network by cytochalasin D diminished the uptake of FD during hypotonicity as did a permanent increase in intracellular calcium induced by ionomycin treatment. During a second osmotic stimulation of IMCD cells preloaded with FD, FD was released in a linear time course reaching a plateau after 1 min. Isotonic ionomycin treatment of preloaded cells also generated a rapid FD release during the first minute but induced a further 2-fold increase during the next 4 min. Under both conditions initial FD release was highly correlated with the simultaneously determined increase in sorbitol permeability. A similar strong correlation was found when different incubation temperatures were used (0 degree C, 15 degrees C, 37 degrees C). These results suggest that during exposure of IMCD cells to hypotonicity the submembranal actin web rapidly disintegrates, and "reserve" vesicles, probably containing sorbitol transporter, move to and fuse with the basal-lateral plasma membrane. The fusion causes a rapid increase in sorbitol permeability. These membrane areas are recovered by internalization, and the transport systems for sorbitol are concomitantly retrieved. In parallel to this internalization the submembranal actin filament network is rearranged. This process seems to be regulated by changes in intracellular calcium.

Compartmentation of intestinal drug sulphoconjugation. Incorporation of luminal and contraluminal [35S]sulphate into 1-naphthol by the isolated mucosa of guinea pig jejunum and colon.

Compartmentation of 1-naphthol metabolism was inferred from the metabolite pattern and distribution in the isolated mucosa of guinea pig intestine mounted in a flux chamber (Sund and Lauterbach, Arch Pharmacol Toxicol 58: 74-83, 1986). To verify the existence of these compartments the dependence of [35S]sulphate incorporation into 1-naphthol sulphate on the side of administration of 1-naphthol and [35S]sulphate was determined. Isolated mucosae were pre-equilibrated with [35S]-sulphate (5 x 10(6) cpm/mumol, 1 mM) for 30 min and subsequently incubated for 15 min with 50 microM 1-naphthol. The three 1-naphthol sulphate fractions (luminal side, blood side and tissue) were assayed by HPLC and liquid scintillation counting; their specific activity was calculated as percentage of the specific activity of the inorganic sulphate administered. 1-Naphthol glucuronide was also measured. In jejunal experiments: after luminal administration of 1-naphthol, 1-naphthol sulphate appeared almost exclusively in the luminal solution; its specific activity approached 70% and 3%, when [35S]sulphate was added to the luminal and blood side, respectively. After introducing 1-naphthol and [35S]sulphate on the blood side, a high and similar specific activity of 50-60% was observed in all three 1-naphthol sulphate fractions (luminal and blood side, tissue) though adding [35S]sulphate to the lumen side decreased the specific activity to 10-20%. In experiments on colonic mucosa: a specific activity both of luminal and blood side 1-naphthol sulphate of more than 50% was observed with blood side [35S]sulphate irrespective of the side of 1-naphthol administration. When [35S]sulphate was placed on the luminal side the specific activity of blood side 1-naphthol sulphate dropped to only 3%, and that of luminal 1-naphthol sulphate ranged between 6% and 20%. Supplementary experiments in which mucosae were exposed to 1-naphthol and [35S]sulphate for 45 min without preincubation showed a tendency to decrease the lumen: blood distribution ratio of 1-naphthol sulphate. However, the general pattern of 1-naphthol sulphate specific activity remained unchanged. The experiments provide further evidence that the jejunal conjugation of phenolic drugs is being performed in two major compartments, which are accessible from the lumen ("luminal compartment") and blood ("systemic compartment") side. The luminal compartment seems practically inaccessible to blood side sulphate as is the systemic compartment for luminal 1-naphthol. In the colonic mucosa, a major "systemic compartment" receiving its sulphate from the blood side is the site for most of the events, but a minor "luminal compartment" seems to be involved as well.

Hypotonicity-evoked release of organic osmolytes from distal renal cells: systems, signals, and sidedness.

After a detailed description of early cellular, membrane and intracellular events in rat renal medullary collecting duct cells when exposed to hypotonicity, a synopsis on organic osmolyte transport properties, possible trigger mechanisms, and the cellular location of transport pathways is given. From the data currently available on renal and nonrenal cells, it is concluded that hypotonicity-evoked efflux of all organic osmolytes appears to be mediated by transport proteins which share a variety of properties more typical for channels than for carriers. A large diversity seems to exist, however, for the signalling mechanisms. Such diversity allows the cells to regulate the intracellular concentration of different organic osmolytes independently of each other, giving flexibility to the spectrum of osmotic responses. The site of release also varies from cell to cell; here conservation of organic osmolytes for future reuptake or further metabolism appears to be the major determinant.