Inhibition of lipoprotein lipase activity by sphingomyelin: role of membrane surface structure.

We have recently shown that sphingomyelin (SM) strongly inhibits lipoprotein lipase (LPL)-mediated lipolysis in monolayers and emulsion particles. To further evaluate how SM modulates LPL activity on the emulsion surface, the relationship between membrane surface structure and LPL activity was investigated. We measured fluorescence anisotropy of 1-palmitoyl-2-[3-(diphenylhexatrienyl)propionyl]-sn-3-phosphati dylcho line, probing surface acyl chain fluidity, and fluorescence lifetime of N-(5-dimethylaminonaphthalene-1-sulfonyl)dipalmitoylphosphatidylethan olamine in H(2)O and D(2)O buffer, assessing the degree of hydration in the head group region. The results revealed that incorporation of egg SM into triolein-egg phosphatidylcholine emulsions markedly increased acyl chain order and decreased head group hydration of the surface monolayers. In contrast, cholesterol was shown to increase head group hydration despite a strong increase in acyl chain order. The close correlation between the apparent K(m) values of LPL and the degree of head group hydration indicated that LPL interacts with the head group region rather than with the hydrophobic interior of the surface monolayers. However, apparent V(max) did not show a simple correlation with any surface structure, and the finding in which SM had no effect on apparent V(max) of medium-chain triglyceride emulsions suggested that the hydrophobic interaction between acyl chains of SM and triglyceride at the emulsion surface is important for determining the apparent V(max). These results showed conclusively that SM inhibits LPL activity mainly by changing the emulsion surface structure and not by a specific interaction between SM and LPL.

W276 mutation in the endothelin receptor subtype B impairs Gq coupling but not Gi or Go coupling.

The mutation of W276 to cysteine within the human endothelin receptor subtype B (ET(B)R) is associated with Hirschsprung's disease, a congenital intestinal disease. The sequence surrounding W276 is highly conserved between the endothelin receptor subtypes A and B. We have introduced sets of mutations into W275 and W276 of the ET(B)R gene, and the corresponding W257 and W258 of the ET(A)R gene, and studied their coupling properties with G(i), G(o), and G(q) in reconstituted phospholipid vesicles. The prepared mutants all showed a similar affinity for endothelin-1. The W276C/ET(B)R and W276A/ET(B)R mutants had reduced activities in G(q) coupling but not in G(i)/G(o) coupling, while the W275A/ET(B)R displayed reduced activities in G(i)/G(q) coupling, with normal G(o) coupling. On the other hand, W257A/ET(A)R and W258A/ET(A)R exhibited wild-type activities in all examined G protein couplings. These results suggest that the defects in the G(q) signaling pathway by the ET(B)R are connected with Hirschsprung's disease and that the two conserved tryptophans play distinct roles in signal transduction by the two receptor subtypes. In addition, W275 and W276, which are thought to be located near the extracellular side of the transmembrane helix 5, play important roles in forming the active structure of ET(B)R.

Interactions of endothelin receptor subtypes A and B with Gi, Go, and Gq in reconstituted phospholipid vesicles.

To understand the biochemical basis for the functional divergence of the human endothelin receptor subtypes A (ETAR) and B (ETBR), they were expressed, purified from insect Sf9 cells, and reconstituted into phospholipid vesicles with the Go, Gq, and Gi proteins. For each G protein, a unique pattern of reactivity was observed with the different receptor subtypes. Both ETAR and ETBR activated Go to a similar maximal extent, and both subtypes activated Gq with similar EC50 values; however, the ETAR displayed a 2-3-fold higher maximal extent of activation. In contrast, both subtypes activated Gi to a similar maximal extent, but the ETAR displayed a 4-fold higher EC50 value as compared to the ETBR. To test whether these coupling specificities are influenced by C-terminal palmitoylation of the receptor, we mutated a cluster of cysteine residues near the end of the seventh transmembrane helix in both receptors. While the cysteine mutations in the ETBR resulted in a partially palmitoylated receptor, the replacement of these cysteine residues in the ETAR yielded a mostly palmitoyl-deficient receptor and had no effect on Go activation, but caused a reduction in the extents of Gi and Gq stimulation. Together, these studies provide important insights into the specificity of G protein coupling in the endothelin receptors. The ability to discriminate between the different G proteins under various physiological conditions may be a key element in the selection of distinct signal transduction pathways by the two receptor subtypes.

Surface composition regulates clearance from plasma and triolein lipolysis of lipid emulsions.

Sphingomyelin (SM) and cholesterol (Chol) are major surface lipid constituents of plasma lipoproteins. We investigated the effects of SM and Chol on the plasma clearance of lipid emulsions as a model for lipoprotein particles in rats. The presence of Chol facilitated the removal of emulsion particles from plasma, whereas SM delayed particle removal. Preinjection of lactoferrin, an inhibitor of the apolipoprotein E (apoE) receptor, revealed that the differences in clearance of emulsions were due to the differences in affinity for the apoE receptor. Measurement of apolipoprotein binding suggested that the balance of apoE and apoC (apoC-II and apoC-III) bound to emulsions caused the difference in plasma clearance of emulsion particles. That is to say, SM in the emulsion surface decreased binding of apoE, which led to a longer circulation of emulsion particles in plasma. Chol, on the other hand, decreased the ratio of apoC to apoE, which may have promoted emulsion uptake through the apoE receptor. We also examined in vitro lipolysis using immobilized lipoprotein lipase (LPL) in a heparin affinity column. Lipolysis rates were significantly reduced by the incorporation of SM into the emulsion surface, but not by the incorporation of Chol, indicating that SM in the lipoprotein surface is an important lipid component regulating LPL-mediated lipolysis. Our results suggest that the presence of SM and Chol in the lipoprotein surface plays an important role in the circulation behavior and LPL-mediated lipolysis of lipid emulsions through their effect on the selectivity of plasma protein binding.

Effects of sphingomyelin and cholesterol on lipoprotein lipase-mediated lipolysis in lipid emulsions.

Lipoprotein lipase (LPL) hydrolyzes triacylglycerol (TG) of TG-rich lipoproteins. We investigated the effects of sphingomyelin (SM) and cholesterol (Chol) on the lipolysis of lipid emulsions by LPL using human apolipoprotein C-II (apoC-II) or plasma as an activator. Kinetic studies of the lipolysis rates clearly demonstrated that the primary effect of the activator on the LPL reaction was not to increase the affinity of LPL for the emulsion surface, but to enhance LPL catalytic activity. Incorporation of SM into the emulsion surface caused increases in Km(app) and decreases in Vmax(app), indicating that SM inhibited lipolysis by decreasing both affinity for substrates and catalytic activity of LPL. SM was also found to affect possible factors related to the lipolysis rates; that is, SM increased TG solubility in surface layers and decreased apoC-II binding to the emulsion surface. Interestingly, Chol did not affect the lipolysis rates even though it decreased TG solubility and apoC-II binding. These results indicated that neither TG solubility nor amount of apoC-II binding were determinate factors in LPL-mediated lipolysis under physiological conditions. Our results suggest that the content of SM in the lipoprotein surface plays an important role by controlling lipoprotein lipase-mediated lipolysis, and that cholesterol enrichment in the lipoprotein surface has no influence on lipolysis, but may affect other metabolic processes such as uptake by the liver through the selectivity of apolipoprotein binding.

Characterization of human endothelin B receptor and mutant receptors expressed in insect cells.

Endothelin type-B receptor (ET(B)R) forms a stable complex with its ligand, endothelin-1. To facilitate biochemical and biophysical studies of human ET(B)R, several ET(B)R mutants carrying a hexahistidine tag sequence at the N or C terminus were expressed in Sf9 cells and were purified by a combination of biotinylated endothelin-1-ligand-affinity and nickel-affinity chromatographies. The ligand-free receptor was purified by dissociating the ligand x receptor complex with 2 M NaSCN, whereas the ligand-bound ET(B)R was purified by the use of thiol-sensitive biotinylated endothelin-1. While the wild-type ET(B)R was expressed at about 100 pmol 125I-endothelin-1-binding activity/mg membrane protein, the deletion of 36 residues from the N-terminus reduced the expressed activity to about 30%. On the other hand, the lack of glycosylation and the replacement of 2-9 residues in the N-terminal tail resulted in a 20-40% reduction in the expressed activity. Among the mutant proteins, [H57-H62, G63-G65]ET(B)R, carrying six His residues in the N-terminal tail, was studied extensively because it was purified most effectively. Ligand-free [H57-H62, G63-G65]ET(B)R, purified in digitonin, retained full ligand-binding activity, while other detergents led to partial denaturation of the receptor after solubilization or after elution with NaSCN. On the other hand, ligand-bound [H57-H62, G63-G65]ET(B)R could be purified in various detergents, such as n-octyl-beta-D-glucopyranoside or n-decyl-beta-D-maltopyranoside. Ligand-free [H57-H62, G63-G65]ET(B)R reconstituted in phospholipid vesicles stimulated the binding of guanosine 5'-3-O-(thio)triphosphate by Gq in the presence of endothelin-1. Ligand-bound [H57-H62, G63-G65]ET(B)R showed similar catalytic activity in nucleotide exchange by Gq. These results indicate that the ligand x receptor complex in a detergent-micellar solution retained the biologically active structure, and that the presence of ligand, endothelin-1, in the receptor molecule reinforces the stable assembly of a helical bundle and therefore the active structure.

Physical states of surface and core lipids in lipid emulsions and apolipoprotein binding to the emulsion surface.

Plasma triglyceride-rich lipoproteins vary in lipid composition during their metabolism. We investigated the effects of the lipid composition of emulsion particles, specifically those of cholesterol enrichment and core replacement (replacing core triglyceride with cholesteryl oleate), on the physical states of surface and core lipids. Steady-state and time-resolved fluorescence anisotropies were measured in lipid emulsions using 1,6-diphenylhexatriene to probe the core and 1,6-diphenylhexatriene analogues for the outer and inner hydrophobic portions of surface phospholipids. In the absence of cholesterol, core replacement had little effect on the surface rigidity, despite the large difference in core mobility. However, core replacement caused a marked increase in surface rigidity in the presence of cholesterol. Quenching experiments using the fluorescent cholesterol analogue, dehydroergosterol, indicated that core replacement allowed surface dehydroergosterol to redistribute from the inner to the outer regions in the emulsion surface. These results indicated that core replacement modulates the surface properties of the emulsion particles through the redistribution of cholesterol in the surface layers. Furthermore, core replacement significantly decreased the binding of apolipoprotein E to the emulsion surface, whereas the binding of apolipoprotein CII responded to the cholesterol enrichment. This binding behavior of exchangeable apolipoproteins may closely correlate with the location of surface cholesterol and the mobility of core lipids.