Differential roles of cysteine residues in the cellular trafficking, dimerization, and function of the high-density lipoprotein receptor, SR-BI

The scavenger receptor, class B, type I (SR-BI) binds high-density lipoprotein (HDL) and mediates selective delivery of cholesteryl esters (CEs) to the liver and steroidogenic cells of the adrenal glands and gonads. Although it is clear that the large extracellular domain (ECD) of SR-BI binds HDL, the role of ECD in the selective HDL-CE transport remains poorly understood. In this study, we used a combination of mutational and chemical approaches to systematically evaluate the contribution of cysteine residues, especially six cysteine residues of ECD, in SR-BI-mediated selective HDL-CE uptake, intracellular trafficking, and SR-BI dimerization. Pretreatment of SR-BI-overexpressing COS-7 cells with a disulfide (S-S) bond reducing agent, β-mercaptoethanol (100 mM) or dithiothreitol (DTT) (10 mM), modestly but significantly impaired SR-BI-mediated selective HDL-CE uptake. Treatment of SR-BI-overexpressing COS-7 cells with the optimal doses of membrane permeant alkyl methanethiosulfonate (MTS) reagents, positively charged MTSEA or neutral MMTS, that specifically react with the free sulfhydryl group of cysteine reduced the rate of SR-BI-mediated selective HDL-CE uptake, indicating that certain intracellular free cysteine residues may also be critically involved in the selective cholesterol transport process. In contrast, use of membrane impermeant MTS reagent, positively charged MTSET and negatively charged MTSES, showed no such effect. Next, the importance of eight cysteine residues in SR-BI expression, cell surface expression, dimer formation, and selective HDL-derived CE transport was evaluated. These cysteine residues were replaced either singly or in pairs with serine, and the mutant SR-BIs were expressed in either COS-7 or CHO cells. Four mutations, C280S, C321S, C323S, and C334S, of the ECD, either singly or in various pair combinations, resulted in significant decreases in SR-BI (HDL) binding activity, selective CE uptake, and trafficking to the cell surface. Surprisingly, we found that mutation of the two remaining cysteine residues, C251 and C384 of the ECD, had no effect on either SR-BI expression or function. Other cysteine mutations and substitutions were also without effect. Western blot data indicated that single and double mutations at C280, C321, C323, and C334 residues strongly favor dimer formation. However, they are rendered nonfunctional presumably because of mutation-induced formation of aberrant disulfide linkages resulting in inhibition of optimal HDL binding and, thus, selective HDL-CE uptake. These results provide novel insights into the functional role of four cysteine residues, C280, C321, C323, and C334, of the SR-BI ECD in SR-BI expression and trafficking to the cell surface, its dimerization, and associated selective CE transport function.     

C323 of SR-BI is required for SR-BI-mediated HDL binding and cholesteryl ester uptake

Scavenger receptor BI (SR-BI) is an HDL receptor. It binds HDL and mediates the uptake of cholesteryl ester from HDL. Early studies have pointed out that the extracellular domain of SR-BI is critical for SR-BI-mediated cholesteryl ester uptake. However, the extracellular loop of SR-BI is large: it contains 403 amino acids. The HDL binding site and the modulation of SR-BI-mediated cholesteryl ester uptake remain to be identified. In this study, using C323G mutant SR-BI, we showed that C323G mutant SR-BI lost its HDL binding and cholesteryl ester uptake activity, indicating that the highly conserved C323 is required for SR-BI-mediated HDL binding and cholesteryl ester uptake. Using a blocking antibody against C323 region, we demonstrated that C323 is directly involved in HDL binding and likely an HDL binding site. Using C323G mutant transgenic mouse model, we further demonstrated that C323 of SR-BI is required for regulating plasma cholesterol levels in vivo. Using redox reagents, we showed that physiological relevant levels of H(2)O(2) upregulated the SR-BI-mediated cholesteryl ester uptake activity by 65%, whereas GSH or DTT significantly downregulated SR-BI-mediated cholesteryl ester uptake activity by 45%. C323 of SR-BI is critical for SR-BI-mediated HDL binding and cholesteryl ester uptake, and changes in redox status may be a regulatory factor modulating SR-BI-mediated cholesterol transport.     

Analysis of chimeric receptors shows that multiple distinct functional activities of scavenger receptor, class B, type I (SR-BI), are localized to the extracellular receptor domain

Scavenger receptor BI (SR-BI) mediates the selective uptake of high-density lipoprotein (HDL) cholesteryl ester (CE), a process by which HDL CE is taken into the cell without degradation of the HDL particle. In addition, SR-BI stimulates the bi-directional flux of free cholesterol (FC) between cells and lipoproteins, an activity that may be responsible for net cholesterol efflux from peripheral cells as well as the rapid hepatic clearance of FC from plasma HDL. SR-BI also increases cellular cholesterol mass and alters cholesterol distribution in plasma membrane domains as judged by the enhanced sensitivity of membrane cholesterol to extracellular cholesterol oxidase. In contrast, CD36, a closely related class B scavenger receptor, has none of these activities despite binding HDL with high affinity. In the present study, analyses of chimeric SR-BI/CD36 receptors and domain-deleted SR-BI have been used to test the various domains of SR-BI for functional activities related to HDL CE selective uptake, bi-directional FC flux, and the alteration of membrane cholesterol mass and distribution. The results show that each of these activities localizes to the extracellular domain of SR-BI. The N-terminal cytoplasmic tail and transmembrane domains appear to play no role in these activities other than targeting the receptor to the plasma membrane. The C-terminal tail of SR-BI is dispensable for activity as well for targeting to the plasma membrane. Thus, multiple distinct functional activities are localized to the SR-BI extracellular domain.     

Separation of lipid transport functions by mutations in the extracellular domain of scavenger receptor class B,type I

Scavenger receptor class B, type I (SR-BI) shows a variety of effects on cellular cholesterol metabolism, including increased selective uptake of high density lipoprotein (HDL) cholesteryl ester, stimulation of free cholesterol (FC) efflux from cells to HDL and phospholipid vesicles, and changes in the distribution of plasma membrane FC as evidenced by increased susceptibility to exogenous cholesterol oxidase. Previous studies showed that these multiple effects require the extracellular domain of SR-BI, but not the transmembrane and cytoplasmic domains. To test whether 1) the extracellular domain of SR-BI mediates multiple activities by virtue of discrete functional subdomains, or 2) the multiple activities are, in fact, secondary to and driven by changes in cholesterol flux, the extracellular domain of SR-BI was subjected to insertional mutagenesis by strategically placing an epitope tag into nine sites. These experiments identified four classes of mutants with disruptions at different levels of function. Class 4 mutants showed a clear separation of function between HDL binding, HDL cholesteryl ester uptake, and HDL-dependent FC efflux on one hand and FC efflux to small unilamellar vesicles and an increased cholesterol oxidase-sensitive pool of membrane FC on the other. Selective disruption of the latter two functions provides evidence for multiple functional subdomains in the extracellular receptor domain. Furthermore, these findings uncover a difference in the SR-BI-mediated efflux pathways for FC transfer to HDL acceptors versus phospholipid vesicles. The loss of the cholesterol oxidase-sensitive FC pool and FC efflux to small unilamellar vesicle acceptors in Class 4 mutants suggests that these activities may be mechanistically related.     

Scavenger receptor BI (SR-BI) mediates free cholesterol flux independently of HDL tethering to the cell surface

In addition to its effect on high density lipoprotein (HDL) cholesteryl ester (CE) uptake, scavenger receptor BI (SR-BI) was recently reported to stimulate free cholesterol (FC) flux from Chinese hamster ovary (CHO) cells stably expressing mouse SR-BI, a novel function of SR-BI that may play a role in cholesterol removal from the vessel wall where the receptor can be found. It is possible that SR-BI stimulates flux simply by tethering acceptor HDL particles in close apposition to the cell surface thereby facilitating the movement of cholesterol between the plasma membrane and HDL. To test this, we used transiently transfected cells and compared the closely related class B scavenger receptors mouse SR-BI and rat CD36 for their ability to stimulate cholesterol efflux as both receptors bind HDL with high affinity. The results showed that, although acceptor binding to SR-BI may contribute to efflux to a modest extent, the major stimulation of FC efflux occurs independently of acceptor binding to cell surface receptors. Instead our data indicate that SR-BI mediates alterations to membrane FC domains which provoke enhanced bidirectional FC flux between cells and extracellular acceptors.     

Scavenger receptor class B type I-mediated cholesteryl ester-selective uptake and efflux of unesterified cholesterol. Influence of high density lipoprotein size and structure

Scavenger receptor (SR)-BI catalyzes the selective uptake of cholesteryl ester (CE) from high density lipoprotein (HDL) by a two-step process that involves the following: 1) binding of HDL to the receptor and 2) diffusion of the CE molecules into the cell plasma membrane. We examined the effects of the size of discoidal HDL particles containing wild-type (WT) apoA-I on selective uptake of CE and efflux of cellular free (unesterified) cholesterol (FC) from COS-7 cells expressing SR-BI to determine the following: 1) the influence of apoA-I conformation on the lipid transfer process, and 2) the contribution of receptor binding-dependent processes to the overall efflux of cellular FC. Large (10 nm diameter) reconstituted HDL bound to SR-BI better (B(max) approximately 420 versus 220 ng of apoA-I/mg cell protein), delivered more CE, and promoted more FC efflux than small ( approximately 8 nm) particles. When normalized to the number of reconstituted HDL particles bound to the receptor, the efficiencies of either CE uptake or FC efflux with these particles were the same indicating that altering the conformation of WT apoA-I modulates binding to the receptor (step 1) but does not change the efficiency of the subsequent lipid transfer (step 2); this implies that binding induces an optimal alignment of the WT apoA-I.SR-BI complex so that the efficiency of lipid transfer is always the same. FC efflux to HDL is affected both by binding of HDL to SR-BI and by the ability of the receptor to perturb the packing of FC molecules in the cell plasma membrane.     

Vulnerability of the cysteine-less proton-coupled folate transporter (PCFT-SLC46A1) to mutational stress associated with the substituted cysteine accessibility method

The proton-coupled transporter (PCFT) mediates intestinal folate absorption and folate transport from blood across the choroid plexus. The membrane topology of PCFT has been defined using the substituted cysteine accessibility method; an intramolecular disulfide bond between the Cys 66 and 298 residues, in the first and fourth extracellular loops, respectively, is present but not essential for function. The current report describes Lys 422 mutations (K422C, K422E) that have no effect on transport activity when introduced into wild-type PCFT but result in a marked loss of activity when introduced into a Cys-less PCFT which is otherwise near-fully functional. The loss of activity of both mutant PCFTs was shown to be due to impaired protein stability and expression. Additional studies were conducted with the K422C mutation in Cys-less PCFT. The impact of re-introduction of one, two, three or five, Cys residues was assessed. While there were some differences in the impact of the different Cys residues re-introduced, restoration was attributed more to a cumulative effect rather than the specific role of individual Cys residues. Preservation of the Cys66-Cys298 intramolecular disulfide bond was not required for stability of the K422C protein. These observations are relevant to studies with Cys-less transporters utilized for the characterization of proteins with the substituted cysteine accessibility method and indicate that functional defects detected in a Cys-less protein, when the tertiary structure of the molecule is stressed, are not necessarily relevant to the wild-type protein.     

The Venus's-flytrap and cysteine-rich domains of the human Ca2+ receptor are not linked by disulfide bonds

The extracellular N-terminal domain of the human Ca(2+) receptor (hCaR) consists of a Venus's-flytrap (VFT) domain and a cysteine-rich (Cys-rich) domain. We have shown earlier that the Cys-rich domain is critical for signal transmission from the VFT domain to the seven-transmembrane domain. The VFT domain contains 10 cysteines: two of them (Cys(129) and Cys(131)) were identified as involved in intermolecular disulfide bonds necessary for homodimerization, and six others (Cys(60)-Cys(101), Cys(358)-Cys(395), and Cys(437)-Cys(449)) are predicted to form three intramolecular disulfide bonds. The Cys-rich domain contains nine cysteines, the involvement of which in disulfide bond formation has not been defined. In this work, we asked whether the remaining cysteines in the hCaR VFT, namely Cys(236) and Cys(482), form disulfide bond(s) with cysteines in the Cys-rich domain. We constructed mutant hCaRs with a unique tobacco etch virus (TEV) protease recognition site inserted between the VFT domain and the Cys-rich domain. These mutant hCaRs remain fully functional compared with the wild type hCaR. After TEV protease digestion of the mutant hCaR proteins, dimers of the VFT were identified on Western blot under nonreducing conditions. We concluded that there is no disulfide bond between the VFT and the Cys-rich domains in the hCaR.     

Hepatic scavenger receptor BI promotes rapid clearance of high density lipoprotein free cholesterol and its transport into bile

The clearance of free cholesterol from plasma lipoproteins by tissues is of major quantitative importance, but it is not known whether this is passive or receptor-mediated. Based on our finding that scavenger receptor BI (SR-BI) promotes free cholesterol (FC) exchange between high density lipoprotein (HDL) and cells, we tested whether SR-BI would effect FC movement in vivo using [(14)C]FC- and [(3)H]cholesteryl ester (CE)-labeled HDL in mice with increased (SR-BI transgenic (Tg)) or decreased (SR-BI attenuated (att)) hepatic SR-BI expression. The initial clearance of HDL FC was increased in SR-BI Tg mice by 72% and decreased in SR-BI att mice by 53%, but was unchanged in apoA-I knockout mice compared with wild-type mice. Transfer of FC to non-HDL and esterification of FC were minor and could not explain differences. The hepatic uptake of FC was increased in SR-BI Tg mice by 34% and decreased in SR-BI att mice by 22%. CE clearance and uptake gave similar results, but with much slower rates. The uptake of HDL FC and CE by SR-BI Tg primary hepatocytes was increased by 2.2- and 2.6-fold (1-h incubation), respectively, compared with control hepatocytes. In SR-BI Tg mice, the initial biliary secretion of [(14)C]FC was markedly increased, whereas increased [(3)H]FC appeared after a slight delay. Thus, in the mouse, a major portion of the clearance of HDL FC from plasma is mediated by SR-BI.     

Glycine 420 near the C-terminal transmembrane domain of SR-BI is critical for proper delivery and metabolism of high density lipoprotein cholesteryl ester

Scavenger receptor BI, SR-BI, is a physiologically relevant receptor for high density lipoprotein (HDL) that mediates the uptake of cholesteryl esters and delivers them to a metabolically active membrane pool where they are subsequently hydrolyzed. A previously characterized SR-BI mutant, A-VI, with an epitope tag inserted into the extracellular domain near the C-terminal transmembrane segment, revealed a separation-of-function between SR-BI-mediated HDL cholesteryl ester uptake and cholesterol efflux to HDL, on one hand, and cholesterol release to small unilamellar phospholipid vesicle acceptors and an increased cholesterol oxidase-sensitive pool of membrane free cholesterol on the other. To further elucidate amino acid residues responsible for this separation-of-function phenotype, we engineered alanine substitutions and point mutations in and around the site of epitope tag insertion, and tested these for various cholesterol transport functions. We found that changing amino acid 420 from glycine to histidine had a profound effect on SR-BI function. Despite the ability to mediate selective HDL cholesteryl ester uptake, the G420H receptor had a greatly reduced ability to: 1) enlarge the cholesterol oxidase-sensitive pool of membrane free cholesterol, 2) mediate cholesterol efflux to HDL, even at low concentrations of HDL acceptor where binding-dependent cholesterol efflux predominates, and 3) accumulate cholesterol mass within the cell. Most importantly, the G420H mutant was unable to deliver the HDL cholesteryl ester to a metabolically active membrane compartment for efficient hydrolysis. These observations have important implications regarding SR-BI function as related to its structure near the C-terminal transmembrane domain.     

Effects of amino acid substitutions at glycine 420 on SR-BI cholesterol transport function

Scavenger receptor class B type I (SR-BI) facilitates the uptake of HDL cholesteryl esters (CEs) in a two-step process involving binding of HDL to its extracellular domain and transfer of HDL core CEs to a metabolically active membrane pool, where they are subsequently hydrolyzed by a neutral CE hydrolase. Recently, we characterized a mutant, G420H, which replaced glycine 420 in the extracellular domain of SR-BI with a histidine residue and had a profound effect on SR-BI function. The G420H mutant receptor exhibited a reduced ability to mediate selective HDL CE uptake and was unable to deliver HDL CE for hydrolysis, despite the fact that it retained the ability to bind HDL. This did not hold true if glycine 420 was replaced with an alanine residue; G420A maintained wild-type HDL binding and cholesterol transport activity. To further understand the role that glycine 420 plays in SR-BI function and why there was a disparity between replacing glycine 420 with a histidine versus an alanine, we generated a battery of point mutants by substituting glycine 420 with amino acids possessing side chains that were charged, hydrophobic, polar, or bulky and tested the resulting mutants for their ability to support HDL binding, HDL cholesterol transport, and delivery for hydrolysis. The results indicated that substitution with a negatively charged residue or a proline impaired cell surface expression of SR-BI or its interaction with HDL, respectively. Furthermore, substitution of glycine 420 with a positively charged residue reduced HDL CE uptake as well as its subsequent hydrolysis.     

Expression of scavenger receptor BI in COS-7 cells alters cholesterol content and distribution

Previous studies have shown that scavenger receptor BI (SR-BI) stimulates the bidirectional flux of free cholesterol (FC) between HDL and SR-BI-expressing cells. A major component of the enhanced FC flux appears to occur independently of HDL binding to SR-BI and may be due to changes in membrane lipid domains resulting from SR-BI expression (1). In the present study, the impact of SR-BI on cellular cholesterol metabolism was determined by examining SR-BI-mediated changes in cellular cholesterol mass, the esterification of HDL-derived FC, and changes in membrane lipid pools. Growth of SR-BI-expressing cells in medium containing HDL led to increased cellular cholesterol mass, most of which accumulated as ester. The esterification of HDL-derived FC was enhanced by SR-BI-expression to a far greater extent than the SR-BI mediated increase in FC uptake, suggesting an SR-BI-mediated effect on cholesterol utilization in the cell. This observation was tested by comparing FC esterification rates in SR-BI positive and negative cells when equivalent amounts of extracellular FC were taken up via cyclodextrins or apolipoprotein AI/phospholipid disks, neither of which contained cholesteryl ester. Under these conditions, SR-BI did not preferentially stimulate cholesterol esterification. These results indicate that the enhanced esterification of HDL-derived FC in SR-BI-expressing cells is due to the expanded pool of cellular FC and not to a specific effect of SR-BI on cholesterol utilization. Two approaches were used to test the effects of SR-BI expression on membrane lipid organization. In the first, the sensitivity of cellular FC to exogenous cholesterol oxidase was tested under conditions in which there is a preferential oxidation of caveolar cholesterol. SR-BI-expression was found to greatly increase the fraction of cellular cholesterol available to the oxidase as compared to either vector-transfected cells or cells expressing the related class B scavenger receptor CD36. These results suggest that SR-BI expression alters the distribution of membrane-free cholesterol to a caveolar fraction or alters the accessibility of this membrane fraction to exogenous cholesterol oxidase. In the second approach, the efflux of cellular FC to high concentrations of cyclodextrins was monitored under conditions where desorption of FC from the plasma membrane is rate limiting for efflux. SR-BI-expressing cells showed a shift in the distribution of FC between two kinetic pools with more FC in the fast pool and less in the slow pool. These data support a model in which SR-BI expression leads to a redistribution of cholesterol to membrane domains that serve to facilitate the flux of FC between cells and lipoproteins.     

Extracellular hydrophobic regions in scavenger receptor BI play a key role in mediating HDL-cholesterol transport

The binding of high density lipoprotein (HDL) to scavenger receptor BI (SR-BI) is responsible for whole-body cholesterol disposal via reverse cholesterol transport. The extracellular domain of SR-BI is required for HDL binding and selective uptake of HDL-cholesterol. We identified six highly hydrophobic regions in this domain that may be important for receptor activity and performed site-directed mutagenesis to investigate the importance of these regions in SR-BI-mediated cholesterol transport. Non-conservative mutation of the regions encompassing V67, L140/L142, V164 or V221 reduced hydrophobicity and impaired the ability of SR-BI to bind HDL, mediate selective uptake of HDL-cholesterol, promote cholesterol efflux, and enlarge the cholesterol oxidase-sensitive pool of membrane free cholesterol. In contrast, conservative mutations at V67, V164 or V221 did not affect the hydrophobicity or these cholesterol transport activities. We conclude that the hydrophobicity of N-terminal extracellular regions of SR-BI is critical for cholesterol transport, possibly by mediating receptor-ligand and/or receptor-membrane interactions.     

Reversible inactivation of AT(2) angiotensin II receptor from cysteine-disulfide bond exchange

Dithiothreitol (DTT) treatment of angiotensin II (Ang II) type 2 (AT(2)) receptor potentiates ligand binding, but the underlying mechanism is not known. Two disulfide bonds proposed in the extracellular domain were examined in this report. Based on the analysis of ligand affinity of cysteine (Cys, C) to alanine (Ala, A) substitution mutants, we provide evidence that Cys(35)-Cys(290) and Cys(117)-Cys(195) disulfide bonds are formed in the wild-type AT(2) receptor. Disruption of the highly conserved Cys(117)-Cys(195) disulfide bond linking the second and third extracellular segments leads to inactivation of the receptor. The Cys(35)-Cys(290) bond is highly sensitive to DTT. Its breakage results in an increased binding affinity for both Ang II and the AT(2) receptor-specific antagonist PD123319. Surprisingly, in the single Cys mutants, C35A and C290A, a labile population of receptors is produced which can be re-folded to high-affinity state by DTT treatment. These results suggest that the free -SH group of Cys(35) or Cys(290) competes with the disulfide bond formation between Cys(117) and Cys(195). This Cys-disulfide bond exchange results in production of the inactive population of the mutant receptors through formation of a non-native disulfide bond.     

Changes in plasma membrane properties and phosphatidylcholine subspecies of insect Sf9 cells due to expression of scavenger receptor class B, type I, and CD36

In mammalian cells scavenger receptor class B, type I (SR-BI), mediates the selective uptake of high density lipoprotein (HDL) cholesteryl ester into hepatic and steroidogenic cells. In addition, SR-BI has a variety of effects on plasma membrane properties including stimulation of the bidirectional flux of free cholesterol (FC) between cells and HDL and changes in the organization of plasma membrane FC as indicated by increased susceptibility to exogenous cholesterol oxidase. Recent studies in SR-BI-deficient mice and in SR-BI-expressing Sf9 insect cells showed that SR-BI has significant effects on plasma membrane ultrastructure. The present study was designed to test the range of SR-BI effects in Sf9 insect cells that typically have very low cholesterol content and a different phospholipid profile compared with mammalian cells. The results showed that, as in mammalian cells, SR-BI expression increased HDL cholesteryl ester selective uptake, cellular cholesterol mass, FC efflux to HDL, and the sensitivity of membrane FC to cholesterol oxidase. These activities were diminished or absent upon expression of the related scavenger receptor CD36. Thus, SR-BI has fundamental effects on cholesterol flux and membrane properties that occur in cells of evolutionarily divergent origins. Profiling of phospholipid species by electrospray ionization mass spectrometry showed that scavenger receptor expression led to the accumulation of phosphatidylcholine species with longer mono- or polyunsaturated acyl chains. These changes would be expected to decrease phosphatidylcholine/cholesterol interactions and thereby enhance cholesterol desorption from the membrane. Scavenger receptor-mediated changes in membrane phosphatidylcholine may contribute to the increased flux of cholesterol and other lipids elicited by these receptors.     

Evidence for difference in the roles of two cysteine residues involved in disulfide bond formation in the folding of human lysozyme

Human lysozyme is made up of 130 amino acid residues and has four disulfide bonds at Cys6-Cys128, Cys30-Cys116, Cys65-Cys81, and Cys77-Cys95. Our previous results using the Saccharomyces cerevisiae secretion system indicate that the individual disulfide bonds of human lysozyme have different functions in the correct in vivo folding and enzymatic activity of the protein (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967). In this paper, we report the results of experiments that were focused on the roles of Cys65 and Cys81 in the folding of human lysozyme protein in yeast. A mutant protein (C81A), in which Cys81 was replaced with Ala, had almost the same enzymatic activity and conformation as those of the native enzyme. On the other hand, another mutant (C65A), in which Cys65 was replaced with Ala, was not found to fold correctly. These results indicate that Cys81 is not a requisite for both correct folding and activity, whereas Cys65 is indispensable. The mutant protein C81A is seen to contain a new, non-native disulfide bond at Cys65-Cys77. The possible occurrence of disulfide bond interchange during our mapping experiments cannot be ruled out by the experimental techniques presently available, but characterization of other mutant proteins and computer analysis suggest that the intramolecular exchange of disulfide bonds is present in the folding pathway of human lysozyme in vivo.     

Functional Characterization of Newly-Discovered Mutations in Human SR-BI

In rodents, SR-BI has been firmly established as a physiologically relevant HDL receptor that mediates removal of HDL-cholesteryl esters (CE). However, its role in human lipoprotein metabolism is less defined. Recently, two unique point mutations in human SR-BI — S112F or T175A — were identified in subjects with high HDL-cholesterol (HDL-C) levels. We hypothesized that mutation of these conserved residues would compromise the cholesterol-transport functions of SR-BI. To test this hypothesis, S112F- and T175A-SR-BI were generated by site-directed mutagenesis. Cell surface expression was confirmed for both mutant receptors in COS-7 cells upon transient transfection, albeit at lower levels for T175A-SR-BI. Both mutant receptors displayed defective HDL binding, selective uptake of HDL-CE and release of free cholesterol (FC) from cells to HDL. Mutant receptors were also unable to re-organize plasma membrane pools of FC. While these impaired functions were independent of receptor oligomerization, inability of T175A-SR-BI to mediate cholesterol-transport functions could be related to altered N-linked glycosylation status. In conclusion, high HDL-C levels observed in carriers of S112F- or T175A-SR-BI mutant receptors are consistent with the inability of these SR-BI receptors to mediate efficient selective uptake of HDL-CE, and suggest that increased plasma HDL concentrations in these settings may not be associated with lower risk of cardiovascular disease.     

Scavenger receptor BI (SR-BI) clustered on microvillar extensions suggests that this plasma membrane domain is a way station for cholesterol trafficking between cells and high-density lipoprotein

Receptor-mediated trafficking of cholesterol between lipoproteins and cells is a fundamental biological process at the organismal and cellular levels. In contrast to the well-studied pathway of LDL receptor-mediated endocytosis, little is known about the trafficking of high-density lipoprotein (HDL) cholesterol by the HDL receptor, scavenger receptor BI (SR-BI). SR-BI mediates HDL cholesteryl ester uptake in a process in which HDL lipids are selectively transferred to the cell membrane without the uptake and degradation of the HDL particle. We report here the cell surface locale where the trafficking of HDL cholesterol occurs. Fluorescence confocal microscopy showed SR-BI in patches and small extensions of the cell surface that were distinct from sites of caveolin-1 expression. Electron microscopy showed SR-BI in patches or clusters primarily on microvillar extensions of the plasma membrane. The organization of SR-BI in this manner suggests that this microvillar domain is a way station for cholesterol trafficking between HDL and cells. The types of phospholipids in this domain are unknown, but SR-BI is not strongly associated with classical membrane rafts rich in detergent-resistant saturated phospholipids. We speculate that SR-BI is in a more fluid membrane domain that will favor rapid cholesterol flux between the membrane and HDL.     

The N-terminal fragment of human parathyroid hormone receptor 1 constitutes a hormone binding domain and reveals a distinct disulfide pattern

The N-terminal extracellular parts of human G-protein coupled receptor class B, for example, receptors for secretin, glucagon, or parathyroid hormone, are involved in ligand binding. To obtain structural and functional information on the N-terminal receptor fragment of human parathyroid hormone receptor 1 (PTHR1), the truncated receptor was expressed in the cytosol of Escherichia coli in the form of inclusion bodies. Oxidative refolding of inclusion body material resulted in stable, soluble, monomeric protein. Ligand binding was proved by surface plasmon resonance spectroscopy and isothermal titration calorimetry. Refolded receptor fragment was able to bind parathyroid hormone with an apparent dissociation constant of 3-5 microM. Far-UV circular dichroism spectra showed that the refolded polypeptide contained approximately 25% alpha-helical and 23% beta-sheet secondary structures. Analysis of the disulfide bond pattern of the refolded receptor fragment revealed disulfide bonds between Cys170 and Cys131, Cys148 and Cys108, and Cys117 and Cys48. These results demonstrate that the extracellular N-terminal domain of the parathyroid hormone receptor (PTHR1) possesses a well-defined, stable conformation, which shows a significant ligand binding activity.