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Recently, a murine scavenger receptor type B class I (SR-BI) was identified that binds high density lipoprotein (HDL) and mediates the selective uptake of cholesterol esters. The human CD36 and LIMPII analogous-1 (CLA-1) receptor shows high sequence homology with SR-BI, but their functional relationship has not been determined. Transfected cells expressing CLA-1 bound HDL with a K d of about 35 μg/ml, similar to the K d for HDL binding to rodent SR-BI. This binding resulted in an intracellular accumulation of HDL-derived 3Hcholesterol esters without internalization or degradation of 125I-apolipoprotein. CLA-1 was strongly expressed in the adrenal gland and was also abundant in liver and testis, suggesting that CLA-1, like SR-BI, could play a role in the metabolism of HDL. However, CLA-1 was also expressed in monocytes and, like SR-BI, recognized modified forms of low density lipoproteins as well as native LDL and anionic phospholipids. These findings suggest that CLA-1 might have additional physiological functions. We found that CLA-1 recognizes apoptotic thymocytes. Our results demonstrate that CLA-1, a close structural homologue of SR-BI, is also functionally related to SR-BI and may play an important role as a “docking receptor” for HDL in connection with selective uptake of cholesterol esters. An additional role in recognition of damaged cells is suggested by these studies. Recently, a murine scavenger receptor type B class I (SR-BI) was identified that binds high density lipoprotein (HDL) and mediates the selective uptake of cholesterol esters. The human CD36 and LIMPII analogous-1 (CLA-1) receptor shows high sequence homology with SR-BI, but their functional relationship has not been determined. Transfected cells expressing CLA-1 bound HDL with a K d of about 35 μg/ml, similar to the K d for HDL binding to rodent SR-BI. This binding resulted in an intracellular accumulation of HDL-derived 3Hcholesterol esters without internalization or degradation of 125I-apolipoprotein. CLA-1 was strongly expressed in the adrenal gland and was also abundant in liver and testis, suggesting that CLA-1, like SR-BI, could play a role in the metabolism of HDL. However, CLA-1 was also expressed in monocytes and, like SR-BI, recognized modified forms of low density lipoproteins as well as native LDL and anionic phospholipids. These findings suggest that CLA-1 might have additional physiological functions. We found that CLA-1 recognizes apoptotic thymocytes. Our results demonstrate that CLA-1, a close structural homologue of SR-BI, is also functionally related to SR-BI and may play an important role as a “docking receptor” for HDL in connection with selective uptake of cholesterol esters. An additional role in recognition of damaged cells is suggested by these studies. Scavenger receptor BI (SR-BI) 1The abbreviations used are: SR-BI, scavenger receptor type B class I; CLA-1, CD36 and LIMPII analogous-1 receptor; HDL, high density lipoprotein; apoA-I, apolipoprotein A-I; apoB, apolipoprotein B; LDL, low density lipoprotein; LIMPII, lysosomal integral membrane protein II; PS, phosphatidylserine; PC, phosphatidylcholine; PI, phosphatidylinositol; PMA, phorbol 12-myristate 13-acetate; CE, cholesterol ester; OxLDL, oxidized LDL; AcLDL, acetylated LDL; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s). was originally cloned on the basis of its ability to bind modified lipoproteins and was therefore classified as a novel scavenger receptor (1Acton S.L. Scherer P.E. Lodish H.F. Krieger M. J. Biol. Chem. 1994; 269: 21003-21009Abstract Full Text PDF PubMed Google Scholar). Its ligand-binding specificity was similar to that of CD36, and thus it was included as a member of the new family of scavenger receptors, designated class B (2Rigotti A. Acton S.L. Krieger M. J. Biol. Chem. 1995; 270: 16221-16224Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar). Subsequent studies showed that SR-BI has a high affinity for the binding of HDL and that it mediates the selective uptake of cholesterol esters from HDL (3Acton S. Rigotti A. Landschulz K.T. Xu S.Z. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (2030) Google Scholar). Furthermore, the tissue distribution of SR-BI, which is predominantly expressed in liver, adrenal gland, and ovary, was compatible with its playing a role in the transport of HDL-derived cholesterol esters to the liver and to steroidogenic tissues (4Landschulz K.T. Pathak R.K. Rigotti A. Krieger M. Hobbs H.H. J. Clin. Invest. 1996; 98: 984-995Crossref PubMed Scopus (475) Google Scholar). More recently, it was reported that mice deficient in apoA-I overexpressed SR-BI, presumably in an effort to compensate for the decreased plasma level of HDL cholesterol and the depleted stores of adrenal cholesterol (5Wang N. Weng W. Breslow J.L. Tall A.R. J. Biol. Chem. 1996; 271: 21001-21004Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). In contrast, transgenic mice deficient in apolipoprotein A-II, apolipoprotein E, the LDL receptor, or cholesterol ester transfer protein did not show any changes of SR-BI expression in either adrenal gland or liver (5Wang N. Weng W. Breslow J.L. Tall A.R. J. Biol. Chem. 1996; 271: 21001-21004Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Previous studies have demonstrated a strong inverse correlation between plasma HDL levels and risk of coronary heart disease (6Buring J.E. O'Connor G.T. Goldhaber S.Z. Rosner B. Herbert P.N. Blum C.B. Breslow J.L. Hennekens C.H. Circulation. 1992; 85: 22-29Crossref PubMed Scopus (207) Google Scholar), possibly because HDL plays a critical role in reverse cholesterol transport, i.e. transport from peripheral tissues to the liver for catabolism (7Miller N.E. La Ville A. Crook D. Nature. 1985; 314: 109-111Crossref PubMed Scopus (160) Google Scholar, 8Franceschini G. Maderna P. Sirtori C.R. Atherosclerosis. 1991; 88: 99-107Abstract Full Text PDF PubMed Scopus (97) Google Scholar, 9Badimon J.J. Fuster V. Badimon L. Circulation. 1992; 86: III86-III94PubMed Google Scholar). Taken together, these results suggest that SR-BI may play a physiologically important role in metabolism of HDL-derived cholesterol. Human CLA-1 was cloned from a cDNA library prepared from differentiated HL-60 cells based on the existence of regions with amino acid sequence highly conserved between CD36 and LIMPII (10Calvo D. Vega M.A. J. Biol. Chem. 1993; 268: 18929-18935Abstract Full Text PDF PubMed Google Scholar). CD36 is an 88-kDa surface glycoprotein present on monocytes, platelets, and endothelial cells (11Greenwalt D.E. Lipsky R.H. Ockenhouse C.F. Ikeda H. Tandon N.N. Jamieson G.A. Blood. 1992; 80: 1105-1115Crossref PubMed Google Scholar), and LIMPII, a structural homologue of CD36, is expressed mainly on lysosomal membranes. Two distinct isoforms of CLA-1 that differ by an insertion of a segment consisting of 100 amino acid residues at the N-terminal region have been identified. Analysis of the CLA-1 cDNAs predicts proteins of 409 and 509 amino acid residues with several potential N-glycosylation sites. Like CD36, which binds to thrombospondin (12Asch A.S. Silbiger S. Heimer E. Nachman R.L. Biochem. Biophys. Res. Commun. 1992; 182: 1208-1217Crossref PubMed Scopus (169) Google Scholar, 13Leung L.L.K. Li W.-X. McGregor J.L. Albrecht G. Howard R.J. J. Biol. Chem. 1992; 267: 18244-18250Abstract Full Text PDF PubMed Google Scholar), collagen (14Tandon N.N. Kralisz U. Jamieson G.A. J. Biol. Chem. 1989; 264: 7576-7583Abstract Full Text PDF PubMed Google Scholar), and erythrocytes infected with Plasmodium falciparum (15Ockenhouse C.F. Tandon N.N. Magowan C. Jamieson G.A. Chulay J.D. Science. 1989; 243: 1469-1471Crossref PubMed Scopus (269) Google Scholar, 16Oquendo P. Hundt E. Lawler J. Seed B. Cell. 1989; 58: 95-101Abstract Full Text PDF PubMed Scopus (412) Google Scholar), CLA-1 is found mainly on the plasma membrane, underlining its potential function as a receptor. The long (509 amino acid residues) form of human CLA-1 shares 81% sequence identity with hamster SR-BI and thus most probably represents the same gene. However, the function of CLA-1 has not been systematically studied. If the primary function of SR-BI in rodents is to facilitate selective uptake of cholesterol esters from HDL, its human homologue CLA-1 might be redundant since human tissues are supplied with cholesterol mainly via LDL. We have generated stably transfected cells to test whether CLA-1 has biological functions similar to those of rodent SR-BI. The current report provides evidence that CLA-1 can function as a receptor for HDL and can mediate selective uptake of cholesterol esters, suggesting that CLA-1 is indeed functionally related to the rodent SR-BI. We find CLA-1 primarily expressed in liver and steroidogenic tissues, like SR-BI. However, we also find it in circulating monocytes and to a lesser extent in fully differentiated macrophages. Preliminary data show also that apoptotic thymocytes can bind to cells transfected with CLA-1. Thus, in addition to its role as receptor for HDL in liver and steroidogenic tissues, CLA-1 may have alternative functions in leukocytes. Cell culture media and G418 sulfate were purchased from Life Technologies Inc. Fetal calf serum was from HyClone Laboratories. Glutathione-Sepharose 4B beads were purchased from Pharmacia Biotech Inc. Carrier-free Na125I was obtained from Amersham and 32PdCTP was from ICN. Nitrocellulose membranes were from Bio-Rad. The phospholipids porcine brain PS, egg yolk PC, and bovine liver PI were obtained from Avanti Polar Lipids. Cholesterol from porcine liver, PMA, and Oil Red O were purchased from Sigma. Human HDL (d = 1.063–1.21 g/ml) and human LDL (d = 1.019–1.063 g/ml) were isolated by preparative ultracentrifugation from fresh plasma collected in EDTA (1 mg/ml) as described (17Havel R.J. Eder H.A. Bragdon J.H. J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6613) Google Scholar). HDL was passed through a heparin-Sepharose affinity column to remove particles containing apolipoprotein E (18Murakami M. Horiuchi S. Takata K. Morino Y. J. Biochem. ( Tokyo ). 1987; 101: 729-741Crossref PubMed Scopus (60) Google Scholar). The isolated lipoproteins were iodinated by the Pierce IODOGEN method to a specific activity of 400–600 cpm/ng protein for HDL and about 200 cpm/ng protein for LDL. For specific uptake studies, the lipid moiety of human HDL was with 3Hcholesterol ester and the apoA-I with The was prepared from 3Hcholesterol and as described J. Biol. Chem. 1987; Full Text PDF PubMed Google Scholar). HDL was with by from particles human transfer protein as the of transfer particles were from the HDL by = Human apoA-I was from HDL, with and the by a at J. Biol. Chem. 1987; Full Text PDF PubMed Google Scholar). The HDL was by = was prepared by LDL for in and was prepared by with as described J.L. U. S. A. PubMed Scopus Google Scholar). Human cells were in with calf 100 and 100 in a containing Human cells were in with containing serum and Human monocytes were isolated from by through and human were prepared by the monocytes in with calf serum for J. Google Scholar). of cells was by the cells for in the of of as membranes were by a with and 1989; Google Scholar). of thymocytes was obtained by tissues by it through a were and as described J.J. J. 1992; PubMed Google Scholar). was by the thymocytes in the of for Cell binding were by apoptotic and thymocytes with transfected cells at for in containing bovine serum of = to remove the of cells binding or thymocytes was determined. In the binding of thymocytes by the transfected cells was in the of consisting of phospholipids and cholesterol. and cholesterol in = = = were and in by were by high of through membranes G. Biophys. 1985; PubMed Scopus Google Scholar). The of in from to 100 were used For of binding by the transfected the were to the to a lipid of The cDNA of the acid form of CLA-1, the sequence for of was by from cDNA as described M. K. Y. J. 1994; PubMed Scopus Google Scholar), and cloned the expression The sequence of CLA-1 cDNA was by cDNA cells were transfected with of as described Biochem. Biophys. Res. Commun. 1991; PubMed Scopus Google Scholar). were by their to G418 sulfate of and a high CLA-1 expression was identified by by transfected cells or cells was at for in the or of a of HDL. was by the cells with containing bovine serum and by an additional with The cells were in and the was to specificity was in of LDL, AcLDL, and test the of phospholipids on HDL binding by CLA-1, containing PS, PI, or were prepared as described and with to the to a lipid of were as described S. D. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar). was isolated from the human cells and several human tissues by acid P. N. Biochem. 1987; PubMed Scopus Google or purchased from cDNA of the acid form of human CLA-1 was by from cells and with 32PdCTP by the method and were as described M. K. Y. J. 1994; PubMed Scopus Google Scholar). the was in at In CLA-1 expression was also by of the the sequence of CLA-1 but with homology with CD36 was used to an was and the of A. J.L. J. PubMed Scopus Google Scholar). An the of CLA-1 between amino acid residues and of the reported sequence of the containing 509 amino acid residues (10Calvo D. Vega M.A. J. Biol. Chem. 1993; 268: 18929-18935Abstract Full Text PDF PubMed Google was The cDNA was from cDNA by The was a and and the protein was expressed The protein was isolated with 4B beads and used to an in The was and used for we used an were with = and in 200 of were used for of cholesterol by a of the method R.L. J. Res. Full Text PDF PubMed Google Scholar). Cell were and with Oil Red O as described J.L. Krieger M. J. Cell Biol. PubMed Scopus Google Scholar). was by the method of R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and was as described Nature. PubMed Scopus Google Scholar). were by of and of proteins from the cells stably expressing CLA-1, the an of CLA-1 described a with an of cells also CLA-1 but at a the role of CLA-1 as a receptor for HDL, we binding The transfected cells expressing CLA-1 bound HDL with high affinity of the binding data d of 35 of HDL and binding of of HDL of However, binding of HDL by CLA-1 was not with any degradation of HDL was the same of lipoprotein to cells transfected with CLA-1. lipoprotein HDL were in the or of of HDL, native LDL, or The used was of In the of the transfected cells bound of of which was as are the of of HDL binding by LDL. The transfected cells were with in the binding of of which was as and of HDL as well as LDL were The of the lipoproteins were based on the of protein for LDL, and protein for HDL. is the of degradation of cells and cells were for at with the The degradation of was as described is the of the the of the are transfected cells expressing CLA-1 showed in their in containing calf Oil Red O that these lipid In contrast, was Oil Red O in cells of CLA-1 accumulation to a level that was about that in cells The cholesterol level was but that the of was we the transfected cells in the level was by about suggesting that lipoproteins in the calf serum were the for the in cholesterol The by which cells cholesterol ester from HDL are not but results from a of a for of J. Biol. Chem. 1987; Full Text PDF PubMed Google Scholar). We whether CLA-1 could in a by the of the uptake of and of in HDL. The binding of to transfected cells expressing CLA-1 a at and to In contrast, 3Hcholesterol ester and was at transfer of the degradation of were in the The cells low levels of either or apolipoprotein on the sequence CLA-1 is to to the same family as CD36 and LIMPII (10Calvo D. Vega M.A. J. Biol. Chem. 1993; 268: 18929-18935Abstract Full Text PDF PubMed Google Scholar, D. J. Vega M.A. 1995; PubMed Scopus Google Scholar). However, its biological function and tissue distribution is We the expression of CLA-1 in several human tissues by The was as a with (10Calvo D. Vega M.A. J. Biol. Chem. 1993; 268: 18929-18935Abstract Full Text PDF PubMed Google Scholar). CLA-1 was strongly expressed in the adrenal gland, and was also expressed in liver, and expression was in tissue and and CLA-1 was in the These findings are with the that CLA-1 in has a role in selective uptake of from HDL in steroidogenic tissues and in reverse cholesterol transport (3Acton S. Rigotti A. Landschulz K.T. Xu S.Z. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (2030) Google K.T. Pathak R.K. Rigotti A. Krieger M. Hobbs H.H. J. Clin. Invest. 1996; 98: 984-995Crossref PubMed Scopus (475) Google Scholar, C. M. D. J. Biol. Chem. 1985; Full Text PDF PubMed Google Scholar). The function of CLA-1 in monocytes, is could have a role as a scavenger receptor, like its test we the expression of CLA-1 in human the and cells expressed the level of CLA-1. The level of expression in these was to that in prepared human However, expression was in human of cells for with 100 resulted in a of CLA-1 This of CLA-1 expression the cells in and addition of at to the tissue culture the that CLA-1 was a plasma protein which was by not with the of CLA-1 protein was also the of cells In on and differentiated we found that cells bound HDL with an affinity similar to that of CLA-1 expressed in transfected of cells with the of HDL binding by about with or of binding affinity The HDL binding cells that CLA-1 could in as a receptor for HDL in monocytes, it is whether is its primary suggested that native LDL and modified LDL and bind to hamster SR-BI (1Acton S.L. Scherer P.E. Lodish H.F. Krieger M. J. Biol. Chem. 1994; 269: 21003-21009Abstract Full Text PDF PubMed Google Scholar). the binding specificity of human CLA-1. The long form of CLA-1 (509 amino acid residues) stably expressed in cells bound HDL, but it also with native and modified LDL the binding of HDL to CLA-1 by about The of and native LDL were However, the lipoproteins were at an of based on their protein The results from a that LDL, present at similar binding of by the cells as as HDL In binding we that CLA-1 recognized native LDL with d of about of LDL However, in to the in HDL, the of native LDL was and The cells were also to mediate LDL possibly through uptake via the receptor. However, expression of CLA-1 in the same that level by about Our findings that CLA-1 is expressed on monocytes to binding studies to phospholipids that are for scavenger B 1996; PubMed Scopus Google Scholar). the binding of HDL by about which was similar to the by In contrast, on HDL by which apoptotic cells is through binding to on the of the plasma membrane J. V. P. C. 1993; Full Text PDF PubMed Scopus Google Scholar). test whether CLA-1 can apoptotic cells via the we cells with apoptotic thymocytes. Analysis of the binding studies showed that CLA-1 recognized apoptotic but not thymocytes The extent of binding of apoptotic cells was highly However, about of the transfected cells showed binding of apoptotic thymocytes. The cells did not bind oxidized cells not The binding of apoptotic thymocytes to CLA-1 was by containing or In contrast, on the that the recognition of apoptotic cells by might be through a possibly CLA-1. However, studies are to of apoptotic thymocytes to of cells binding or cells cells or apoptotic thymocytes were to the transfected cells expressing CLA-1 at a of = and for at In the the phospholipids were as = prepared as described at a lipid of cells were and the thymocytes to the transfected cells were The results are expressed as the of or cells binding or thymocytes. the of from the binding to transfected cells from the binding to CLA-1 transfected cells without in a new or apoptotic thymocytes were to the transfected cells expressing CLA-1 at a of = and for at In the the phospholipids were as = prepared as described at a lipid of cells were and the thymocytes to the transfected cells were The results are expressed as the of or cells binding or thymocytes. the of from the binding to transfected cells from the binding to CLA-1 transfected cells without These studies show that the acid of CLA-1, like its close rodent SR-BI, binds HDL in a and can mediate the selective uptake of cholesterol esters from HDL. or not role of the receptor is important in lipid transport in to be determined. studies suggest that the of cholesterol for in is LDL, but it is to the of LDL and HDL in in LDL are low in adrenal function to to these selective cholesterol ester transport from HDL via CLA-1 might physiologically The role of SR-BI in the selective uptake of HDL-derived cholesterol esters has been demonstrated in studies (4Landschulz K.T. Pathak R.K. Rigotti A. Krieger M. Hobbs H.H. J. Clin. Invest. 1996; 98: 984-995Crossref PubMed Scopus (475) Google Scholar, N. Weng W. Breslow J.L. Tall A.R. J. Biol. Chem. 1996; 271: 21001-21004Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). CLA-1 not a high sequence but as results it is also functionally related to SR-BI. a similar ligand-binding specificity and show an tissue that in CLA-1 may have a function similar to that of SR-BI in binding demonstrated that CLA-1 can function as receptor for HDL. The binding of HDL was with accumulation of HDL-derived without any degradation of In contrast, the transfected cells not bound LDL, but also and its CLA-1 binds LDL with high affinity d = of but its as a receptor for LDL is In in the receptor to a in plasma LDL suggesting that the receptor is the LDL receptor J.L. Cell Biol. 1985; PubMed Scopus Google and that CLA-1 might not be in LDL The high level of expression of CLA-1 in circulating human monocytes to that found in human was it is not monocytes selective uptake of HDL cholesterol esters, since as as we cholesterol to any important However, and of monocytes, the for cholesterol is high and might the from LDL. the cells in the are may of cholesterol at high and HDL via selective cholesterol ester uptake might The is that CLA-1, to bind apoptotic may play a role in the of damaged a function of CLA-1 as a scavenger receptor is the that its expression is the a However, low levels of CLA-1 were in human CLA-1 expression and HDL binding was in cells differentiated with B and and selective uptake of cholesterol esters was demonstrated in human S. E. H. Atherosclerosis. 1994; Full Text PDF PubMed Scopus Google Scholar). In contrast, expression of CD36, and a identified receptor for and possibly apoptotic is in the which is for that are in of damaged cells P. C. Krieger M. U. S. A. 85: PubMed Scopus Google Scholar, J.L. R.L. Blood. 1996; PubMed Google Scholar, J. S. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, W. J.L. D. U. S. A. 1995; PubMed Scopus Google Scholar, V. N. D. U. S. A. 1996; PubMed Scopus Google Scholar). might that CLA-1 on the might play a role in the selective of cells the but is evidence for of the by which apoptotic cells is through for J. V. P. C. 1993; Full Text PDF PubMed Scopus Google Scholar). cells and cells the membrane in which anionic phospholipids are to the of the plasma a on the of the like and bound and also recognized apoptotic thymocytes. which are by through a presumably D. U. S. A. 1995; PubMed Scopus Google Scholar), were not recognized by the transfected cells not that several distinct for might the for oxidized cells is and on of the plasma membrane in addition to on the D. U. S. A. 1995; PubMed Scopus Google Scholar). The for the recognition of these additional may not be present on the which therefore not bind oxidized phospholipids not the binding of apoptotic thymocytes by CLA-1, but are also for the binding of HDL. The on the phospholipids as well as their on the have been suggested to play an important role in that (2Rigotti A. Acton S.L. Krieger M. J. Biol. Chem. 1995; 270: 16221-16224Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar). Taken together, these data suggest that several by which monocytes could apoptotic of which CLA-1. The not but may a CLA-1 in and expression of for similar role has been suggested for CD36, which may a to of monocytes and thus may have functions and C. P. Google Scholar). In we have demonstrated like SR-BI, CLA-1 can function as a receptor for HDL and mediate the selective uptake of the HDL cholesterol esters. is expressed mainly in liver and steroidogenic In CLA-1 like SR-BI, a specificity that is for scavenger its expression is in fully differentiated macrophages. The role of CLA-1 in cholesterol as well as its function as a receptor on monocytes, We W. and J. L. for and E. and J. for their
Murao et al. (Tue,) studied this question.