The Processing of Ligands by the Class A Scavenger Receptor Is Dependent on Signal Information Located in the Cytoplasmic Domain (1999)

The mechanisms that regulate the transport of the macrophage class A scavenger receptor during ligand uptake were investigated. Kinetic analysis of the changes in receptor phosphorylation demonstrated that serine phosphorylation increased during the internalization of acetyl-low density lipoproteins (LDL) by macrophages. The increase was maximal at about 2.5 min after the initiation of ligand uptake. Oxidized LDL also stimulated serine phosphorylation, but the relative increase was smaller and the time to maximum was shorter. Receptor mutants expressed in Chinese hamster ovary and COS cells showed that elimination of the potential phosphorylation site at Ser21 increased acetyl-LDL metabolism, whereas inactivation of the site at Ser49reduced acetyl-LDL uptake. The increase in uptake by the Ser21 mutant was due to an increase in surface receptor expression. In contrast, elimination of the site at Ser49did not affect receptor expression but slowed receptor internalization. To identify potential internalization signal sequences, β-turn structure in the cytosolic domain was targeted for mutagenesis. Disruption of one region near Asp25 inhibited receptor activity. The studies support a model whereby receptor internalization requires the presence of an internalization signal motif but that the rate of receptor internalization is governed by the pattern of receptor phosphorylation induced by the ligand. The mechanisms that regulate the transport of the macrophage class A scavenger receptor during ligand uptake were investigated. Kinetic analysis of the changes in receptor phosphorylation demonstrated that serine phosphorylation increased during the internalization of acetyl-low density lipoproteins (LDL) by macrophages. The increase was maximal at about 2.5 min after the initiation of ligand uptake. Oxidized LDL also stimulated serine phosphorylation, but the relative increase was smaller and the time to maximum was shorter. Receptor mutants expressed in Chinese hamster ovary and COS cells showed that elimination of the potential phosphorylation site at Ser21 increased acetyl-LDL metabolism, whereas inactivation of the site at Ser49reduced acetyl-LDL uptake. The increase in uptake by the Ser21 mutant was due to an increase in surface receptor expression. In contrast, elimination of the site at Ser49did not affect receptor expression but slowed receptor internalization. To identify potential internalization signal sequences, β-turn structure in the cytosolic domain was targeted for mutagenesis. Disruption of one region near Asp25 inhibited receptor activity. The studies support a model whereby receptor internalization requires the presence of an internalization signal motif but that the rate of receptor internalization is governed by the pattern of receptor phosphorylation induced by the ligand. low density lipoproteins Dulbecco's modified Eagle's medium fetal calf serum dimethyl sulfoxide chloramphenicol acetyltransferase Chinese hamster ovary Nu-Serum phosphate-buffered saline The predominant feature of the fatty streak lesion of atherosclerosis is the presence of lipid-engorged cells, called foam cells, in the arterial intima. The appearance of fatty streaks in all models of lipid-induced atherosclerosis predicts that they are part of the early events and are important to explore the mechanism by which they are formed. The major cellular events that precede fatty streak formation are fairly consistent between different atherosclerosis models; however, it is clear from the various transgenic and knockout mouse models that the transition to the fatty streak can be initiated by a number of metabolic alterations. This supports the view that it is unlikely that a single pathogenic event accounting for fatty streak lesion formation under all circumstances will be identified, and the net accumulation of cholesterol in the artery wall, in any particular model, will be determined by the balance between the processes that promote cholesterol deposition and those that oppose it. One component of the formulation is likely to be the modified low density lipoprotein (LDL)1-scavenger receptor pathway (reviewed in Refs. 1Steinberg D. Parthasarathy S. Carew T.E. Khoo J.C. Witztum J.L. N. Engl. J. Med. 1989; 320: 915-924Crossref PubMed Google Scholar and 2Steinberg D. J. Biol. Chem. 1997; 272: 20963-20966Abstract Full Text Full Text PDF PubMed Scopus (1445) Google Scholar). The hypothesis proposes that LDL that has entered the subendothelial space is oxidized by artery wall cells, producing a modified lipoprotein exhibiting biological properties that promote atherosclerotic lesion development. One of these is avid metabolism by monocyte-derived macrophages leading to the excessive accumulation of lipid and foam cell formation (3Henriksen T. Mahoney E.M. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6499-6503Crossref PubMed Scopus (810) Google Scholar, 4Heinecke J.W. Baker L. Rosen H. Chait A. J. Clin. Invest. 1986; 77: 757-761Crossref PubMed Scopus (429) Google Scholar). This is supported by the demonstration that modified forms of LDL are located in regions of atherosclerosis (5Rosenfeld M.E. Palinski W. Yla-Herttuala S. Butler S. Witztum J.L. Arteriosclerosis. 1990; 10: 336-349Crossref PubMed Scopus (411) Google Scholar, 6Haberland M.E. Fong D. Cheng L. Science. 1988; 241: 215-218Crossref PubMed Scopus (610) Google Scholar), and arterial wall foam cells express receptors that recognize oxidized LDL (7Rosenfeld M.E. Khoo J.C. Miller E. Parthasarathy S. Palinski W. Witztum J.L. J. Clin. Invest. 1991; 87: 90-99Crossref PubMed Scopus (179) Google Scholar). Macrophages express multiple receptors on their cell surface that bind modified forms of LDL, including the class A scavenger receptor (8Brown M.S. Basu S.K. Falck J.R. Ho Y.K. Goldstein J.L. J. Supermol. Struct. 1980; 13: 67-81Crossref PubMed Scopus (351) Google Scholar), CD36 (9Endemann G. Stanton L.W. Madden K.S. Bryant C.M. White R.T. Protter A.A. J. Biol. Chem. 1993; 268: 11811-11816Abstract Full Text PDF PubMed Google Scholar), macrosialin (10Ramprasad M.P. Fischer W. Witztum J.L. Sambrano G.R. Quehenberger O. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9580-9584Crossref PubMed Scopus (296) Google Scholar), the Fc receptor (11Stanton L.W. White R.T. Bryant C.M. Protter A.A. J. Biol. Chem. 1992; 267: 22446-22451Abstract Full Text PDF PubMed Google Scholar), the class B scavenger receptor (12Acton S.L. Scherer P.E. Lodish H.F. Krieger M. J. Biol. Chem. 1994; 269: 21003-21009Abstract Full Text PDF PubMed Google Scholar), and macrophage receptor with a collagenous structure (13Elomaa O. Kangas M. Sahlberg C. Tuukkanen J. Sormunen R. Liakka A. Thesleff I. Kraal G. Tryggvason K. Cell. 1995; 80: 603-609Abstract Full Text PDF PubMed Scopus (406) Google Scholar). Their relative importance to foam cell formation is unclear; however, the most compelling evidence thus far establishes a significant role for the class A scavenger receptor. Kodama and colleagues (14Suzuki H. Kurihara Y. Takeya M. Kamada N. Kataoka M. Jishage K. Ueda O. Sakaguchi H. Higashi T. Suzuki T. Takashima Y. Kawabe Y. Cynshi O. Wada Y. Doi T. Matsumoto A. Azuma S. Noda T. Toyada Y. Itakura H. Yazaki Y. Horiuchi S. Takahashi K. Steinbrecher U.P. Ishibashi S. Maeda N. Gordon S. Kodama T. Nature. 1997; 386: 292-296Crossref PubMed Scopus (994) Google Scholar) generated, by targeted gene disruption, a mouse line that does not express the scavenger receptor. When scavenger receptor knockout mice were crossed with apoE-deficient mice, lesion formation was decreased by 58% (14Suzuki H. Kurihara Y. Takeya M. Kamada N. Kataoka M. Jishage K. Ueda O. Sakaguchi H. Higashi T. Suzuki T. Takashima Y. Kawabe Y. Cynshi O. Wada Y. Doi T. Matsumoto A. Azuma S. Noda T. Toyada Y. Itakura H. Yazaki Y. Horiuchi S. Takahashi K. Steinbrecher U.P. Ishibashi S. Maeda N. Gordon S. Kodama T. Nature. 1997; 386: 292-296Crossref PubMed Scopus (994) Google Scholar). This is quite remarkable considering that apoE deficiency produces plasma cholesterol levels in excess of 500 mg/dl and extensive atherosclerosis. The processing of modified lipoproteins by the class A scavenger receptor (scavenger receptor A) follows a specific itinerary (8Brown M.S. Basu S.K. Falck J.R. Ho Y.K. Goldstein J.L. J. Supermol. Struct. 1980; 13: 67-81Crossref PubMed Scopus (351) Google Scholar,15Naito M. Kodama T. Akiyo M. Doi T. Takahashi K. Am. J. Pathol. 1991; 139: 1411-1423PubMed Google Scholar, 16Mori T. Takahashi K. Naito M. Kodama T. Hakamata H. Sakai M. Miyazaki A. Horiuchi M. Ando M. Lab. Invest. 1994; 71: 409-416PubMed Google Scholar, 17Doi T. Kurasawa M. Higashino K. Imanishi T. Mori T. Naito M. Takahashi K. Kawabe Y. Wada Y. Matsumoto A. Kodama T. J. Biol. Chem. 1994; 269: 25598-25604Abstract Full Text PDF PubMed Google Scholar, 18Fong L.G. Fong T.A.T. Cooper A.D. J, Biol, Chem. 1990; 265: 11751-11760Abstract Full Text PDF PubMed Google Scholar, 19Robenek H. Schmitz G. Assman G. J. Histochem. Cytochem. 1984; 32: 1017-1027Crossref PubMed Scopus (23) Google Scholar, 20Mommas-Kienhuis A.M. van der Schroeff J.G. Wijsman M.C. Daems W.T. Vermeer B.J. Histochemistry. 1985; 83: 29-35Crossref PubMed Scopus (17) Google Scholar, 21Fukuda S. Horiuchi S. Tomita K. Murakami M. Morino Y. Takahashi K. Virchow's Arch. B Cell Pathol. Incl. Mol. Pathol. 1986; 52: 1-13Crossref PubMed Scopus (27) Google Scholar). After binding to surface receptors, the receptor-ligand complexes concentrate in coated pits and are rapidly internalized into endosomes. The ligand dissociates from the receptor and is transported within endosomes to lysosomes where it is metabolized while the receptor recycles back to the cell surface. The process is highly efficient and can lead to foam cell formation in vitro. The receptor does not appear to cycle continuously through the metabolic pathway since, in the absence of suitable ligands, receptors are not present in coated pits. This suggests that the process depends upon a stimulus and thus it is regulated. In support of this, pharmacological studies have shown that receptor transport can be modulated. Alterations of calcium flux, protein phosphorylation, or protein glycosylation have been reported to affect scavenger receptor transport (22Fong L.G. J. Lipid Res. 1996; 37: 574-587Abstract Full Text PDF PubMed Google Scholar, 23Bernini F. Scurati N. Bonfadin G. Fumagalli E. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1352-1358Crossref PubMed Scopus (38) Google Scholar, 24Van Berkel T.J.C. Nagelkerke J.F. Kruijt J.K. FEBS Lett. 1981; 132: 61-66Crossref PubMed Scopus (19) Google Scholar, 25Sulistiyani R.W. St C. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1691-1700Crossref PubMed Google Scholar). The basis for their actions or the precise step(s) affected has not yet been established. The most direct evidence that receptor transport is tightly controlled is based on the observation of Doi and colleagues (17Doi T. Kurasawa M. Higashino K. Imanishi T. Mori T. Naito M. Takahashi K. Kawabe Y. Wada Y. Matsumoto A. Kodama T. J. Biol. Chem. 1994; 269: 25598-25604Abstract Full Text PDF PubMed Google Scholar). They showed that the substitution of His260 in the α-helical coiled-coil domain of the receptor with a leucine prevents the acid pH-induced dissociation of ligands from the scavenger receptor. When cells expressing this mutant receptor were incubated with ligand, the binding and internalization of ligand proceeded normally; however, after their internalization, the receptors did not recycle back to the cell surface. Recent studies have begun to map regions of the receptor important for ligand binding (26Andersson L. Freeman M.W. J. Biol. Chem. 1998; 273: 19592-19601Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The present study focuses on the processes likely to control scavenger receptor internalization. There are two of potential importance; the expression of an internalization signal motif and receptor phosphorylation (reviewed in Refs. 27Trowbridge I.S. Collawn J.F. Hopkins C.R. Annu. Rev. Cell Biol. 1993; 9: 129-161Crossref PubMed Scopus (700) Google Scholar and 28Pearse B.M.F. Robinson M.S. Annu. Rev. Cell Biol. 1990; 6: 151-171Crossref PubMed Scopus (534) Google Scholar). The signal motifs are thought to define a three-dimensional conformation and chemistry. These conformations are thought to provide the appropriate signal for the association of the cytoplasmic domain of receptors with components of clathrin-coated pits (28Pearse B.M.F. Robinson M.S. Annu. Rev. Cell Biol. 1990; 6: 151-171Crossref PubMed Scopus (534) Google Scholar, 29Collawn J.F. Stangel M. Kuhn L.A. Esekogwu V. Jing S. Trowbridge I.S. Tainer J.A. Cell. 1990; 63: 1061-1072Abstract Full Text PDF PubMed Scopus (388) Google Scholar, 30Keen J.H. Annu. Rev. Biochem. 1990; 59: 415-438Crossref PubMed Scopus (170) Google Scholar), thereby providing an efficient mechanism for the selective concentration of receptors at sites of internalization. An internalization signal sequence has not been identified for the scavenger receptor; however, the targeting of scavenger receptor-ligand complexes to coated pits strongly suggests that one may be present. Changes in receptor phosphorylation can also modulate receptor internalization. This has been documented for several receptors that are internalized at coated pits including the asialoglycoprotein receptor (31Fallon R.J. Danaher R.L. Saylor R.L. Saxena A. J. Biol. Chem. 1994; 269: 11011-11017Abstract Full Text PDF PubMed Google Scholar), epidermal growth factor receptor (32Wiley H.S. Herbst J.J. Walsh B.J. Lauffenburger D.A. Rosenfeld M.G. Gill G.N. J. Biol. Chem. 1991; 266: 11083-11094Abstract Full Text PDF PubMed Google Scholar), and the insulin receptor (33Carpentier J.L. Paccaud J.P. Baecker J. Gilbert A. Orci L. Kahn C.R. J. Cell Biol. 1993; 122: 1243-1252Crossref PubMed Scopus (49) Google Scholar). The scavenger receptor contains three conserved phosphorylation sites in the cytoplasmic domain of the human, mouse, bovine, and rabbit receptors (34Kodama T. Freeman M. Rohrer L. Zabrecky J. Matsudaira P. Krieger M. Nature. 1990; 343: 531-535Crossref PubMed Scopus (833) Google Scholar, 35Ashkenas J. Penman M. Vasile E. Acton S. Freeman M. Krieger M. J. Lipid Res. 1993; 34: 983-1000Abstract Full Text PDF PubMed Google Scholar, 36Matsumoto A. Naito M. Itakura H. Ikemoto S. Asaoka H. Hayakawa I. Kanamori H. Aburatani H. Takaku F. Suzuki H. Kobari Y. Miyai T. Takahashi K. Cohen E.H. Wydro R. Housman D.E. Kodama T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9133-9137Crossref PubMed Scopus (301) Google Scholar, 37Bickel P.E. Freeman M.W.. J. Clin. Invest. 1992; 90: 1450-1457Crossref PubMed Scopus (106) Google Scholar) located at Ser21, Thr30, and Ser49. We have shown previously that the mouse macrophage scavenger receptor is phosphorylated in situ (22Fong L.G. J. Lipid Res. 1996; 37: 574-587Abstract Full Text PDF PubMed Google Scholar), but the relation of this to receptor transport has not yet been established. This report investigates the role of receptor phosphorylation and potential internalization motifs in the control of receptor function during modified LDL uptake. The results show that the function of the mouse scavenger receptor is dependent on both receptor phosphorylation and one or more internalization signal motifs. The presence of phosphorylation sites in the cytoplasmic domain that affect receptor function both in a positive and negative fashion predict that the level of receptor activity will depend on which sites are phosphorylated during the metabolic cycle. However, the dependence on phosphorylation is not absolute. It is proposed that receptor phosphorylation acts as a modulator of receptor activity by governing the accessibility of one or more internalization signal sequences in the receptor cytoplasmic domain with specific components of clathrin-coated pit structures. Carrier-free Na125I and [14C]chloramphenicol were purchased from Amersham Pharmacia Biotech. Endotoxin tested Dulbecco's modified Eagle's medium (DMEM) and fetal calf serum (FCS) were purchased from Life Technologies, Inc. FCS was heat-inactivated before use (56 °C for 30 min). Nu-Serum (NS) was obtained from Becton Dickenson (Bedford, MA). Butyryl-CoA, chloramphenicol, dimethyl sulfoxide (Me2SO), DEAE-dextran (M r 500,000), chloroquine, phosphoserine, phosphothreonine, and phosphotyrosine were purchased from Sigma. COS-7 and CHO K-1 cells were obtained from American Type Culture Collection (Manassas, VA). Synthetic oligonucleotides were purchased from Genemed Biotechnologies (South San Francisco, CA) or Genosys (Woodlands, TX). Human LDL (d 1.019–1.063 g/ml) was isolated from EDTA-treated plasma by density gradient ultracentrifugation (38Havel R.J. Eder H.A. Bragdon J.H. J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6449) Google Scholar). LDL was radiolabeled with Na125I using IODO-GEN (Pierce) to specific activities of 200–400 cpm/ng. The acetylation and oxidation of LDL were performed as described previously (18Fong L.G. Fong T.A.T. Cooper A.D. J, Biol, Chem. 1990; 265: 11751-11760Abstract Full Text PDF PubMed Google Scholar, 22Fong L.G. J. Lipid Res. 1996; 37: 574-587Abstract Full Text PDF PubMed Google Scholar). The class A type I mouse scavenger receptor cDNA was kindly provided by Dr. Larry Stanton at Scios Nova (Mountain View, CA). This was subcloned into pcDNA 3.1 (Invitrogen) using the restriction enzyme sites BamHI andNotI. The mutagenesis was performed using the Stratagene QuickchangeTM method that is based on the incorporation of a mutation using Pfu DNA polymerase and complementary mutagenic oligonucleotide primers. The native receptor mutant construct (50 ng) was incubated with 125 ng of each mutagenic oligonucleotide primer (32–38 bases in length) and Pfu DNA polymerase (2.5 units). The temperature cycling parameters followed the recommendations suggested by the manufacturer. The reaction mixture was incubated withDpnI (20 units) to digest the parental DNA template and then used to transform Top10F′ cells (Invitrogen). DNA from selected bacterial colonies was isolated by phenol/chloroform extraction (39Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning. A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.1989: 1.25-1.28Google Scholar) and analyzed by manual dideoxynucleotide sequencing (Promega Fmol DNA Sequencing System). DNAs containing the correct mutations were identified, and large plasmid preparations were isolated by ion exchange chromatography (Qiagen; Valencia, CA). For a selected number of receptor constructs, the scavenger receptor cDNA was subcloned into the mammalian expression vector pCMV-Tag4 (Stratagene). This produces a fusion protein that contains an extra 10 amino acids (LEDYKDDDDK) located at the carboxyl terminus of the receptor protein. The stop codon in the scavenger receptor cDNA was eliminated and substituted with an XhoI site by polymerase chain reaction using the native scavenger receptor cDNA in pcDNA3.1 as the template and the primers 5′-CAAGCGCACGTGGAACAGGAAGTAAAACAG-3′ and 5′-GATCTCGAGTGAAGTACAAGTGACCCCAGC-3′. The polymerase chain reaction fragment was subcloned into each of the pcDNA 3.1 receptor mutant constructs using the unique restriction enzyme sites Bbr andXhoI. Sequencing confirmed that the epitope DNA sequence was in frame with the receptor cDNA sequence. The entire receptor cDNA was then subcloned into pCMV-Tag4 using the restriction enzyme sites BamHI and XhoI. COS-7 cells were transfected with plasmid DNA using DEAE-dextran (40Aruffo A. Current Protocols in Molecular Biology. 2. Greene Publishing Associates and Wiley-Interscience, New York1991: 16.13.1-16.13.7Google Scholar). COS cells were plated at a density of 4 × 105 cells/10-cm tissue culture dish and incubated overnight in DMEM supplemented with 10% FCS. The following day the cells were washed twice with DMEM containing 10% NS and incubated with the different plasmid DNAs. Prior to their addition to the cells, the DNA preparations were pretreated. The scavenger receptor DNA preparations (7.5 μg) were diluted in Tris-EDTA buffer (20 μl) and added to 15-ml culture tubes. To this was added 6 ml of a freshly containing chloroquine, and of the gene construct in 10% These were incubated at temperature for min and then added to the washed After at the cells were incubated with containing 10% at temperature for The cells were washed with 10% FCS and incubated in the The day the cells were by in ml of 10% and added to 4 of a and of a The metabolism of modified LDL and activities was CHO cells were transfected using calcium CHO cells were at a density of 4 × 105 tissue culture dish and overnight in medium supplemented with 10% FCS. The following day the culture medium was with medium and the cells were incubated for The calcium was by plasmid DNA (7.5 each of the receptor DNA construct and with 125 and DNA buffer and 6 in a of ml to the This was added to the cells and incubated for The culture medium was and the cells were incubated with ml of in DNA buffer at temperature for saline was added and incubated at temperature for 4 The cells were with and incubated overnight with tissue culture The day the cells were plated and analyzed as described for transfected COS cells in were with 10% FCS and incubated with modified To lipoprotein the cells were incubated with modified LDL in the medium at The of was determined by the of and in the and the of cell lipoprotein was determined by the of with an of a cell (18Fong L.G. Fong T.A.T. Cooper A.D. J, Biol, Chem. 1990; 265: 11751-11760Abstract Full Text PDF PubMed Google Scholar, 22Fong L.G. J. Lipid Res. 1996; 37: 574-587Abstract Full Text PDF PubMed Google Scholar). The of specific lipoprotein metabolism uptake was using to the of lipoprotein The results are expressed as the of or in cell protein. To lipoprotein the cells were incubated with modified LDL in 10% FCS with 10 at 4 °C for lipoprotein was by the cells three with and the was determined by the of The of specific binding was and expressed as described To correct for in the cells were with and the results based on the of activity J. Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley-Interscience, New Scholar). in culture were washed with and with of containing The cell was to a and at The was for and the protein concentration of the was using An containing 6 of protein for COS cells and for CHO cells was diluted to a of 10 with buffer and incubated with an of at °C for The and 4 [14C]chloramphenicol The reaction mixture was in a and then with of The was to and twice with of The of radiolabeled chloramphenicol in of the was then by these the of chloramphenicol to chloramphenicol was between and mouse macrophages in tissue culture were with as described previously (22Fong L.G. J. Lipid Res. 1996; 37: 574-587Abstract Full Text PDF PubMed Google Scholar). The cells were washed three with 10% FCS with 10 and incubated with lipoprotein (50 at 4 °C for lipoprotein was by with medium and then incubated at °C from to The cells were on and washed with that were not incubated at °C but on were also to protein phosphorylation at time The cells were and of cell protein were incubated with a receptor to the receptor protein (22Fong L.G. J. Lipid Res. 1996; 37: 574-587Abstract Full Text PDF PubMed Google Scholar). The were by and to by The of the receptor was identified by and the of containing the receptor was The were in of 6 at °C for and the was using a The was in of buffer containing each of phosphoserine, and An μl) of the was and the amino acids were using the der P. K. T. Cell A Laboratory for Human Scholar). in the was at in buffer for min and the at in buffer for The was with to the acid The radiolabeled were using a and the of for each acid was determined using a and using from three were and from the for each The results were expressed relative to time that was an of A maximum of can be analyzed on one to on an the number of for each was to and they were analyzed on the was by the method of R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar) or by the protein method (Pierce) using as a analysis was using The cytoplasmic domain of the scavenger receptor A contains three phosphorylation sites that are conserved all for which protein sequence is They are located at Ser21, Thr30, and Ser49. The mouse receptor contains a site at that is unique to the mouse protein. To receptor phosphorylation and receptor activity be the level of receptor phosphorylation during the internalization of a ligand of the scavenger receptor by mouse macrophages was To the of changes in phosphorylation, the internalization of surface receptors was This the number of surface receptors that are at the of the internalization were incubated with acetyl-LDL at 4 °C to surface binding and then washed to The cells were then incubated at °C to receptor-ligand internalization, and the level of receptor phosphorylation was by acid To the level of phosphorylation at one of cells was on and in the line was a low level of receptor phosphorylation on serine This was present cells were in the absence of lipoprotein the internalization of was an increase in the phosphorylation of serine but phosphorylation of or The absence of