Abstract
Macrophage scavenger receptors have been implicated in various macrophage-associated processes, including atherosclerosis and clearance of bacterial endotoxin. They bind to a wide variety of polyanionic ligands and display complex binding characteristics. cDNAs from the murine macrophage-like cell line P388D1 encoding the full-length type I and type II murine scavenger receptors were cloned, sequenced, and expressed in Chinese hamster ovary cells. A fragment of the corresponding murine genomic DNA was also cloned, partially sequenced, and the positions of the cloned intron/exon boundaries were determined. Comparisons of the murine scavenger receptors' sequences with the bovine, rabbit, and human sequences were used to refine a multidomain model of these trimeric, fibrous, membrane receptors. Metabolic labeling/immunoprecipitation experiments showed that most of the macrophage scavenger receptor protein expressed by P388D1 cells was the N-glycosylated type II receptor; only small amounts of type I receptor were detected. Analysis of the binding properties of the receptors provided evidence that such differential expression of the type I and type II forms may have functional significance. There were substantial receptor-type (I vs. II), as well as receptor-species (bovine vs. murine), differences in the inhibition of 125I-labeled AcLDL (acetylated low density lipoprotein) binding by ReLPS, a form of bacterial endotoxin. These differences arose, in part, because these receptors exhibited both high (Kd1(4 degrees C) = 0.05-0.2 micrograms protein/ml) and low (Kd2(4 degrees C) = 2.5-12.8 micrograms protein/ml) affinity binding of 125I-labeled AcLDL. The ability of ReLPS (1 mg/ml) to inhibit either or both of these two classes of binding interactions varied depending on the species and type of receptor.
Highlights
Macrophage scavenger receptors have been implicated in various macrophage-associated processes, including atherosclerosis and clearance of bacterial endotoxin
-described the partial purification and characterization of 260 kD scavenger receptors from P388D1-derived tumors, and we have reported the cloning and sequencing of portions of the cDNAs encoding the C-terminal ends of the type I and type I1 murine scavenger receptors [13]
These C-terminal sequences were used in conjunction with the polymerase chain reaction (PCR) technique and standard hybridization screening to clone cDNAs encoding the full-length type I and type I1 polypeptides from a library generated from P388D1 cells, a murine macrophage-like cell line [37]
Summary
Maintained as stock cultures in medium E. A XDASH (Stratagene) genomic library derived from murine D3 cells, which was generously provided by Doug Gray and Rudolf Jaenisch, was screened by hybridization using as a probe a mixture of DNAs containing the murine scavenger receptor type I (bp -17 to 1440 in Fig. 1A) and type I1 (-17 to 1175 in Fig. 1B) cDNA sequences. O n day 1, each monolayer was rinsed twice with 1 ml of Dulbecco's phosphate-buffered saline (PBS), pulse-labeled for 15 min with 0.5 ml of methionine-free medium A containing 3% (v/v) newborn calf lipoprotein-deficient serum and 300 pCilml of 35Slabeled protein labeling mix (DuPont-New England Nuclear), washed once with medium C and chased for the indicated times in medium C supplemented with 1 mM unlabeled methionine. The immunoprecipitated proteins were solubilized by boiling in reducing sample buffer (0.2% SDS, 0.1 mg/ml bromphenol blue, 10% (v/v) glycerol, 0.5% (vlv) 6-mercaptoethanol, 62.5 mM Tris-HC1, pH 6.8) and subjected to SDS gel electrophoresis (3-10% polyacrylamide gradients) followed by fixation, staining, and autoradiography, as previously described [33]
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