The type I and type II bovine scavenger receptors expressed in Chinese hamster ovary cells are trimeric proteins with collagenous triple helical domains comprising noncovalently associated monomers and Cys83-disulfide-linked dimers.
Scavenger receptors have been implicated in the development of atherosclerosis and other macrophage-associated functions. The structures and processing of type I and type II bovine macrophage scavenger receptors were examined using polyclonal anti-receptor antibodies. Pulse/chase metabolic labeling experiments showed that both types of scavenger receptors expressed in Chinese hamster ovary (CHO) cells behaved as typical cell surface membrane glycoproteins. They were synthesized as endoglycosidase H-sensitive precursors which were converted to endoglycosidase H-resistant mature forms expressed on the cell surface. The reduced precursor and mature forms were doublets on sodium dodecyl sulfate-gel electrophoresis, primarily because of heterogeneous N-glycosylation. The approximate molecular sizes were: type I precursor, 65/63 kDa; type I mature, 82/76 kDa; type II precursor, 57/53 kDa; and type II mature, 72/65 kDa. During post-translational processing, the cysteine-rich C terminus (SRCR domain) of some of the type I receptors was proteolytically removed to form a relatively stable, approximately 69-kDa degradation product. Type II receptors differ from type I receptors in that they do not have SRCR domains and an analogous proteolytic cleavage was not observed. Several experiments provided strong evidence that the Gly-X-Y-repeat domains in the scavenger receptors oligomerize into collagenous triple helices. For example, alpha,alpha'-dipyridyl, an inhibitor of the collagen-modifying enzymes prolyl and lysyl hydroxylases, interfered with both the kinetics and nature of post-translational receptor processing, and both precursor and mature forms of the receptors in intact cells could be cross-linked with difluorodinitrobenzene into reduction-resistant trimers. In intact cells, precursor receptor trimers (type I, 198 kDa; type II, 176 kDa) were assembled in the endoplasmic reticulum by the noncovalent association of monomers and Cys83-disulfide-linked dimers (type I, 129 kDa; type II, 119 kDa). When cells were lysed in the absence of the sulfhydryl trapping agent iodoacetamide, oxidation of the side chain of Cys17 in the cytoplasmic domain leads to the artifactual formation of reduction-sensitive covalently linked trimers. The approximate masses of the mature dimer and trimer forms were 162 and 237 kDa for type I receptors and 147 and 219 kDa for type II receptors. Cys83-disulfide-linked dimer formation was not required for function because mutant receptors (Cys83----Gly83) assembled into trimers of noncovalently associated monomers and exhibited normal receptor activity. Treatment of cells with difluorodinitrobenzene cross-linked some of the receptors into complexes larger than trimers, raising the possibility that the trimers may assemble into higher order oligomers.
Penman
M
M
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Lux
A
A
Freedman
N J
NJ
Rohrer
L
L
Ekkel
Y
Y
McKinstry
H
H
Resnick
D
D
Krieger
M
M
D015082:2,2'-Dipyridyl / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002417:Cattle; D006224:Cricetinae; D003545:Cysteine; D004220:Disulfides; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011971:Receptors, Immunologic / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018110:Receptors, Lipoprotein; D051116:Receptors, Scavenger; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D051122:Scavenger Receptors, Class B; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D004220:Disulfides; D046911:Macromolecular Substances; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D011971:Receptors, Immunologic; D018110:Receptors, Lipoprotein; D051116:Receptors, Scavenger; D011994:Recombinant Proteins; C454739:Scarb1 protein, mouse; D051122:Scavenger Receptors, Class B; D014415:Tunicamycin; D015082:2,2'-Dipyridyl; D003545:Cysteine; D007460:Iodoacetamide