Expression of a functional asialoglycoprotein receptor through transfection of a cloned cDNA that encodes a macrophage lectin.
The Gal/GalNAc-specific lectin on rat peritoneal macrophages (macrophage asialoglycoprotein binding protein, M-ASGP-BP) is structurally similar to rat hepatic asialoglycoprotein-binding protein (ASGP-BP) or rat hepatic lectin (RHL) and is highly homologous with the major component of RHL, RHL-1 (Ii, M, Kurata, H., Itoh, N., Yamashina, I., and Kawasaki, T. (1990) J. Biol. Chem. 265, 11295-11298). We found in this study that transfection with a cDNA clone that encodes a single polypeptide, M-ASGP-BP, was sufficient for the expression of an endocytic receptor for asialoorosomucoid (ASOR) on the COS-1 cell surface. The Kuptake value for ASOR for the transfected cells was 12.5 nM, which is similar to that for peritoneal macrophages (23 nM), and the number of ASOR bound on the cell surface was 1-8 x 10(5)/cell, this value being hundreds of times larger than that for peritoneal macrophages. 125I-ASOR bound on the surfaces of the transfected cells was rapidly internalized on incubation at 37 degrees C, and after 90 min of incubation, most of the radioactivity was recovered in acid-soluble degraded products from the medium. These results confirmed that the cDNA cloned in our previous study does in fact encode M-ASGP-BP and also that the single polypeptide chain can form a homooligomeric receptor (probably a hexamer or octamer) exhibiting high affinity for ASOR. The latter property was distinct from that of the hepatic ASGP-BP in that simultaneous transfection of two cloned cDNAs that encode RHL-1 and RHL-2/3 was required to produce an active ASOR receptor (McPhaul, M., and Berg, P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8863-8867). This M-ASGP-BP expression system may serve as a simple model with which to investigate the molecular mechanisms underlying carbohydrate-mediated endocytosis.
Ozaki
K
K
Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Japan.
Ii
M
M
Itoh
N
N
Kawasaki
T
T
D000818:Animals; D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins / Q000378:metabolism*; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D004247:DNA; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D037102:Lectins / Q000235:genetics*; D008264:Macrophages / Q000378:metabolism*; D011233:Precipitin Tests; D051381:Rats; D011971:Receptors, Immunologic / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D037102:Lectins; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; D004247:DNA