Expression of human cation-independent mannose 6-phosphate receptor cDNA in receptor-negative mouse P388D1 cells following gene transfer.
We recently reported the cDNA cloning, sequence, and expression of the human cation-independent mannose 6-phosphate receptor (hCI-MPR) (Oshima, A., Nolan, C. M., Kyle, J. W., Grubb, J. H., and Sly, W. S. (1988) J. Biol. Chem. 263, 2553-2562). The sequence of the hCI-MPR was virtually identical to that of the human insulin-like growth factor II receptor cDNA (Morgan, D. O., Edman, J. C., Standring, D. N., Fried, V. A., Smith, M. C., Roth, R. A., and Rutter, W. J. (1987) Nature 329, 301-307). To test the role of the putative bifunctional receptor in intracellular sorting of acid hydrolases, we studied its effect on lysosomal enzyme transport following gene transfer to receptor-negative cells. Receptor-negative mouse P388D1 cells were transfected with a cDNA construct containing the entire coding sequence of hCI-MPR under the control of the mouse metallothionine I promoter. Stable transformants were isolated and characterized. The expressed hCI-MPR was localized in membranes including the plasma membrane, bound mannose 6-phosphate containing ligands, and mediated endocytosis which could be specifically blocked by mannose 6-phosphate. We next measured the effect of the expressed hCI-MPR on intracellular and secreted acid hydrolases. The intracellular activity of the lysosomal marker enzymes beta-glucuronidase and beta-hexosaminidase increased up to 2-fold following transformation. In addition, expression of the receptor greatly reduced the fraction of acid hydrolases secreted. These phenotypic changes in the transformed cell lines support the proposed role of the cation-independent mannose 6-phosphate receptor in intracellular sorting and targeting of lysosomal enzymes.
Kyle
J W
JW
E. A. Doisy Department of Biochemistry, St. Louis University School of Medicine, Missouri 64104.
Nolan
C M
CM
Oshima
A
A
Sly
W S
WS
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002460:Cell Line; D002846:Chromatography, Affinity; D004247:DNA / Q000235:genetics*; D004705:Endocytosis; D005796:Genes; D005966:Glucuronidase / Q000378:metabolism; D006801:Humans; D008360:Mannosephosphates / Q000378:metabolism; D051379:Mice; D017527:Receptor, IGF Type 2; D014162:Transfection
D002352:Carrier Proteins; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; D004247:DNA; D005966:Glucuronidase