Synthesis of a truncated Mr 46,000 mannose 6-phosphate receptor that is secreted and retains ligand binding.
The Mr 46,000 mannose 6-phosphate receptor is an integral membrane protein with its ligand-binding site in the ectoplasmic domain. By site-directed mutagenesis, a stop codon was introduced in the receptor cDNA at the border between the ectoplasmic and membrane-spanning domain. The truncated receptor was expressed in three different systems, Xenopus oocytes, COS cells and BHK-21 cells. In all three systems the truncated receptor behaved as a soluble protein. In oocytes only small amounts of the truncated receptor were secreted within 48 h after synthesis. Accumulation of endoglucosaminidase H-sensitive forms of the truncated receptor in oocytes suggested that exit from the endoplasmic reticulum was slowed down. In COS and BHK-21 cells, the truncated receptor was secreted and, as for wild-type receptor, most of the N-linked oligosaccharides were processed to complex forms. Both the intracellularly-retained (oocytes) and the secreted (COS and BHK-21 cells) truncated receptors bound to phosphomannan-Sepharose in a mannose-6-phosphate-dependent manner. Using chemical cross-linking, the truncated receptor was shown to be secreted as a homodimer.
Wendland
M
M
Universität Göttingen, Biochemie II, Gosslerstrasse 12d, Federal Republic of Germany.
Hille
A
A
Nagel
G
G
Waheed
A
A
von Figura
K
K
Pohlmann
R
R
D000818:Animals; D002478:Cells, Cultured; D006600:Hexosephosphates / Q000378:metabolism*; D008024:Ligands; D008360:Mannosephosphates / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D011487:Protein Conformation; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000378:metabolism; D014982:Xenopus laevis
D006600:Hexosephosphates; D008024:Ligands; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface