Structural requirements for glycosyl-phosphatidylinositol-anchor attachment in the cellular receptor for urokinase plasminogen activator.
The urokinase-plasminogen-activator receptor (u-PAR) is a glycosyl-phosphatidylinositol(glycosyl-PtdIns)-anchored membrane protein. Using site-directed mutagenesis, we have studied features in the u-PAR sequence important for successful glycosyl-PtdIns attachment. Two critical sequence elements were identified. In the sequence Ser282-Gly283-Ala284, simultaneous substitution of all of these residues prevented membrane anchoring. Individual substitution of each of the residues indicated that Gly283 is the more critical residue and the likely attachment site. However, it was unexpectedly found that mutation of this residue gave rise only to a partial impairment of glycosyl-PtdIns attachment. We therefore propose that more than one residue within this sequence can be utilized as glycosyl-PtdIns-attachment site. In the last eight COOH-terminal amino acids encoded in u-PAR cDNA, deletion of this sequence (residues 306-313) completely prevented glycosyl-PtdIns attachment. However, the remaining COOH-terminal region proved still to possess a potential glycosyl-PtdIns signal activity; it could be converted to a new functional glycosyl-PtdIns signal by substitution of a single positively charged residue (Arg304). Substitution of Arg304 by Leu converted this truntaced u-PAR to a glycosyl-PtdIns-anchored protein, indistinguishable from the wild type. Substitution of Arg304 by a negatively charged residue (Glu) led to a partial acquisition of the glycosyl-PtdIns-anchoring ability. These findings show that charged amino acids placed in the COOH-terminus interfere negatively with glycosyl-PtdIns-anchoring, and, furthermore, that this effect is more pronounced for positively charged than for negatively charged amino acid residues.
Møller
L B
LB
Institute of Microbiology, University of Copenhagen, Denmark.
Ploug
M
M
Blasi
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000235:genetics; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006017:Glycolipids / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D010716:Phosphatidylinositols / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator; D013329:Structure-Activity Relationship; D014162:Transfection
D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D010446:Peptide Fragments; D010716:Phosphatidylinositols; C524899:Plaur protein, mouse; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D004247:DNA