Carboxy-terminal processing of the urokinase receptor: Implications for substrate recognition and glycosylphosphatidplinositol anchor addition

Proteins linked to cell membranes by a glycosylphosphatidylinositol (GPI) a nchor must first undergo cleavage by a putative transamidase between the om ega and omega + 1 positions within a proposed small amino acid (SAD) domain in the carboxy terminus of the nascent polypeptide. The requirements for s uch processing, defined in an engineered placental alkaline phosphatase con struct (miniPLAP), suggest the SAD domain functions as an autonomous unit w ithin the context of an otherwise permissive carboxyterminal sequence with only certain amino acids tolerated at the omega, omega + 1, and omega + 2 p ositions. To test whether this hypothesis could be generalized, we engineer ed a chimeric molecule containing the extracellular domain of miniPLAP and the carboxy-terminal portion of the urokinase receptor (MP/uPAR) into which various amino acid substitutions were introduced. The variant proteins wer e translated and metabolically labeled in vitro using a cell-free translati on system that contains the enzymatic machinery required for carboxy-termin al processing and GPI anchor addition. The results of this study indicate t hat the SAD domain functions as an independent, but not an autonomous, unit . The requirements fur processing in miniPLAP and MP/uPAR differed markedly in some respects, in part due to the influence of the amino acid at the om ega + 4 position which both modified cleavage between the omega and omega 1 positions and permitted a second cleavage site to be generated in some c ases. In addition, substitution of bulky hydrophobic amino acids in series at the omega + 2 and omega + 3 positions inhibited carboxy-terminal process ing in a dose-dependent manner, suggesting the presence of a critical docki ng site adjacent to the cleavage site. These results suggest the carboxy-te rminal transamidase recognizes a more extended structure similar to the mec hanism proposed for serine proteases. Further, the data provide a potential means for isolating the transamidase.