title: Opposite modulation of cell migration by distinct subregions of urokinase connecting peptide.
abstract: Functional analysis of isolated protein domains may uncover cryptic activities otherwise missed. The serine protease urokinase (uPA) has a clear-cut motogen activity that is catalytically independent and resides in its amino-terminal growth factor domain (GFD, residues 1-49) and connecting peptide region (CP, residues 132-158). To functionally dissect the CP region, we analysed the biological activity of two synthetic peptides corresponding to the N-terminal [uPA-(135-143), residues 135-143] and C-terminal [uPA-(144-158), residues 144-158] CP subregions. Most of the chemotactic activity of connecting peptide-derived peptide (CPp, [uPA-(135-158)]) for embryonic kidney HEK293/uPAR-25 cells is retained by uPA-(144-158) at nanomolar concentrations. In contrast, uPA-(135-143) inhibits basal, CPp -, vitronectin- and fibronectin-induced cell migration. Radioreceptor binding assays on intact HEK293 cells revealed that uPA-(135-143) and uPA-(144-158) are both able to compete with [(125)I]-CPp, albeit with different binding affinities. The consequences of phospho-mimicking, S138E substitution, were studied using [138E]uPA-(135-158) and [138E]uPA-(135-143) peptides. Unlike CPp, [138E]uPA-(135-158) and [138E]uPA-(135-143) exhibit remarkable inhibitory properties. Finally, analysis of the conformational preferences of the peptides allowed to identify secondary structure elements exclusively characterising the stimulatory CPp and uPA-(144-158) versus the inhibitory uPA-(135-143), [138E]uPA-(135-158) and [138E]uPA-(135-143) peptides. In conclusion, these data shed light on the cryptic activities of uPA connecting peptide, revealing the occurrence of two adjacent regions, both competing for binding to cell surface but conveying opposite signalling on cell migration.
authors.affiliation: Institute of Genetics and Biophysics Adriano Buzzati-Traverso, National Research Council, Via Castellino 111, Naples, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Franco
authors.forename: Alfonso
authors.initials: A
authors.lastname: Carotenuto
authors.forename: Cristina
authors.initials: C
authors.lastname: Marcozzi
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Votta
authors.forename: Ciro
authors.initials: C
authors.lastname: Sarno
authors.forename: Ingram
authors.initials: I
authors.lastname: Iaccarino
authors.forename: Diego
authors.initials: D
authors.lastname: Brancaccio
authors.forename: Anna
authors.initials: A
authors.lastname: De Vincenzo
authors.forename: Ettore
authors.initials: E
authors.lastname: Novellino
authors.forename: Paolo
authors.initials: P
authors.lastname: Grieco
authors.forename: Maria Patrizia
authors.initials: MP
authors.lastname: Stoppelli
mesh_terms: D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry* / Q000378:metabolism
chemical_list: D010455:Peptides; D014568:Urokinase-Type Plasminogen Activator