Urokinase (uPA) domain specificity for smooth muscle cell migration

S. Mukhina, V. Stepanova, D. Traktouev, A. Polyakov, R. Bibilashvily, V. Tkachuk

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Molecular mechanisms and the role of urokinase (uPA) domains in urokinase-stimulated cell migration were studied on human smooth muscle cells. uPA contains three domains: protease domain (PD), growth factorlike domain (GFD) mediating binding to the specific uPA receptor uPAR/CD87 and kringle domain (KD) with yet unknown function. We constructed and produced in coli the following uPA-derivatives: uPA with wild-type structure (r-uPAwt), proteolytically inactive uPA with substitution of His24 for Gin (r-uPA'Q), proteolitically inactive uPA lacking GFD (r-uPA GFD), separate protease domain (r-LMW) and kringle domain (r-KD). Using Boyden chamber migration assay we demonstrated that kringle domain is essential for uPA-induced chemotaxis since (i) uPAderivatives containing kringle domain (glycosylated uPA from urine, r-uPAwt, r-uPAH/Q and r-uPAH/Q-GFD) stimulated cell migration in similar dose-dependent manner in 0.5-40 nM concentration range with halfmaximal effect - 2 nM, while r-LMW did not stimulate chemotaxis, (ii) separate r-KD was able to induce cell migration, while denaturated r-KD had no effect, (iii) antibody against kringle domain blocked cell migration induced by r-uPAwt, r-KD and r-uPAH -GFD. UPAR/CD87 was involved in migration induced only by the full-length uPA, but not by r-KD, since antibody blocked interaction of uPA with uPAR/CD87 significantly diminished chemotaxis stimulated by r-uPAwt, but had no effect on r-KDstimulated cell migration. Binding of 125I-r-uPAwt to human smooth muscle cells was characterized by high affinity binding (Ku =1.51 +/-0.06 nM, Bmax1 = 140000 +/- 11000 sites/cell) mediated by UPAR/CD87 via GFD, and low affinity binding to an unknown binding site (Kj, = 20.27+/-1.01 nM, Bmax2 = 815000 +/- 133000 sites/cell) via uPA kringle domain. Analysis of binding parameters for 125I-r-uPAH/Q-GFD revealed only one type of binding sites (K = 20.29+/-2.22 nM, B = 1 028 000 +/- 129 000 sites/cell) that matched the characteristics of the low affinity site for fulllength uPA. In competition experiments glycosylated uPA, r-uPAwt, rUPAH/Q-GFD and r-KD were able to compete for binding with 125I-r-uPAH/Q -GFD, while r-LMW, denaturated r-KD and other kringle-containing proteins such as tissue plasminogen activator and plasminogen had no effect. Interaction of uPA via kringle domain with kringle-binding target was sufficient for induction of chemotaxis, since human embryonic kidney cells HEK 293 deficient in UPAR/CD87 were able to bind 125I-rUPAH/Q-GFD (Kd, - 11 nM, Bmax - 46 000 sites/cell) and migrate in response to this uPA-derivate. r-uPAH/Q also induced chemotaxis of HEK 293 cells, but with lower efficiency. These data demonstrate that urokinase kringle domain mediates the uPA-induced chemotaxis. Interaction with kringlebinding target is specific for uPA kringle domain and seems to be necessary and sufficient for the uPA-stimulated cell migration. However, binding to UPAR/CD87 is necessary for more efficient chemotaxis induced by the full-length uPA.

Original languageEnglish (US)
Number of pages1
JournalFibrinolysis and Proteolysis
Issue numberSUPPL. 1
StatePublished - Dec 1 2000
Externally publishedYes

ASJC Scopus subject areas

  • Hematology

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    Mukhina, S., Stepanova, V., Traktouev, D., Polyakov, A., Bibilashvily, R., & Tkachuk, V. (2000). Urokinase (uPA) domain specificity for smooth muscle cell migration. Fibrinolysis and Proteolysis, 14(SUPPL. 1).