Urokinase (uPA) domain specificity for smooth muscle cell migration

S. Mukhina, V. Stepanova, Dmitry Traktuev, A. Polyakov, R. Bibilashvily, V. Tkachuk

Research output: Contribution to journalArticle

Abstract

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
Volume14
Issue numberSUPPL. 1
StatePublished - Dec 1 2000
Externally publishedYes

Fingerprint

Kringles
Urokinase-Type Plasminogen Activator
Smooth Muscle Myocytes
Cell Movement
Chemotaxis
Growth
HEK293 Cells
Peptide Hydrolases
Binding Sites

ASJC Scopus subject areas

  • Hematology

Cite this

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

Urokinase (uPA) domain specificity for smooth muscle cell migration. / Mukhina, S.; Stepanova, V.; Traktuev, Dmitry; Polyakov, A.; Bibilashvily, R.; Tkachuk, V.

In: Fibrinolysis and Proteolysis, Vol. 14, No. SUPPL. 1, 01.12.2000.

Research output: Contribution to journalArticle

Mukhina, S, Stepanova, V, Traktuev, D, Polyakov, A, Bibilashvily, R & Tkachuk, V 2000, 'Urokinase (uPA) domain specificity for smooth muscle cell migration', Fibrinolysis and Proteolysis, vol. 14, no. SUPPL. 1.
Mukhina S, Stepanova V, Traktuev D, Polyakov A, Bibilashvily R, Tkachuk V. Urokinase (uPA) domain specificity for smooth muscle cell migration. Fibrinolysis and Proteolysis. 2000 Dec 1;14(SUPPL. 1).
Mukhina, S. ; Stepanova, V. ; Traktuev, Dmitry ; Polyakov, A. ; Bibilashvily, R. ; Tkachuk, V. / Urokinase (uPA) domain specificity for smooth muscle cell migration. In: Fibrinolysis and Proteolysis. 2000 ; Vol. 14, No. SUPPL. 1.
@article{3a6ab65144ea45cd972a108a011ce3a7,
title = "Urokinase (uPA) domain specificity for smooth muscle cell migration",
abstract = "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.",
author = "S. Mukhina and V. Stepanova and Dmitry Traktuev and A. Polyakov and R. Bibilashvily and V. Tkachuk",
year = "2000",
month = "12",
day = "1",
language = "English (US)",
volume = "14",
journal = "Fibrinolysis and Proteolysis",
issn = "1369-0191",
publisher = "Churchill Livingstone",
number = "SUPPL. 1",

}

TY - JOUR

T1 - Urokinase (uPA) domain specificity for smooth muscle cell migration

AU - Mukhina, S.

AU - Stepanova, V.

AU - Traktuev, Dmitry

AU - Polyakov, A.

AU - Bibilashvily, R.

AU - Tkachuk, V.

PY - 2000/12/1

Y1 - 2000/12/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=33846954808&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33846954808&partnerID=8YFLogxK

M3 - Article

VL - 14

JO - Fibrinolysis and Proteolysis

JF - Fibrinolysis and Proteolysis

SN - 1369-0191

IS - SUPPL. 1

ER -