Abstract
Urokinase plasminogen activator (uPA) is thought to exert its effects on cell growth, adhesion, and migration by mechanisms involving proteolysis and interaction with its cell surface receptor (uPAR). The functional properties of uPA and the significance of its various domains for chemotactic activity were analyzed using human airway smooth muscle cells (hAWSMC). The wild-type uPA (r-uPAwt), inactive urokinase with single mutation (His204 to Gln) (r- uPA(H/Q)), urokinase with mutation of His204 to Gln together with a deletion of growth factor-like domain (r-uPA(H/Q)-GFD), the catalytic domain of urokinase (r-uPA(LMW)), and its kringle domain (r-KD) were expressed in Escherichia coli. We demonstrate that glycosylated uPA, r-uPAwt, r-uPA(H/Q), and r-uPA(H/Q)-GFD elicited similar chemotactic effects. Half-maximal chemotaxis (EC50) were apparent at approximately 2 nM with all the uPA variants. The kringle domain induced cell migration with an EC50 of about 6 nM, whereas the denaturated r-KD and r-uPA(LMW) were without effect. R-uPAwt- induced chemotaxis was dependent on an association with uPAR and a uPA- kringle domain-binding site, determined using a monoclonal uPAR antibody to prevent the uPA-uPAR interaction, and a monoclonal antibody to the uPA- kringle domain. The binding of iodinated r-uPAwt with hAWSMC was due to interaction with a high affinity binding site on the uPAR, and a lower affinity binding site on an unidentified cell surface target, which was mediated exclusively through the kringle domain of urokinase. Specific binding of r-uPAP(H/Q)-GFD to hAWSMC involved an interaction with a single site whose characteristics were similar to those of the low affinity site of r-uPAwt binding to hAWSMC, uPAR-deficient HEK 293 cells specifically bound r- uPAwt and r-uPAP(H/Q)-GFD via a single, similar type of binding site. These cells migrated when stimulated by r-uPA(H/Q)-GFD and uPAwt, but not r- uPA(LMW). HEK 293 cells transfected with the uPAR cDNA expressed two classes of sites that bound r-uPAwt; however, only a single site was responsible for the binding of r-uPA(H/Q)-GFD. Together, these findings indicate that uPA- induced chemotaxis is dependent on the binding of the uPA-kringle to the membrane surface of cells and the association of uPA with uPAR.
Original language | English (US) |
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Pages (from-to) | 16450-16458 |
Number of pages | 9 |
Journal | Journal of Biological Chemistry |
Volume | 275 |
Issue number | 22 |
DOIs | |
State | Published - Jun 2 2000 |
Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Cell Biology
Cite this
The chemotactic action of urokinase on smooth muscle cells is dependent on its kringle domain. Characterization of interactions and contribution to chemotaxis. / Mukhina, Svetlana; Stepanova, Victoria; Traktuev, Dmitry; Poliakov, Alexei; Beabealashvilly, Robert; Gursky, Yaroslav; Minashkin, Mikhail; Shevelev, Alexander; Tkachuk, Vsevolod.
In: Journal of Biological Chemistry, Vol. 275, No. 22, 02.06.2000, p. 16450-16458.Research output: Contribution to journal › Article
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TY - JOUR
T1 - The chemotactic action of urokinase on smooth muscle cells is dependent on its kringle domain. Characterization of interactions and contribution to chemotaxis
AU - Mukhina, Svetlana
AU - Stepanova, Victoria
AU - Traktuev, Dmitry
AU - Poliakov, Alexei
AU - Beabealashvilly, Robert
AU - Gursky, Yaroslav
AU - Minashkin, Mikhail
AU - Shevelev, Alexander
AU - Tkachuk, Vsevolod
PY - 2000/6/2
Y1 - 2000/6/2
N2 - Urokinase plasminogen activator (uPA) is thought to exert its effects on cell growth, adhesion, and migration by mechanisms involving proteolysis and interaction with its cell surface receptor (uPAR). The functional properties of uPA and the significance of its various domains for chemotactic activity were analyzed using human airway smooth muscle cells (hAWSMC). The wild-type uPA (r-uPAwt), inactive urokinase with single mutation (His204 to Gln) (r- uPA(H/Q)), urokinase with mutation of His204 to Gln together with a deletion of growth factor-like domain (r-uPA(H/Q)-GFD), the catalytic domain of urokinase (r-uPA(LMW)), and its kringle domain (r-KD) were expressed in Escherichia coli. We demonstrate that glycosylated uPA, r-uPAwt, r-uPA(H/Q), and r-uPA(H/Q)-GFD elicited similar chemotactic effects. Half-maximal chemotaxis (EC50) were apparent at approximately 2 nM with all the uPA variants. The kringle domain induced cell migration with an EC50 of about 6 nM, whereas the denaturated r-KD and r-uPA(LMW) were without effect. R-uPAwt- induced chemotaxis was dependent on an association with uPAR and a uPA- kringle domain-binding site, determined using a monoclonal uPAR antibody to prevent the uPA-uPAR interaction, and a monoclonal antibody to the uPA- kringle domain. The binding of iodinated r-uPAwt with hAWSMC was due to interaction with a high affinity binding site on the uPAR, and a lower affinity binding site on an unidentified cell surface target, which was mediated exclusively through the kringle domain of urokinase. Specific binding of r-uPAP(H/Q)-GFD to hAWSMC involved an interaction with a single site whose characteristics were similar to those of the low affinity site of r-uPAwt binding to hAWSMC, uPAR-deficient HEK 293 cells specifically bound r- uPAwt and r-uPAP(H/Q)-GFD via a single, similar type of binding site. These cells migrated when stimulated by r-uPA(H/Q)-GFD and uPAwt, but not r- uPA(LMW). HEK 293 cells transfected with the uPAR cDNA expressed two classes of sites that bound r-uPAwt; however, only a single site was responsible for the binding of r-uPA(H/Q)-GFD. Together, these findings indicate that uPA- induced chemotaxis is dependent on the binding of the uPA-kringle to the membrane surface of cells and the association of uPA with uPAR.
AB - Urokinase plasminogen activator (uPA) is thought to exert its effects on cell growth, adhesion, and migration by mechanisms involving proteolysis and interaction with its cell surface receptor (uPAR). The functional properties of uPA and the significance of its various domains for chemotactic activity were analyzed using human airway smooth muscle cells (hAWSMC). The wild-type uPA (r-uPAwt), inactive urokinase with single mutation (His204 to Gln) (r- uPA(H/Q)), urokinase with mutation of His204 to Gln together with a deletion of growth factor-like domain (r-uPA(H/Q)-GFD), the catalytic domain of urokinase (r-uPA(LMW)), and its kringle domain (r-KD) were expressed in Escherichia coli. We demonstrate that glycosylated uPA, r-uPAwt, r-uPA(H/Q), and r-uPA(H/Q)-GFD elicited similar chemotactic effects. Half-maximal chemotaxis (EC50) were apparent at approximately 2 nM with all the uPA variants. The kringle domain induced cell migration with an EC50 of about 6 nM, whereas the denaturated r-KD and r-uPA(LMW) were without effect. R-uPAwt- induced chemotaxis was dependent on an association with uPAR and a uPA- kringle domain-binding site, determined using a monoclonal uPAR antibody to prevent the uPA-uPAR interaction, and a monoclonal antibody to the uPA- kringle domain. The binding of iodinated r-uPAwt with hAWSMC was due to interaction with a high affinity binding site on the uPAR, and a lower affinity binding site on an unidentified cell surface target, which was mediated exclusively through the kringle domain of urokinase. Specific binding of r-uPAP(H/Q)-GFD to hAWSMC involved an interaction with a single site whose characteristics were similar to those of the low affinity site of r-uPAwt binding to hAWSMC, uPAR-deficient HEK 293 cells specifically bound r- uPAwt and r-uPAP(H/Q)-GFD via a single, similar type of binding site. These cells migrated when stimulated by r-uPA(H/Q)-GFD and uPAwt, but not r- uPA(LMW). HEK 293 cells transfected with the uPAR cDNA expressed two classes of sites that bound r-uPAwt; however, only a single site was responsible for the binding of r-uPA(H/Q)-GFD. Together, these findings indicate that uPA- induced chemotaxis is dependent on the binding of the uPA-kringle to the membrane surface of cells and the association of uPA with uPAR.
UR - http://www.scopus.com/inward/record.url?scp=0034595990&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0034595990&partnerID=8YFLogxK
U2 - 10.1074/jbc.M909080199
DO - 10.1074/jbc.M909080199
M3 - Article
C2 - 10749881
AN - SCOPUS:0034595990
VL - 275
SP - 16450
EP - 16458
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 22
ER -