The chemotactic action of urokinase on smooth muscle cells is dependent on its kringle domain. Characterization of interactions and contribution to chemotaxis

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.

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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 -