Differential regulation of urokinase-type-1 inhibitor complex endocytosis by phorbol esters in different cell lines is associated with differential regulation of α2-macroglobulin receptor and urokinase receptor expression

Kjøller, Lars; Simonsen, Anna Carina Wiborg; Ellgaard, Lars; Andreasen, Peter A.

doi:10.1016/0303-7207(95)03504-z

Cited by 17 publications

(14 citation statements)

References 30 publications

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“…6). For LRP‐1A‐mediated endocytosis, we used COS‐1 cells, in which the only RAP‐binding receptor detectable by RAP ligand blotting analysis is LRP‐1A [29]. In both cell lines, the receptor‐mediated degradation of wild‐type and variant complexes was reduced largely in parallel with receptor binding (Fig.…”

Section: Resultsmentioning

confidence: 99%

Binding areas of urokinase‐type plasminogen activator–plasminogen activator inhibitor‐1 complex for endocytosis receptors of the low‐density lipoprotein receptor family, determined by site‐directed mutagenesis

Skeldal¹,

Larsen²,

Pedersen³

et al. 2006

The FEBS Journal

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The low-density lipoprotein receptor (LDLR) family of endocytosis receptors has been implicated in binding and endocytosis of a large number of structurally unrelated proteins, including apolipoproteins, proteaseinhibitor complexes, extracellular matrix proteins, and hormone carriers. In mammals, this receptor family includes LDLR itself, low-density lipoprotein receptorrelated protein-1A (LRP-1A), LRP-1B, megalin or LRP-2, very-low-density lipoprotein receptor (VLDLR), and apolipoprotein E receptor-2. These Keywords low-density lipoprotein receptor-related protein; plasminogen activator inhibitor 1; sorting protein-related receptor; urokinase plasminogen activator; very-low-density lipoprotein receptor Some endocytosis receptors related to the low-density lipoprotein receptor, including low-density lipoprotein receptor-related protein-1A, very-lowdensity lipoprotein receptor, and sorting protein-related receptor, bind protease-inhibitor complexes, including urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and the uPA-PAI-1 complex. The unique capacity of these receptors for high-affinity binding of many structurally unrelated ligands renders mapping of receptor-binding surfaces of serpin and serine protease ligands a special challenge. We have mapped the receptor-binding area of the uPA-PAI-1 complex by site-directed mutagenesis. Substitution of a cluster of basic residues near the 37-loop and 60-loop of uPA reduced the receptor-binding affinity of the uPA-PAI-1 complex approximately twofold. Deletion of the N-terminal growth factor domain of uPA reduced the affinity 2-4-fold, depending on the receptor, and deletion of both the growth factor domain and the kringle reduced the affinity sevenfold. The binding affinity of the uPA-PAI-1 complex to the receptors was greatly reduced by substitution of basic and hydrophobic residues in a-helix D and a-helix E of PAI-1. The localization of the implicated residues in the 3D structures of uPA and PAI-1 shows that they form a continuous receptor-binding area spanning the serpin as well as the A-chain and the serine protease domain of uPA. Our results suggest that the 10-100-fold higher affinity of the uPA-PAI-1 complex compared with the free components depends on the bonus effect of bringing the binding areas on uPA and PAI-1 together on the same binding entity.Abbreviations a 1 -PI, a 1 -antiproteinase inhibitor; CTR, complement type repeat; HEK293T, human embryonic kidney cell line 293T; LDLR, low-density lipoprotein receptor; LRP, low-density lipoprotein receptor-related protein; PAI-1, plasminogen activator inhibitor 1; RAP, receptor-associated protein; RCL, reactive centre loop; sorLA, sorting protein-related receptor; SPD, serine protease domain; tPA, tissue-type plasminogen activator; uPA, urokinase-type plasminogen activator; uPAR, uPA receptor; VLDLR, very-low-density lipoprotein receptor.

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Section: Resultsmentioning

confidence: 99%

Binding areas of urokinase‐type plasminogen activator–plasminogen activator inhibitor‐1 complex for endocytosis receptors of the low‐density lipoprotein receptor family, determined by site‐directed mutagenesis

Skeldal¹,

Larsen²,

Pedersen³

et al. 2006

The FEBS Journal

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“…In our studies, the role of LRP/␣ 2 MR in the regulation of cell surface plasminogen activator activity was investigated by inhibiting the function of the endocytic receptor using RAP (9,14,41,42). This strategy avoids the difficulty of identifying agonists that selectively influence LRP/␣ 2 MR expression and/or activity without altering that of other secreted and/or cell-associated proteins involved in plasminogen activation (65). Our premise that the effects of RAP on the uptake and clearance of 125 I-pro-UK (or 125 I-tc-u-PA⅐PAI complexes) and cell surface plasminogen activator activity were mediated through interactions with LRP/␣ 2 MR is supported by the observation that these effects were mimicked by anti-LRP/␣ 2 MR antibodies.…”

Section: Discussionmentioning

confidence: 99%

The Low Density Lipoprotein Receptor-related Protein/α2-Macroglobulin Receptor Regulates Cell Surface Plasminogen Activator Activity on Human Trophoblast Cells

Zhang¹,

Sakthivel²,

Kniss³

et al. 1998

Journal of Biological Chemistry

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The low density lipoprotein receptor-related protein/ ␣ 2 -macroglobulin receptor (LRP/␣ 2 MR) mediates the internalization of numerous ligands, including prourokinase (pro-UK) and complexes between two-chain urokinase (tc-u-PA) and plasminogen activator inhibitor type-1 (PAI-1). It has been suggested that through its ability to internalize these ligands, LRP/␣ 2 MR may regulate the expression of plasminogen activator activity on cell surfaces; this hypothesis, however, has not been experimentally confirmed. To address this issue, we assessed the ability of LRP/␣ 2 MR to regulate plasminogen activator activity on human trophoblast cells, which express both LRP/␣ 2 MR and the urokinase receptor (uPAR). Trophoblasts internalized and degraded exogenous 125 I-pro-UK (primarily following its conversion to tc-u-PA and incorporation into tc-u-PA⅐PAI complexes) in an LRP/␣ 2 MR-dependent manner, which was inhibited by the LRP/␣ 2 MR receptor-associated protein. Receptor-associated protein also caused a ϳ50% reduction in cell surface plasminogen activator activity and delayed the regeneration of unoccupied uPAR by cells on which uPAR were initially saturated with pro-UK. Identical effects were caused by anti-LRP/␣ 2 MR antibodies. These results demonstrate that LRP/␣ 2 MR promotes the expression of cell surface plasminogen activator activity on trophoblasts by facilitating the clearance of tc-u-PA⅐PAI complexes and regeneration of unoccupied cell surface uPAR.

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“…There is evidence indicating that in COS cells, such non‐specific endo‐cytosis is present to a much lesser extent than in other cell types ( Gerard & Gerard 1994). Activation of PKC only slightly stimulates ( Gerard & Gerard 1994) or even inhibits ( Kjøller et al . 1995 ) endo‐cytosis in COS cells.…”

Section: Discussionmentioning

confidence: 99%

Forskolin–induced down–regulation of Na⁺,K⁺–ATPase activity is not associated with internalization of the enzyme

Andersson

Cheng

Aperia

1998

Acta Physiologica Scandinavica

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Activation by protein kinase A by forskolin phosphorylates and inactivates Na+,K(+)-ATPase in COS-7 cells (Cheng et al. 1997b). In this study we show, using [3H]ouabain binding, that forskolin-induced inhibition of Na+,K(+)-ATPase activity is not because of internalization of the enzyme. The effect of forskolin on Na+,K(+)-ATPase activity was examined by two independent methods, ouabain-sensitive 86Rb+ uptake in intact cells and ATP hydrolysis in microsomal preparations from cells. The change in number of functional pumps on cell surface before and after protein kinase A activation was assessed by [3H]ouabain binding measured under equilibrium conditions. Cells, which had been ATP-depleted by antimycin A and 2-deoxyglucose treatment, served as a positive control for the internalization of Na+,K(+)-ATPase. Activation of protein kinase A with forskolin in combination with the phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine, inhibited Na+,K(+)-ATPase activity, but this treatment had no effect on specific ouabain binding. No change in ouabain binding was found following activation of protein kinase C by phorbol ester or diacyl glycerol analogue treatment in cells. These data suggest that protein kinase A phosphorylation and inhibition of Na+,K(+)-ATPase activity does not lead to any internalization of the enzyme in COS-7 cells.

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Differential regulation of urokinase-type-1 inhibitor complex endocytosis by phorbol esters in different cell lines is associated with differential regulation of α2-macroglobulin receptor and urokinase receptor expression

Cited by 17 publications

References 30 publications

Binding areas of urokinase‐type plasminogen activator–plasminogen activator inhibitor‐1 complex for endocytosis receptors of the low‐density lipoprotein receptor family, determined by site‐directed mutagenesis

Binding areas of urokinase‐type plasminogen activator–plasminogen activator inhibitor‐1 complex for endocytosis receptors of the low‐density lipoprotein receptor family, determined by site‐directed mutagenesis

The Low Density Lipoprotein Receptor-related Protein/α2-Macroglobulin Receptor Regulates Cell Surface Plasminogen Activator Activity on Human Trophoblast Cells

Forskolin–induced down–regulation of Na⁺,K⁺–ATPase activity is not associated with internalization of the enzyme

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Differential regulation of urokinase-type-1 inhibitor complex endocytosis by phorbol esters in different cell lines is associated with differential regulation of α2-macroglobulin receptor and urokinase receptor expression

Cited by 17 publications

References 30 publications

Binding areas of urokinase‐type plasminogen activator–plasminogen activator inhibitor‐1 complex for endocytosis receptors of the low‐density lipoprotein receptor family, determined by site‐directed mutagenesis

Binding areas of urokinase‐type plasminogen activator–plasminogen activator inhibitor‐1 complex for endocytosis receptors of the low‐density lipoprotein receptor family, determined by site‐directed mutagenesis

The Low Density Lipoprotein Receptor-related Protein/α2-Macroglobulin Receptor Regulates Cell Surface Plasminogen Activator Activity on Human Trophoblast Cells

Forskolin–induced down–regulation of Na+,K+–ATPase activity is not associated with internalization of the enzyme

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Forskolin–induced down–regulation of Na⁺,K⁺–ATPase activity is not associated with internalization of the enzyme