Analysis of a Structural Determinant in Thrombin-Protease Nexin 1 Complexes That Mediates Clearance by the Low Density Lipoprotein Receptor-related Protein
1999; Elsevier BV; Volume: 274; Issue: 1 Linguagem: Inglês
10.1074/jbc.274.1.275
ISSN1083-351X
AutoresMary F. Knauer, Robert Crisp, Steven J. Kridel, Daniel J. Knauer,
Tópico(s)Signaling Pathways in Disease
ResumoWe recently identified a synthetic peptide, Pro47–Ile58, derived from the mature protease nexin 1 (PN1) sequence, that inhibited the low density lipoprotein receptor-related protein (LRP)-mediated internalization of thrombin-PN1 (Th-PN1) complexes. Presently, we have analyzed this sequence in Th-PN1 complex catabolism using two independent approaches: 1) An antibody was generated against Pro47–Ile58, which inhibited complex degradation by 70% but had no effect on the binding of the complexes to cell surface heparins. This places the structural determinant in PN1 mediating complex internalization by the LRP outside of the heparin-binding site. 2) Site-directed genetic variants of PN1 with a single Ala substitution at His48, or two Ala substitutions, one at His48 and another at Asp49, were expressed in Sf9 insect cells. The catabolic rate of complexes formed between Th and the singly substituted and doubly substituted variants was lowered to 50 and 15%, respectively, when compared with the catabolic rate of native Th-PN1 complexes. This is the first analysis of a structural determinant in a serineprotease inhibitor (SERPIN) required for LRP-mediated internalization and in part may explain the cryptic nature of this site in the unreacted serine protease inhibitor. We recently identified a synthetic peptide, Pro47–Ile58, derived from the mature protease nexin 1 (PN1) sequence, that inhibited the low density lipoprotein receptor-related protein (LRP)-mediated internalization of thrombin-PN1 (Th-PN1) complexes. Presently, we have analyzed this sequence in Th-PN1 complex catabolism using two independent approaches: 1) An antibody was generated against Pro47–Ile58, which inhibited complex degradation by 70% but had no effect on the binding of the complexes to cell surface heparins. This places the structural determinant in PN1 mediating complex internalization by the LRP outside of the heparin-binding site. 2) Site-directed genetic variants of PN1 with a single Ala substitution at His48, or two Ala substitutions, one at His48 and another at Asp49, were expressed in Sf9 insect cells. The catabolic rate of complexes formed between Th and the singly substituted and doubly substituted variants was lowered to 50 and 15%, respectively, when compared with the catabolic rate of native Th-PN1 complexes. This is the first analysis of a structural determinant in a serineprotease inhibitor (SERPIN) required for LRP-mediated internalization and in part may explain the cryptic nature of this site in the unreacted serine protease inhibitor. protease nexin I serine protease inhibitor thrombin urinary plasminogen activator antithrombin III low density lipoprotein receptor-related protein phosphate buffered saline human foreskin fibroblasts polyacrylamide gel electrophoresis vitronectin receptor-associated protein-glutathione S-transferase. Protease nexin 1 (PN1)1is a member of the SERPIN super-family (1Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (829) Google Scholar, 2Scott R.W. Bergman B.L. Bajpai A. Hersh R.T. Rodriguez H. Jones B.N. Barreda C. Watts S. Baker J.B. J. Biol. Chem. 1985; 260: 7029-7034Abstract Full Text PDF PubMed Google Scholar), and an important physiological regulator of thrombin (Th) and urinary plasminogen activator (uPA) (2Scott R.W. Bergman B.L. Bajpai A. Hersh R.T. Rodriguez H. Jones B.N. Barreda C. Watts S. Baker J.B. J. Biol. Chem. 1985; 260: 7029-7034Abstract Full Text PDF PubMed Google Scholar). PN1 forms stoichiometric complexes with both Th and uPA (3Baker J.B. Low D.A. Simmer R.L. Cunningham D.D. Cell. 1980; 21: 37-45Abstract Full Text PDF PubMed Scopus (290) Google Scholar) that ultimately results in their removal by cellular endocytosis and degradation (4Low D.A. Baker J.B. Koonce W.C. Cunningham D.D. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2340-2344Crossref PubMed Scopus (117) Google Scholar). The precise biochemical nature of the complexes is still not completely clear, but they are extremely stable and possibly covalent (5Cohen A.B. Gruenke L.D. Craig J.C. Geczy D. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 4311-4314Crossref PubMed Scopus (11) Google Scholar, 6Lawrence D.A. Ginsburg D. Day D.E. Berkenpas M.B. Verhamme I.M. Kvassman J.O. Shore J.D. J. Biol. Chem. 1995; 270: 25309-25312Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 7Shieh B.H. Potempa J. Travis J. J. Biol. Chem. 1989; 264: 13420-13423Abstract Full Text PDF PubMed Google Scholar). When complexes are formed between the SERPIN and its target protease, there is an accompanying conformational change in the SERPIN that either unmasks or causes the formation of a new binding site in the complexed SERPIN that is not present in the free SERPIN (8Howard E.W. Knauer D.J. J. Cell. Physiol. 1987; 131: 276-283Crossref PubMed Scopus (20) Google Scholar, 9Perlmutter D.H. Travis J. Punsal P.I. J. Clin. Invest. 1988; 81: 1774-1780Crossref PubMed Scopus (64) Google Scholar). The cryptic nature of the LRP-binding site in the free SERPIN makes sense biologically. It ensures that SERPINs will remain extracellular, either in plasma or in tissues near cell surfaces, until they have formed an irreversible complex with a protease.The list of SERPINs dependent on the LRP for cellular internalization includes, protease nexin 1 (PN1) (10Knauer M.F. Hawley S.B. Knauer D.J. J. Biol. Chem. 1997; 272: 12261-12264Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 11Conese M. Olson D. Blasi F. J. Biol. Chem. 1994; 269: 17886-17892Abstract Full Text PDF PubMed Google Scholar), heparin cofactor-II (12Kounnas M.Z. Church F.C. Argraves W.S. Strickland D.K. J. Biol. Chem. 1996; 271: 6523-6529Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), antithrombin III (ATIII) (12Kounnas M.Z. Church F.C. Argraves W.S. Strickland D.K. J. Biol. Chem. 1996; 271: 6523-6529Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) and α-1-antitrypsin (12Kounnas M.Z. Church F.C. Argraves W.S. Strickland D.K. J. Biol. Chem. 1996; 271: 6523-6529Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). It is interesting to note that although the LRP acts as the internalization receptor, other cellular components are most likely required for the efficient catabolism of the SERPIN-Protease complexes (12Kounnas M.Z. Church F.C. Argraves W.S. Strickland D.K. J. Biol. Chem. 1996; 271: 6523-6529Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 13Wells M.J. Hatton M.W. Hewlett B. Podor T.J. Sheffield W.P. Blajchman M.A. J. Biol. Chem. 1997; 272: 28574-28581Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 14Wells M.J. Blajchman M.A. J. Biol. Chem. 1998; 273: 23440-23447Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 15Knauer M.F. Kridel S.J. Hawley S.B. Knauer D.J. J. Biol. Chem. 1997; 272: 29039-29045Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In the case of the plasma SERPINs these components remain to be identified. However PN1, which is primarily restricted to tissues, utilizes at least two different cell surface molecules to assist LRP-mediated internalization. When PN1 forms complexes with uPA, the uPA receptor is required for efficient concentration at the cell surface of uPA-PN1 complexes and subsequent internalization via the LRP (11Conese M. Olson D. Blasi F. J. Biol. Chem. 1994; 269: 17886-17892Abstract Full Text PDF PubMed Google Scholar, 16Conese M. Nykjaer A. Petersen C.M. Cremona O. Pardi R. Andreasen P.A. Gliemann J. Christensen E.I. Blasi F. J. Cell Biol. 1995; 131: 1609-1622Crossref PubMed Scopus (196) Google Scholar). In contrast, when PN1 is in complex with thrombin, heparin chains present at the cell surface greatly facilitate the uptake and turnover of thrombin-PN1 complexes (15Knauer M.F. Kridel S.J. Hawley S.B. Knauer D.J. J. Biol. Chem. 1997; 272: 29039-29045Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), and uPA receptor is not involved (11Conese M. Olson D. Blasi F. J. Biol. Chem. 1994; 269: 17886-17892Abstract Full Text PDF PubMed Google Scholar, 16Conese M. Nykjaer A. Petersen C.M. Cremona O. Pardi R. Andreasen P.A. Gliemann J. Christensen E.I. Blasi F. J. Cell Biol. 1995; 131: 1609-1622Crossref PubMed Scopus (196) Google Scholar). Thus, in the case of PN1, the nature of the target protease directly plays a role in the clearance mechanism.In a recent study using a synthetic peptide library strategy, a putative LRP-binding site was identified in PN1 (10Knauer M.F. Hawley S.B. Knauer D.J. J. Biol. Chem. 1997; 272: 12261-12264Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The library consisted of peptides 12 amino acids in length, and spanned nearly the entire PN1 sequence. A single peptide in the library,47PHDNIVISPHGI58 was identified as a potent inhibitor of Th-PN1 internalization and degradation. Using α-1-antitrypsin structure and sequence alignments, this sequence is predicted to be a transition sequence that occurs just after helix A and continues to form the sixth strand of sheet B (1Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (829) Google Scholar). Consequently, this site meets the criteria of being at least partially buried in the intact SERPIN, with the potential of becoming more exposed after complex formation with thrombin. There is no direct evidence, however, to distinguish this possibility from a simple conformational change in which the exposure of this site remains constant.In the present studies we further investigate the potential role of this putative site in LRP-mediated internalization using two different approaches. In the first approach, a polyclonal IgG was generated against Pro47–Ile58 with a cysteine residue added after the Ile to facilitate haptenization to ovalbumin. We demonstrate that this antibody specifically and selectively inhibits the binding of Th-PN1 complexes to the LRP, but does not affect the interaction of the complexes with cell surface heparins. In the second approach we utilize site-directed mutagenesis and baculovirus-driven expression in insect cells. Two variant forms of PN1 were expressed; one with an alanine substitution at the position of His 48 (H48A), and another with alanine substitutions at the positions of His 48 and Asp 49 (H48A,D49A). Each PN1 variant was characterized biochemically by determining the k assoc value for thrombin inhibition and ability to form SDS-resistant complexes with thrombin. Additionally, the PN1 variant-thrombin complexes and native PN1-thrombin complexes were assayed for their capacity to bind to cell surface heparins. While the PN1 variants were found to be very similar to native PN1 in their ability to inactivate thrombin and bind to cell surface heparins, complexes made with each of the PN1 variants showed decreased rates of catabolism. These experiments define a critical role for the structural determinant, Pro47–Ile58, in the LRP-mediated internalization of Th-PN1 complexes. These data also demonstrate that with the use of anti-(Pro47–Ile58) antibody, cell surface heparin binding and LRP-mediated internalization of Th-PN1 complexes can be studied as independent events, even though they act cooperatively to facilitate complex catabolism.DISCUSSIONThe present studies were undertaken to further probe the potential role of the PN1 peptide sequence,47PHDNIVISPHGI58, in the LRP-mediated clearance of Th-PN1 complexes and to determine what structural features of this sequence might be important in this process. The potential importance of this sequence was discovered using a synthetic peptide library of PN1 sequences and screening for peptides that inhibited the catabolism of complexes by HF cells (10Knauer M.F. Hawley S.B. Knauer D.J. J. Biol. Chem. 1997; 272: 12261-12264Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The requirement for heparin-mediated binding of the complexes to the cell surface to promote an efficient interaction between the complexes and the LRP (15Knauer M.F. Kridel S.J. Hawley S.B. Knauer D.J. J. Biol. Chem. 1997; 272: 29039-29045Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) precluded more detailed studies of this sequence using a synthetic peptide approach, because the sites in PN1 that interact with heparin and the LRP are distinct. In the present studies, two different approaches have been used to more clearly define the role of the structural determinant, Pro47–Ile58, in Th-PN1 complex catabolism and to identify critical residues within this determinant.To independently confirm and more narrowly define the role of this structural determinant in Th-PN1 catabolism, an anti-(Pro47–Ile58) polyclonal IgG was generated. This antibody specifically inhibited the internalization and subsequent degradation of Th-PN1 complexes but had no effect on the binding of complexes to cell surface heparins. These data independently confirm the role of Pro47–Ile58 in LRP-mediated internalization, which had previously been based solely on synthetic peptide competition studies (10Knauer M.F. Hawley S.B. Knauer D.J. J. Biol. Chem. 1997; 272: 12261-12264Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). In addition, they strongly suggest that the LRP and heparin-binding determinants in PN1 are structurally distinct, despite the fact that they act cooperatively to promote efficient complex catabolism (15Knauer M.F. Kridel S.J. Hawley S.B. Knauer D.J. J. Biol. Chem. 1997; 272: 29039-29045Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar).In addition to the antibody studies, a genetic approach was used in which alanine substitutions were introduced at positions His48 and Asp49 of the intact PN1 protein. The rationale for these choices is derived from the predicted structural location of 47PHDNIVISPHGI58 in PN1 based on its homology to α-1-antitrypsin (1Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (829) Google Scholar). This sequence is predicted to be at least partially interior to the protein surface. Pro47most likely represents the beginning of the transition sequence between helix A and strand 6B(1). His48 and Asp49 are transition amino acids, and Asn50 through Pro55become strand 6 of sheet B. Given this structural information, we hypothesized that the charged residues, His48 and Asp49, might be important for either direct interaction with the LRP receptor or assisting in the attainment of the active conformation of this structural determinant when fully exposed to the hydrophilic exterior of the molecule.To evaluate the role of these specific amino acids we generated two different PN1 variants: one in which only His48 was replaced by Ala and another where both His48 and Asp49 were replaced by Ala. Replacement of the first charged residue, His48 by Ala, had a significant effect on the catabolism of PN1(H48A) complexes, reducing it by 50%. The additional substitution of Ala for Asp49, reduced complex catabolism by 85%. In both cases this was demonstrated to be due to a decreased rate of LRP-mediated internalization. Control studies revealed that the substitutions had no effect on the heparin-mediated binding of the complexes to the cell surface nor on the biochemical characteristics of the PN1 variant complexes with thrombin. Taken together, it is clear that both His48 and Asp49play very important roles in the interaction of the PN1 structural determinant, 47PHDNIVISPHGI58, with the LRP. Whether this is due to a direct interaction of these residues with the LRP or because these residues are required to establish a particular structural conformation of this determinant is presently unknown. Given the overall hydrophobic character of Pro47–Ile58, (PHDNIVISPHGI), it is likely that removal of the charged residues affects the solubility of the sequence. The genetic evidence using PN1 variants with point substitutions and the anti-(Pro47–Ile58) antibody experiments represent two important and independent lines of evidence that support and extend the original observation that the sequence Pro47–Ile58 in PN1 is required for the LRP-mediated clearance of Th-PN1 complexes.A common pathway for many LRP internalized ligands seems to be emerging that involves cell surface proteoglycans and perhaps other accessory proteins in many cases. Several of the ligands first bind to cell surface heparin sulfate proteoglycans and are subsequently internalized by the LRP (15Knauer M.F. Kridel S.J. Hawley S.B. Knauer D.J. J. Biol. Chem. 1997; 272: 29039-29045Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 25Mikhailenko I. Krylov D. Argraves K.M. Roberts D.D. Liau G. Strickland D.K. J. Biol. Chem. 1997; 272: 6784-6791Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 26Nielsen M.S. Brejning J. Garcı́a R. Zhang H. Hayden M.R. Vilar S. Gliemann J. J. Biol. Chem. 1997; 272: 5821-5827Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 27Stefansson S. Muhammad S. Cheng X.F. Battey F.D. Strickland D.K. Lawrence D.A. J. Biol. Chem. 1998; 273: 6358-6366Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Even Th-ATIII complexes, which display a negligible affinity for heparin, use hepatic heparin sulfate proteoglycans as a part of their clearance mechanism but do so by an association with vitronectin (Vn) (14Wells M.J. Blajchman M.A. J. Biol. Chem. 1998; 273: 23440-23447Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Th-ATIII complexes first bind to Vn and then utilize the heparin-binding site in Vn, which is exposed only after it binds to Th-ATIII complexes (14Wells M.J. Blajchman M.A. J. Biol. Chem. 1998; 273: 23440-23447Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Recently, another accessory molecule, cytokeratin 18, has been shown to play an important role in the clearance of Th-ATIII-Vn ternary complexes (13Wells M.J. Hatton M.W. Hewlett B. Podor T.J. Sheffield W.P. Blajchman M.A. J. Biol. Chem. 1997; 272: 28574-28581Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Antibodies specific for cytokeratin 18 were shown to markedly reduce the LRP-mediated internalization of Th-ATIII-Vn ternary complexes. Although the heparin-mediated pathway appears to be common for many LRP internalized ligands, there is at least one example where the involvement of heparin has not yet been documented (27Stefansson S. Muhammad S. Cheng X.F. Battey F.D. Strickland D.K. Lawrence D.A. J. Biol. Chem. 1998; 273: 6358-6366Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The uPA receptor binds complexes between high molecular weight urokinase and plasminogen activator inhibitor 1 or PN1 prior to LRP-mediated internalization (27Stefansson S. Muhammad S. Cheng X.F. Battey F.D. Strickland D.K. Lawrence D.A. J. Biol. Chem. 1998; 273: 6358-6366Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). There are data suggesting that the uPA receptor, as well as its bound ligand, is co-internalized along with the LRP (16Conese M. Nykjaer A. Petersen C.M. Cremona O. Pardi R. Andreasen P.A. Gliemann J. Christensen E.I. Blasi F. J. Cell Biol. 1995; 131: 1609-1622Crossref PubMed Scopus (196) Google Scholar). Because plasminogen activator inhibitor 1 does contain a heparin-binding site, however, the potential involvement of heparin in this pathway should be examined more carefully.Structural information on binding sites in ligands that interact with the LRP is, however, limited. The highest resolution studies have been done on the LRP-binding site in activated α2-macroglobulin, which identified lysine residues 1370 and 1374 as essential for binding to the LRP (28Nielsen K.L. Holtet T.L. Etzerodt M. Moestrup S.K. Gliemann J. Sottrup-Jensen L. Thogersen H.C. J. Biol. Chem. 1996; 271: 12909-12912Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The LRP-binding site in thrombospondin 1 has been localized to the same amino-terminal fragment that contains the heparin-binding domain (25Mikhailenko I. Krylov D. Argraves K.M. Roberts D.D. Liau G. Strickland D.K. J. Biol. Chem. 1997; 272: 6784-6791Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), and the carboxyl-terminal folding domain of lipoprotein lipase, which also contains the heparin-binding domain, has been implicated in binding to the LRP (26Nielsen M.S. Brejning J. Garcı́a R. Zhang H. Hayden M.R. Vilar S. Gliemann J. J. Biol. Chem. 1997; 272: 5821-5827Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Most recently, studies on the binding of plasminogen activator inhibitor 1-protease complexes to the LRP have implicated two residues located in the heparin-binding site in LRP binding (27Stefansson S. Muhammad S. Cheng X.F. Battey F.D. Strickland D.K. Lawrence D.A. J. Biol. Chem. 1998; 273: 6358-6366Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). These data may be subject to alternate interpretations, because the opposite charge nature of the amino acid substitutions in plasminogen activator inhibitor 1 variants that resulted in a decreased LRP affinity may impart structural changes in the general region that are not necessarily part of the heparin-binding site (27Stefansson S. Muhammad S. Cheng X.F. Battey F.D. Strickland D.K. Lawrence D.A. J. Biol. Chem. 1998; 273: 6358-6366Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). In addition, data supporting any type of a universal structural motif shared by all LRP ligands are not very compelling, because the ligands are diverse, and many of the ligands do not cross-compete for binding (28Nielsen K.L. Holtet T.L. Etzerodt M. Moestrup S.K. Gliemann J. Sottrup-Jensen L. Thogersen H.C. J. Biol. Chem. 1996; 271: 12909-12912Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Although the binding of several LRP ligands to cell surface heparins has been shown to play an important role in their clearance by the LRP, it remains to be determined whether the heparin and the LRP-binding sites overlap in some cases. The data in the present report, along with previously published studies using synthetic peptides strongly argue against this in the case of PN1. Protease nexin 1 (PN1)1is a member of the SERPIN super-family (1Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (829) Google Scholar, 2Scott R.W. Bergman B.L. Bajpai A. Hersh R.T. Rodriguez H. Jones B.N. Barreda C. Watts S. Baker J.B. J. Biol. Chem. 1985; 260: 7029-7034Abstract Full Text PDF PubMed Google Scholar), and an important physiological regulator of thrombin (Th) and urinary plasminogen activator (uPA) (2Scott R.W. Bergman B.L. Bajpai A. Hersh R.T. Rodriguez H. Jones B.N. Barreda C. Watts S. Baker J.B. J. Biol. Chem. 1985; 260: 7029-7034Abstract Full Text PDF PubMed Google Scholar). PN1 forms stoichiometric complexes with both Th and uPA (3Baker J.B. Low D.A. Simmer R.L. Cunningham D.D. Cell. 1980; 21: 37-45Abstract Full Text PDF PubMed Scopus (290) Google Scholar) that ultimately results in their removal by cellular endocytosis and degradation (4Low D.A. Baker J.B. Koonce W.C. Cunningham D.D. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2340-2344Crossref PubMed Scopus (117) Google Scholar). The precise biochemical nature of the complexes is still not completely clear, but they are extremely stable and possibly covalent (5Cohen A.B. Gruenke L.D. Craig J.C. Geczy D. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 4311-4314Crossref PubMed Scopus (11) Google Scholar, 6Lawrence D.A. Ginsburg D. Day D.E. Berkenpas M.B. Verhamme I.M. Kvassman J.O. Shore J.D. J. Biol. Chem. 1995; 270: 25309-25312Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 7Shieh B.H. Potempa J. Travis J. J. Biol. Chem. 1989; 264: 13420-13423Abstract Full Text PDF PubMed Google Scholar). When complexes are formed between the SERPIN and its target protease, there is an accompanying conformational change in the SERPIN that either unmasks or causes the formation of a new binding site in the complexed SERPIN that is not present in the free SERPIN (8Howard E.W. Knauer D.J. J. Cell. Physiol. 1987; 131: 276-283Crossref PubMed Scopus (20) Google Scholar, 9Perlmutter D.H. Travis J. Punsal P.I. J. Clin. Invest. 1988; 81: 1774-1780Crossref PubMed Scopus (64) Google Scholar). The cryptic nature of the LRP-binding site in the free SERPIN makes sense biologically. It ensures that SERPINs will remain extracellular, either in plasma or in tissues near cell surfaces, until they have formed an irreversible complex with a protease. The list of SERPINs dependent on the LRP for cellular internalization includes, protease nexin 1 (PN1) (10Knauer M.F. Hawley S.B. Knauer D.J. J. Biol. Chem. 1997; 272: 12261-12264Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 11Conese M. Olson D. Blasi F. J. Biol. Chem. 1994; 269: 17886-17892Abstract Full Text PDF PubMed Google Scholar), heparin cofactor-II (12Kounnas M.Z. Church F.C. Argraves W.S. Strickland D.K. J. Biol. Chem. 1996; 271: 6523-6529Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), antithrombin III (ATIII) (12Kounnas M.Z. Church F.C. Argraves W.S. Strickland D.K. J. Biol. Chem. 1996; 271: 6523-6529Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) and α-1-antitrypsin (12Kounnas M.Z. Church F.C. Argraves W.S. Strickland D.K. J. Biol. Chem. 1996; 271: 6523-6529Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). It is interesting to note that although the LRP acts as the internalization receptor, other cellular components are most likely required for the efficient catabolism of the SERPIN-Protease complexes (12Kounnas M.Z. Church F.C. Argraves W.S. Strickland D.K. J. Biol. Chem. 1996; 271: 6523-6529Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 13Wells M.J. Hatton M.W. Hewlett B. Podor T.J. Sheffield W.P. Blajchman M.A. J. Biol. Chem. 1997; 272: 28574-28581Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 14Wells M.J. Blajchman M.A. J. Biol. Chem. 1998; 273: 23440-23447Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 15Knauer M.F. Kridel S.J. Hawley S.B. Knauer D.J. J. Biol. Chem. 1997; 272: 29039-29045Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In the case of the plasma SERPINs these components remain to be identified. However PN1, which is primarily restricted to tissues, utilizes at least two different cell surface molecules to assist LRP-mediated internalization. When PN1 forms complexes with uPA, the uPA receptor is required for efficient concentration at the cell surface of uPA-PN1 complexes and subsequent internalization via the LRP (11Conese M. Olson D. Blasi F. J. Biol. Chem. 1994; 269: 17886-17892Abstract Full Text PDF PubMed Google Scholar, 16Conese M. Nykjaer A. Petersen C.M. Cremona O. Pardi R. Andreasen P.A. Gliemann J. Christensen E.I. Blasi F. J. Cell Biol. 1995; 131: 1609-1622Crossref PubMed Scopus (196) Google Scholar). In contrast, when PN1 is in complex with thrombin, heparin chains present at the cell surface greatly facilitate the uptake and turnover of thrombin-PN1 complexes (15Knauer M.F. Kridel S.J. Hawley S.B. Knauer D.J. J. Biol. Chem. 1997; 272: 29039-29045Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), and uPA receptor is not involved (11Conese M. Olson D. Blasi F. J. Biol. Chem. 1994; 269: 17886-17892Abstract Full Text PDF PubMed Google Scholar, 16Conese M. Nykjaer A. Petersen C.M. Cremona O. Pardi R. Andreasen P.A. Gliemann J. Christensen E.I. Blasi F. J. Cell Biol. 1995; 131: 1609-1622Crossref PubMed Scopus (196) Google Scholar). Thus, in the case of PN1, the nature of the target protease directly plays a role in the clearance mechanism. In a recent study using a synthetic peptide library strategy, a putative LRP-binding site was identified in PN1 (10Knauer M.F. Hawley S.B. Knauer D.J. J. Biol. Chem. 1997; 272: 12261-12264Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The library consisted of peptides 12 amino acids in length, and spanned nearly the entire PN1 sequence. A single peptide in the library,47PHDNIVISPHGI58 was identified as a potent inhibitor of Th-PN1 internalization and degradation. Using α-1-antitrypsin structure and sequence alignments, this sequence is predicted to be a transition sequence that occurs just after helix A and continues to form the sixth strand of sheet B (1Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (829) Google Scholar). Consequently, this site meets the criteria of being at least partially buried in the intact SERPIN, with the potential of becoming more exposed after complex formation with thrombin. There is no direct evidence, however, to distinguish this possibility from a simple conformational change in which the exposure of this site remains constant. In the present studies we further investigate the potential role of this putative site in LRP-mediated internalization using two different approaches. In the first approach, a polyclonal IgG was generated against Pro47–Ile58 with a cysteine residue added after the Ile to facilitate haptenization to ovalbumin. We demonstrate that this antibody specifically and selectively inhibits the binding of Th-PN1 complexes to the LRP, but does not affect the interaction of the complexes with cell surface heparins. In the second approach we utilize site-directed mutagenesis and baculovirus-driven expression in insect cells. Two variant forms of PN1 were expressed; one with an alanine substitution at the position of His 48 (H48A), and another with alanine substitutions at the positions of His 48 and Asp 49 (H48A,D49A). Each PN1 variant was characterized biochemically by determining the k assoc value for thrombin inhibition and ability to form SDS-resistant complexes with thrombin. Additionally, the PN1 variant-thrombin complexes and native PN1-thrombin complexes were assayed for their capacity to bind to cell surface heparins. While the PN1 variants were found to be very similar to native PN1 in their ability to inactivate thrombin and bind to cell surface heparins, complexes made with each of the PN1 variants showed decreased rates of catabolism. These experiments define a critical role for the structural determinant, Pro47–Ile58, in the LRP-mediated internalization of Th-PN1 complexes. These data also demonstrate that with the use of anti-(Pro47–Ile58) antibody, cell surface heparin binding and LRP-mediated internalization of Th-PN1 complexes can be studied as independent events, even though they act cooperatively to facilitate complex catabolism. DISCUSSIONThe present studies were undertaken to further probe the potential role of the PN1 peptide sequence,47PHDNIVISPHGI58, in the LRP-mediated clearance of Th-PN1 complexes and to determine what structural features of this sequence might be important in this process. The potential importance of this sequence was discovered using a synthetic peptide library of PN1 sequences and screening for peptides that inhibited the catabolism of complexes by HF cells (10Knauer M.F. Hawley S.B. Knauer D.J. J. Biol. Chem. 1997; 272: 12261-12264Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The requirement for heparin-mediated binding of the complexes to the cell surface to promote an efficient interaction between the complexes and the LRP (15Knauer M.F. Kridel S.J. Hawley S.B. Knauer D.J. J. Biol. Chem. 1997; 272: 29039-29045Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) precluded more detailed studies of this sequence using a synthetic peptide approach, because the sites in PN1 that interact with heparin and the LRP are distinct. In the present studies, two different approaches have been used to more clearly define the role of the structural determinant, Pro47–Ile58, in Th-PN1 complex catabolism and to identify critical residues within this determinant.To independently confirm and more narrowly define the role of this structural determinant in Th-PN1 catabolism, an anti-(Pro47–Ile58) polyclonal IgG was generated. This antibody specifically inhibited the internalization and subsequent degradation of Th-PN1 complexes but had no effect on the binding of complexes to cell surface heparins. These data independently confirm the role of Pro47–Ile58 in LRP-mediated internalization, which had previously been based solely on synthetic peptide competition studies (10Knauer M.F. Hawley S.B. Knauer D.J. J. Biol. Chem. 1997; 272: 12261-12264Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). In addition, they strongly suggest that the LRP and heparin-binding determinants in PN1 are structurally distinct, despite the fact that they act cooperatively to promote efficient complex catabolism (15Knauer M.F. Kridel S.J. Hawley S.B. Knauer D.J. J. Biol. Chem. 1997; 272: 29039-29045Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar).In addition to the antibody studies, a genetic approach was used in which alanine substitutions were introduced at positions His48 and Asp49 of the intact PN1 protein. The rationale for these choices is derived from the predicted structural location of 47PHDNIVISPHGI58 in PN1 based on its homology to α-1-antitrypsin (1Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (829) Google Scholar). This sequence is predicted to be at least partially interior to the protein surface. Pro47most likely represents the beginning of the transition sequence between helix A and strand 6B(1). His48 and Asp49 are transition amino acids, and Asn50 through Pro55become strand 6 of sheet B. Given this structural information, we hypothesized that the charged residues, His48 and Asp49, might be important for either direct interaction with the LRP receptor or assisting in the attainment of the active conformation of this structural determinant when fully exposed to the hydrophilic exterior of the molecule.To evaluate the role of these specific amino acids we generated two different PN1 variants: one in which only His48 was replaced by Ala and another where both His48 and Asp49 were replaced by Ala. Replacement of the first charged residue, His48 by Ala, had a significant effect on the catabolism of PN1(H48A) complexes, reducing it by 50%. The additional substitution of Ala for Asp49, reduced complex catabolism by 85%. In both cases this was demonstrated to be due to a decreased rate of LRP-mediated internalization. Control studies revealed that the substitutions had no effect on the heparin-mediated binding of the complexes to the cell surface nor on the biochemical characteristics of the PN1 variant complexes with thrombin. Taken together, it is clear that both His48 and Asp49play very important roles in the interaction of the PN1 structural determinant, 47PHDNIVISPHGI58, with the LRP. Whether this is due to a direct interaction of these residues with the LRP or because these residues are required to establish a particular structural conformation of this determinant is presently unknown. Given the overall hydrophobic character of Pro47–Ile58, (PHDNIVISPHGI), it is likely that removal of the charged residues affects the solubility of the sequence. The genetic evidence using PN1 variants with point substitutions and the anti-(Pro47–Ile58) antibody experiments represent two important and independent lines of evidence that support and extend the original observation that the sequence Pro47–Ile58 in PN1 is required for the LRP-mediated clearance of Th-PN1 complexes.A common pathway for many LRP internalized ligands seems to be emerging that involves cell surface proteoglycans and perhaps other accessory proteins in many cases. Several of the ligands first bind to cell surface heparin sulfate proteoglycans and are subsequently internalized by the LRP (15Knauer M.F. Kridel S.J. Hawley S.B. Knauer D.J. J. Biol. Chem. 1997; 272: 29039-29045Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 25Mikhailenko I. Krylov D. Argraves K.M. Roberts D.D. Liau G. Strickland D.K. J. Biol. Chem. 1997; 272: 6784-6791Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 26Nielsen M.S. Brejning J. Garcı́a R. Zhang H. Hayden M.R. Vilar S. Gliemann J. J. Biol. Chem. 1997; 272: 5821-5827Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 27Stefansson S. Muhammad S. Cheng X.F. Battey F.D. Strickland D.K. Lawrence D.A. J. Biol. Chem. 1998; 273: 6358-6366Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Even Th-ATIII complexes, which display a negligible affinity for heparin, use hepatic heparin sulfate proteoglycans as a part of their clearance mechanism but do so by an association with vitronectin (Vn) (14Wells M.J. Blajchman M.A. J. Biol. Chem. 1998; 273: 23440-23447Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Th-ATIII complexes first bind to Vn and then utilize the heparin-binding site in Vn, which is exposed only after it binds to Th-ATIII complexes (14Wells M.J. Blajchman M.A. J. Biol. Chem. 1998; 273: 23440-23447Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Recently, another accessory molecule, cytokeratin 18, has been shown to play an important role in the clearance of Th-ATIII-Vn ternary complexes (13Wells M.J. Hatton M.W. Hewlett B. Podor T.J. Sheffield W.P. Blajchman M.A. J. Biol. Chem. 1997; 272: 28574-28581Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Antibodies specific for cytokeratin 18 were shown to markedly reduce the LRP-mediated internalization of Th-ATIII-Vn ternary complexes. Although the heparin-mediated pathway appears to be common for many LRP internalized ligands, there is at least one example where the involvement of heparin has not yet been documented (27Stefansson S. Muhammad S. Cheng X.F. Battey F.D. Strickland D.K. Lawrence D.A. J. Biol. Chem. 1998; 273: 6358-6366Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The uPA receptor binds complexes between high molecular weight urokinase and plasminogen activator inhibitor 1 or PN1 prior to LRP-mediated internalization (27Stefansson S. Muhammad S. Cheng X.F. Battey F.D. Strickland D.K. Lawrence D.A. J. Biol. Chem. 1998; 273: 6358-6366Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). There are data suggesting that the uPA receptor, as well as its bound ligand, is co-internalized along with the LRP (16Conese M. Nykjaer A. Petersen C.M. Cremona O. Pardi R. Andreasen P.A. Gliemann J. Christensen E.I. Blasi F. J. Cell Biol. 1995; 131: 1609-1622Crossref PubMed Scopus (196) Google Scholar). Because plasminogen activator inhibitor 1 does contain a heparin-binding site, however, the potential involvement of heparin in this pathway should be examined more carefully.Structural information on binding sites in ligands that interact with the LRP is, however, limited. The highest resolution studies have been done on the LRP-binding site in activated α2-macroglobulin, which identified lysine residues 1370 and 1374 as essential for binding to the LRP (28Nielsen K.L. Holtet T.L. Etzerodt M. Moestrup S.K. Gliemann J. Sottrup-Jensen L. Thogersen H.C. J. Biol. Chem. 1996; 271: 12909-12912Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The LRP-binding site in thrombospondin 1 has been localized to the same amino-terminal fragment that contains the heparin-binding domain (25Mikhailenko I. Krylov D. Argraves K.M. Roberts D.D. Liau G. Strickland D.K. J. Biol. Chem. 1997; 272: 6784-6791Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), and the carboxyl-terminal folding domain of lipoprotein lipase, which also contains the heparin-binding domain, has been implicated in binding to the LRP (26Nielsen M.S. Brejning J. Garcı́a R. Zhang H. Hayden M.R. Vilar S. Gliemann J. J. Biol. Chem. 1997; 272: 5821-5827Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Most recently, studies on the binding of plasminogen activator inhibitor 1-protease complexes to the LRP have implicated two residues located in the heparin-binding site in LRP binding (27Stefansson S. Muhammad S. Cheng X.F. Battey F.D. Strickland D.K. Lawrence D.A. J. Biol. Chem. 1998; 273: 6358-6366Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). These data may be subject to alternate interpretations, because the opposite charge nature of the amino acid substitutions in plasminogen activator inhibitor 1 variants that resulted in a decreased LRP affinity may impart structural changes in the general region that are not necessarily part of the heparin-binding site (27Stefansson S. Muhammad S. Cheng X.F. Battey F.D. Strickland D.K. Lawrence D.A. J. Biol. Chem. 1998; 273: 6358-6366Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). In addition, data supporting any type of a universal structural motif shared by all LRP ligands are not very compelling, because the ligands are diverse, and many of the ligands do not cross-compete for binding (28Nielsen K.L. Holtet T.L. Etzerodt M. Moestrup S.K. Gliemann J. Sottrup-Jensen L. Thogersen H.C. J. Biol. Chem. 1996; 271: 12909-12912Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Although the binding of several LRP ligands to cell surface heparins has been shown to play an important role in their clearance by the LRP, it remains to be determined whether the heparin and the LRP-binding sites overlap in some cases. The data in the present report, along with previously published studies using synthetic peptides strongly argue against this in the case of PN1. The present studies were undertaken to further probe the potential role of the PN1 peptide sequence,47PHDNIVISPHGI58, in the LRP-mediated clearance of Th-PN1 complexes and to determine what structural features of this sequence might be important in this process. The potential importance of this sequence was discovered using a synthetic peptide library of PN1 sequences and screening for peptides that inhibited the catabolism of complexes by HF cells (10Knauer M.F. Hawley S.B. Knauer D.J. J. Biol. Chem. 1997; 272: 12261-12264Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The requirement for heparin-mediated binding of the complexes to the cell surface to promote an efficient interaction between the complexes and the LRP (15Knauer M.F. Kridel S.J. Hawley S.B. Knauer D.J. J. Biol. Chem. 1997; 272: 29039-29045Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) precluded more detailed studies of this sequence using a synthetic peptide approach, because the sites in PN1 that interact with heparin and the LRP are distinct. In the present studies, two different approaches have been used to more clearly define the role of the structural determinant, Pro47–Ile58, in Th-PN1 complex catabolism and to identify critical residues within this determinant. To independently confirm and more narrowly define the role of this structural determinant in Th-PN1 catabolism, an anti-(Pro47–Ile58) polyclonal IgG was generated. This antibody specifically inhibited the internalization and subsequent degradation of Th-PN1 complexes but had no effect on the binding of complexes to cell surface heparins. These data independently confirm the role of Pro47–Ile58 in LRP-mediated internalization, which had previously been based solely on synthetic peptide competition studies (10Knauer M.F. Hawley S.B. Knauer D.J. J. Biol. Chem. 1997; 272: 12261-12264Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). In addition, they strongly suggest that the LRP and heparin-binding determinants in PN1 are structurally distinct, despite the fact that they act cooperatively to promote efficient complex catabolism (15Knauer M.F. Kridel S.J. Hawley S.B. Knauer D.J. J. Biol. Chem. 1997; 272: 29039-29045Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In addition to the antibody studies, a genetic approach was used in which alanine substitutions were introduced at positions His48 and Asp49 of the intact PN1 protein. The rationale for these choices is derived from the predicted structural location of 47PHDNIVISPHGI58 in PN1 based on its homology to α-1-antitrypsin (1Huber R. Carrell R.W. Biochemistry. 1989; 28: 8951-8966Crossref PubMed Scopus (829) Google Scholar). This sequence is predicted to be at least partially interior to the protein surface. Pro47most likely represents the beginning of the transition sequence between helix A and strand 6B(1). His48 and Asp49 are transition amino acids, and Asn50 through Pro55become strand 6 of sheet B. Given this structural information, we hypothesized that the charged residues, His48 and Asp49, might be important for either direct interaction with the LRP receptor or assisting in the attainment of the active conformation of this structural determinant when fully exposed to the hydrophilic exterior of the molecule. To evaluate the role of these specific amino acids we generated two different PN1 variants: one in which only His48 was replaced by Ala and another where both His48 and Asp49 were replaced by Ala. Replacement of the first charged residue, His48 by Ala, had a significant effect on the catabolism of PN1(H48A) complexes, reducing it by 50%. The additional substitution of Ala for Asp49, reduced complex catabolism by 85%. In both cases this was demonstrated to be due to a decreased rate of LRP-mediated internalization. Control studies revealed that the substitutions had no effect on the heparin-mediated binding of the complexes to the cell surface nor on the biochemical characteristics of the PN1 variant complexes with thrombin. Taken together, it is clear that both His48 and Asp49play very important roles in the interaction of the PN1 structural determinant, 47PHDNIVISPHGI58, with the LRP. Whether this is due to a direct interaction of these residues with the LRP or because these residues are required to establish a particular structural conformation of this determinant is presently unknown. Given the overall hydrophobic character of Pro47–Ile58, (PHDNIVISPHGI), it is likely that removal of the charged residues affects the solubility of the sequence. The genetic evidence using PN1 variants with point substitutions and the anti-(Pro47–Ile58) antibody experiments represent two important and independent lines of evidence that support and extend the original observation that the sequence Pro47–Ile58 in PN1 is required for the LRP-mediated clearance of Th-PN1 complexes. A common pathway for many LRP internalized ligands seems to be emerging that involves cell surface proteoglycans and perhaps other accessory proteins in many cases. Several of the ligands first bind to cell surface heparin sulfate proteoglycans and are subsequently internalized by the LRP (15Knauer M.F. Kridel S.J. Hawley S.B. Knauer D.J. J. Biol. Chem. 1997; 272: 29039-29045Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 25Mikhailenko I. Krylov D. Argraves K.M. Roberts D.D. Liau G. Strickland D.K. J. Biol. Chem. 1997; 272: 6784-6791Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 26Nielsen M.S. Brejning J. Garcı́a R. Zhang H. Hayden M.R. Vilar S. Gliemann J. J. Biol. Chem. 1997; 272: 5821-5827Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 27Stefansson S. Muhammad S. Cheng X.F. Battey F.D. Strickland D.K. Lawrence D.A. J. Biol. Chem. 1998; 273: 6358-6366Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Even Th-ATIII complexes, which display a negligible affinity for heparin, use hepatic heparin sulfate proteoglycans as a part of their clearance mechanism but do so by an association with vitronectin (Vn) (14Wells M.J. Blajchman M.A. J. Biol. Chem. 1998; 273: 23440-23447Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Th-ATIII complexes first bind to Vn and then utilize the heparin-binding site in Vn, which is exposed only after it binds to Th-ATIII complexes (14Wells M.J. Blajchman M.A. J. Biol. Chem. 1998; 273: 23440-23447Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Recently, another accessory molecule, cytokeratin 18, has been shown to play an important role in the clearance of Th-ATIII-Vn ternary complexes (13Wells M.J. Hatton M.W. Hewlett B. Podor T.J. Sheffield W.P. Blajchman M.A. J. Biol. Chem. 1997; 272: 28574-28581Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Antibodies specific for cytokeratin 18 were shown to markedly reduce the LRP-mediated internalization of Th-ATIII-Vn ternary complexes. Although the heparin-mediated pathway appears to be common for many LRP internalized ligands, there is at least one example where the involvement of heparin has not yet been documented (27Stefansson S. Muhammad S. Cheng X.F. Battey F.D. Strickland D.K. Lawrence D.A. J. Biol. Chem. 1998; 273: 6358-6366Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The uPA receptor binds complexes between high molecular weight urokinase and plasminogen activator inhibitor 1 or PN1 prior to LRP-mediated internalization (27Stefansson S. Muhammad S. Cheng X.F. Battey F.D. Strickland D.K. Lawrence D.A. J. Biol. Chem. 1998; 273: 6358-6366Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). There are data suggesting that the uPA receptor, as well as its bound ligand, is co-internalized along with the LRP (16Conese M. Nykjaer A. Petersen C.M. Cremona O. Pardi R. Andreasen P.A. Gliemann J. Christensen E.I. Blasi F. J. Cell Biol. 1995; 131: 1609-1622Crossref PubMed Scopus (196) Google Scholar). Because plasminogen activator inhibitor 1 does contain a heparin-binding site, however, the potential involvement of heparin in this pathway should be examined more carefully. Structural information on binding sites in ligands that interact with the LRP is, however, limited. The highest resolution studies have been done on the LRP-binding site in activated α2-macroglobulin, which identified lysine residues 1370 and 1374 as essential for binding to the LRP (28Nielsen K.L. Holtet T.L. Etzerodt M. Moestrup S.K. Gliemann J. Sottrup-Jensen L. Thogersen H.C. J. Biol. Chem. 1996; 271: 12909-12912Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The LRP-binding site in thrombospondin 1 has been localized to the same amino-terminal fragment that contains the heparin-binding domain (25Mikhailenko I. Krylov D. Argraves K.M. Roberts D.D. Liau G. Strickland D.K. J. Biol. Chem. 1997; 272: 6784-6791Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), and the carboxyl-terminal folding domain of lipoprotein lipase, which also contains the heparin-binding domain, has been implicated in binding to the LRP (26Nielsen M.S. Brejning J. Garcı́a R. Zhang H. Hayden M.R. Vilar S. Gliemann J. J. Biol. Chem. 1997; 272: 5821-5827Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Most recently, studies on the binding of plasminogen activator inhibitor 1-protease complexes to the LRP have implicated two residues located in the heparin-binding site in LRP binding (27Stefansson S. Muhammad S. Cheng X.F. Battey F.D. Strickland D.K. Lawrence D.A. J. Biol. Chem. 1998; 273: 6358-6366Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). These data may be subject to alternate interpretations, because the opposite charge nature of the amino acid substitutions in plasminogen activator inhibitor 1 variants that resulted in a decreased LRP affinity may impart structural changes in the general region that are not necessarily part of the heparin-binding site (27Stefansson S. Muhammad S. Cheng X.F. Battey F.D. Strickland D.K. Lawrence D.A. J. Biol. Chem. 1998; 273: 6358-6366Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). In addition, data supporting any type of a universal structural motif shared by all LRP ligands are not very compelling, because the ligands are diverse, and many of the ligands do not cross-compete for binding (28Nielsen K.L. Holtet T.L. Etzerodt M. Moestrup S.K. Gliemann J. Sottrup-Jensen L. Thogersen H.C. J. Biol. Chem. 1996; 271: 12909-12912Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Although the binding of several LRP ligands to cell surface heparins has been shown to play an important role in their clearance by the LRP, it remains to be determined whether the heparin and the LRP-binding sites overlap in some cases. The data in the present report, along with previously published studies using synthetic peptides strongly argue against this in the case of PN1.
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