Molecular Analysis of Ligand Binding to the Second Cluster of Complement-type Repeats of the Low Density Lipoprotein Receptor-related Protein

Abstract

The low density lipoprotein receptor-related protein (LRP), a member of the low density lipoprotein receptor gene family, mediates the cellular uptake of a diversity of ligands. A folding chaperone, the 39-kDa receptor-associated protein (RAP) that resides in the early compartments of the secretory pathway inhibits the binding of all ligands to the receptor and may serve to prevent premature binding of ligands to the receptor during the trafficking to the cell surface. To elucidate the molecular interactions that underlie the interplay between the receptor, RAP, and the ligands, we have analyzed and delineated the binding sites of plasminogen activator inhibitor-1 (PAI-1), tissue-type plasminogen activator (t-PA)·PAI-1 complexes, RAP, and the anti-LRP Fab fragment Fab A8. To that end, we have generated a series of soluble recombinant fragments spanning the second cluster of complement-type repeats (C3-C10) and the amino-terminal flanking epidermal growth factor repeat (E4) of LRP (E4-C10; amino acids 787–1165). All fragments were expressed by stably transfected baby hamster kidney cells and purified by affinity chromatography. A detailed study of ligand binding to the fragments using surface plasmon resonance revealed the presence of three distinct, Ca2+-dependent ligand binding sites in the cluster II domain (Cl-II) of LRP. t-PA·PAI-1 complexes as well as PAI-1 bind to a domain located in the amino-terminal portion of Cl-II, spanning repeats E4-C3-C7. Adjacent to this site and partially overlapping is a high affinity RAP-binding site located on repeats C5-C7. Fab A8, a pseudo-ligand of the receptor, binds to a third Ca2+-dependent binding site on repeats C8-C10 at the carboxyl-terminal end of Cl-II. Next, we studied the RAP-mediated inhibition of ligand binding to LRP and to Cl-II. As expected, we observed a strong inhibition of t-PA·PAI-1 complex and Fab A8 binding to LRP by RAP (IC50 ≅ 0.3 nm), whereas in the reverse experiment, competition of t-PA·PAI-1 complexes and Fab A8 for RAP binding to LRP could only be shown at high concentrations of competitors (≥1 μm). Interestingly, even though the equilibrium dissociation constants for the binding of RAP to LRP and to Cl-II are similar, the binding of the ligands to Cl-II is only prevented by RAP at concentrations that are at least 2 orders of magnitude higher than those required for inhibition of ligand binding to LRP. Our results favor models that propose RAP-induced allosteric inhibition of ligand binding to LRP that may require LRP moieties that are located outside Cl-II of the receptor. The low density lipoprotein receptor-related protein (LRP), a member of the low density lipoprotein receptor gene family, mediates the cellular uptake of a diversity of ligands. A folding chaperone, the 39-kDa receptor-associated protein (RAP) that resides in the early compartments of the secretory pathway inhibits the binding of all ligands to the receptor and may serve to prevent premature binding of ligands to the receptor during the trafficking to the cell surface. To elucidate the molecular interactions that underlie the interplay between the receptor, RAP, and the ligands, we have analyzed and delineated the binding sites of plasminogen activator inhibitor-1 (PAI-1), tissue-type plasminogen activator (t-PA)·PAI-1 complexes, RAP, and the anti-LRP Fab fragment Fab A8. To that end, we have generated a series of soluble recombinant fragments spanning the second cluster of complement-type repeats (C3-C10) and the amino-terminal flanking epidermal growth factor repeat (E4) of LRP (E4-C10; amino acids 787–1165). All fragments were expressed by stably transfected baby hamster kidney cells and purified by affinity chromatography. A detailed study of ligand binding to the fragments using surface plasmon resonance revealed the presence of three distinct, Ca2+-dependent ligand binding sites in the cluster II domain (Cl-II) of LRP. t-PA·PAI-1 complexes as well as PAI-1 bind to a domain located in the amino-terminal portion of Cl-II, spanning repeats E4-C3-C7. Adjacent to this site and partially overlapping is a high affinity RAP-binding site located on repeats C5-C7. Fab A8, a pseudo-ligand of the receptor, binds to a third Ca2+-dependent binding site on repeats C8-C10 at the carboxyl-terminal end of Cl-II. Next, we studied the RAP-mediated inhibition of ligand binding to LRP and to Cl-II. As expected, we observed a strong inhibition of t-PA·PAI-1 complex and Fab A8 binding to LRP by RAP (IC50 ≅ 0.3 nm), whereas in the reverse experiment, competition of t-PA·PAI-1 complexes and Fab A8 for RAP binding to LRP could only be shown at high concentrations of competitors (≥1 μm). Interestingly, even though the equilibrium dissociation constants for the binding of RAP to LRP and to Cl-II are similar, the binding of the ligands to Cl-II is only prevented by RAP at concentrations that are at least 2 orders of magnitude higher than those required for inhibition of ligand binding to LRP. Our results favor models that propose RAP-induced allosteric inhibition of ligand binding to LRP that may require LRP moieties that are located outside Cl-II of the receptor. The low density lipoprotein receptor-related protein (LRP), 1The abbreviations used are: LRP, low density lipoprotein receptor-related protein; C, complement-type repeat; Cl-II, second cluster of complement-type repeats; E, epidermal growth factor-type repeat; mAb, monoclonal antibody; PAI-1, plasminogen activator inhibitor type-1; RAP, receptor-associated protein; SPR, surface plasmon resonance; t-PA, tissue-type plasminogen activator; ELISA, enzyme-linked immunosorbent assay; HBS, HEPES-buffered saline. 1The abbreviations used are: LRP, low density lipoprotein receptor-related protein; C, complement-type repeat; Cl-II, second cluster of complement-type repeats; E, epidermal growth factor-type repeat; mAb, monoclonal antibody; PAI-1, plasminogen activator inhibitor type-1; RAP, receptor-associated protein; SPR, surface plasmon resonance; t-PA, tissue-type plasminogen activator; ELISA, enzyme-linked immunosorbent assay; HBS, HEPES-buffered saline. a 600-kDa glycoprotein, is a multifunctional endocytic receptor (1Krieger M. Herz J. Annu. Rev. Biochem. 1994; 63: 601-637Google Scholar, 2Moestrup S.K. Biochim. Biophys. Acta. 1994; 1197: 197-213Google Scholar, 3Strickland D.K. Kounnas M.Z. Williams S.E. Argraves W.S. Fibrinolysis. 1994; 8: 204-215Google Scholar). LRP binds and internalizes a broad spectrum of structurally unrelated ligands including two important components of the fibrinolytic system, tissue-type plasminogen activator (t-PA) and its specific inhibitor plasminogen activator inhibitor type-1 (PAI-1) (4Orth K. Madison E.L. Gething M.J. Sambrook J.F. Herz J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7422-7426Google Scholar, 5Bu G. Williams S.E. Strickland D.K. Schwartz A.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7427-7431Google Scholar). In rodents, LRP was shown to be one of the in vivo receptor systems involved in the clearance of t-PA (6Warshawsky I. Bu G. Schwartz A.L. J. Clin. Invest. 1993; 92: 937-944Google Scholar, 7Narita M. Bu G. Herz J. Schwartz A.L. J. Clin. Invest. 1995; 96: 1164-1168Google Scholar). LRP is synthesized as a 600-kDa precursor protein that is subsequently processed in the trans-Golgi compartment to a 515-kDa α-chain and a 85-kDa β-chain. Both chains remain associated in an unusual noncovalent fashion (8Willnow T.E. Moehring J.M. Inocencio N.M. Moehring T.J. Herz J. Biochem. J. 1996; 313: 71-76Google Scholar). Ligand binding by the receptor is thought to be mediated by the so-called complement-type repeats. These 40-residue-long repeats are characterized by a highly conserved spacing pattern of six cysteine residues, and they are found in all other members of the low density lipoprotein receptor gene family (1Krieger M. Herz J. Annu. Rev. Biochem. 1994; 63: 601-637Google Scholar). In the low density lipoprotein receptor, these complement-type repeats were shown to mediate ligand binding (9Esser V. Limbird L.E. Brown M.S. Goldstein J.L. Russell D.W. J. Biol. Chem. 1988; 263: 13282-13290Google Scholar, 10Russell D.W. Brown M.S. Goldstein J.L. J. Biol. Chem. 1989; 264: 21682-21688Google Scholar). The α-chain of LRP contains four potential ligand-binding domains assembled as sequential series of 2, 8, 10, and 11 complement-type repeats (cluster I, II, III, and IV). The first evidence for a role of the cluster II domain (Cl-II) in ligand binding was presented by Moestrup et al., who demonstrated the binding of α2-macroglobulin-complexes, urokinase-type plasminogen activator· PAI-1 complexes, and receptor-associated protein (RAP) to a 75-kDa CNBr-generated LRP fragment, spanning Cl-II, on a ligand blot (11Moestrup S.K. Holtet T.L. Etzerodt M. Thøgersen H.C. Nykjær A. Andreasen P.A. Rasmussen H.H. Sottrup-Jensen L. Gliemann J. J. Biol. Chem. 1993; 268: 13691-13696Google Scholar). Using a recombinant DNA approach, Willnow et al. expressed LRP minireceptors and showed t-PA·PAI-1 complex and RAP binding to Cl-II and a low affinity binding of RAP to a truncated receptor harboring the cluster IV domain (12Willnow T.E. Orth K. Herz J. J. Biol. Chem. 1994; 269: 15827-15832Google Scholar). In a previous study, we provided evidence for an additional ligand binding site in Cl-II by the selection of a high affinity Cl-II-binding Fab fragment (Fab A8) that bound the receptor in a Ca2+-dependent fashion and completely inhibited the binding of pro-urokinase to LRP (13Horn I.R. Moestrup S.K. van den Berg B.M.M. Pannekoek H. Nielsen M.S. van Zonneveld A.-J. J. Biol. Chem. 1995; 270: 11770-11775Google Scholar). The 39-kDa folding chaperone RAP is a molecule with a high affinity for LRP that resides in the endoplasmic reticulum and thecis-Golgi compartments (14Bu G. Geuze H.J. Strous G.J. Schwartz A.L. EMBO J. 1995; 14: 2269-2280Scopus (268) Google Scholar). RAP inhibits the binding of all known ligands to LRP, and it has been proposed that this activity may serve to prevent premature binding of ligands to the receptor during the maturation and trafficking of LRP (14Bu G. Geuze H.J. Strous G.J. Schwartz A.L. EMBO J. 1995; 14: 2269-2280Scopus (268) Google Scholar, 15Willnow T.E. Rohlmann A. Horton J. Otani H. Braun J.R. Hammer R.E. Herz J. EMBO J. 1996; 15: 2632-2639Google Scholar). Little is known about the mechanism by which RAP inhibits the binding of the diversity of structurally unrelated ligands. One model assumes a close spatial association of a RAP binding site to each of the independent LRP ligand-binding sites (16Iadonato S.P. Bu G. Maksymovitch E.A. Schwartz A.L. Biochem. J. 1993; 296: 867-875Google Scholar, 17Bu G. Warshawsky I. Schwartz A.L. Blood. 1994; 83: 3427-3436Google Scholar). Consequently, RAP would inhibit ligand binding by steric hindrance of each of the LRP ligands. An alternative model for the regulation of ligand binding by RAP proposes a RAP-induced conformational change in the LRP molecule (18Willnow T.E. Goldstein J.L. Orth K. Brown M.S. Herz J. J. Biol. Chem. 1992; 267: 26172-26180Google Scholar). A unique feature of RAP is its repeated triplicate structure, endowing the protein with three potential LRP-binding sites as determined by cross-linking (14Bu G. Geuze H.J. Strous G.J. Schwartz A.L. EMBO J. 1995; 14: 2269-2280Scopus (268) Google Scholar). with amino-terminal and carboxyl-terminal fragments of RAP have that at least two high affinity LRP binding sites are in RAP I. Bu G. Schwartz A.L. J. Biol. Chem. 1994; 269: Scholar). The presence of RAP-binding sites in the of LRP (11Moestrup S.K. Holtet T.L. Etzerodt M. Thøgersen H.C. Nykjær A. Andreasen P.A. Rasmussen H.H. Sottrup-Jensen L. Gliemann J. J. Biol. Chem. 1993; 268: 13691-13696Google Scholar, T.E. Orth K. Herz J. J. Biol. Chem. 1994; 269: 15827-15832Google Scholar, G. S. J. Biol. Chem. 1996; as well as the presence of sites in RAP, may a model in which RAP binds to a conformational change in LRP that would be with ligand a model would that competition by RAP for ligand binding is on the of the the LRP In this study, we have a detailed of ligand-binding sites in the cluster II domain of the receptor. Using recombinant fragments spanning Cl-II, we that Cl-II contains three binding sites for t-PA·PAI-1 complexes, RAP, and the pseudo-ligand Fab A8. In we evidence for an allosteric in RAP-mediated inhibition of ligand binding to LRP. were and other were All other used were LRP was provided by S. K. Moestrup of of RAP was provided by J. of The t-PA was and recombinant t-PA was PAI-1 were as D.W. J. Biochem. 1994; M. J. van Zonneveld J. Pannekoek H. J. Biol. Chem. 1996; Scholar). t-PA·PAI-1 complexes were as t-PA was with an of PAI-1 for 2 at t-PA was the of PAI-1 by affinity using bound to the were with and by with and with and were by in a high at for at using and subsequently and 2 revealed a protein on a and were at of the monoclonal Fab fragment Fab A8 as well as the of the monoclonal were as (13Horn I.R. Moestrup S.K. van den Berg B.M.M. Pannekoek H. Nielsen M.S. van Zonneveld A.-J. J. Biol. Chem. 1995; 270: 11770-11775Google Scholar, J. M. van Zonneveld H. Pannekoek H. Blood. Scholar). The and including an and were recombinant LRP Cl-II fragments amino acids amino acids amino acids and amino acids to Herz et al. J. U. S. H. EMBO J. 1988; were by using a the LRP J. of as a cluster II fragments were using LRP and LRP and and LRP and LRP and for LRP Cl-II fragments Cl-II, and were with and subsequently with a fragment, the and of t-PA acids to Proc. Natl. Acad. Sci. U. S. A. by a that contains the of the A. van K. Biochem. J. 1989; Scholar). DNA Cl-II fragments were the using and using DNA and the site of D.W. 1992; to LRP Cl-II fragments Cl-II, and and were by of for Cl-II and using and and the DNA All were by DNA using an DNA hamster kidney cells were to in and were transfected with of DNA using the as J. A Scholar). were and the cells were for with were to in for and subsequently for on with were and for the of recombinant LRP cluster II fragments by using a to LRP S. K. Cl-II were to in Next, the cells were for in and were Cl-II fragments were purified the by affinity using were with HEPES-buffered and with affinity was using Fab These were with and with Cl-II were in a HEPES-buffered by in a high at for at using and and 2 All Cl-II fragment were analyzed by ELISA, and with and anti-LRP Cl-II fragments were analyzed by a To the of to the observed of the the Cl-II fragment were with the protein were for at in the were with and with of for 2 at were and analyzed on a The was to a using the the of the at a high density of to binding of the of A on the was and using of The binding of protein to this was specific To the system, Cl-II fragment with known concentrations were the at at a of using as of the surface was with each the of the binding was determined by using the Using the the was to Cl-II fragment LRP was on a by at a low density of Using as RAP and Fab A8 were three with LRP and one at at a of was at least in triplicate at concentrations in the constants were determined by to a site model site model the to the is the at is the are the at equilibrium for the first and second are the observed for the first and the second The are subsequently of the are by the of the by the 2 The dissociation of the was to the is the at is the of the dissociation and binding to receptor sites and 2, and are dissociation constants for the binding sites and 2, A site model was used to all other the of this model is that of the model the of a site model Cl-II interactions were as RAP, purified t-PA·PAI-1 complexes, PAI-1, and Fab A8 were to the at low were at at a of were at least at four constants were by to site models the as and and as The were by to the of Biochem. Sci. 1996; Scholar). were at concentrations of ligands LRP Cl-II. To of LRP of Cl-II was to for at were with for at and with a of ligand concentrations in were using The for t-PA·PAI-1 complexes, Fab A8, and was as (13Horn I.R. Moestrup S.K. van den Berg B.M.M. Pannekoek H. Nielsen M.S. van Zonneveld A.-J. J. Biol. Chem. 1995; 270: 11770-11775Google Scholar). were subsequently and were in In a previous study, we and the evidence for the of Cl-II for ligand binding by the and of a Fab fragment (Fab A8) (13Horn I.R. Moestrup S.K. van den Berg B.M.M. Pannekoek H. Nielsen M.S. van Zonneveld A.-J. J. Biol. Chem. 1995; 270: 11770-11775Google Scholar). Fab fragment a pseudo-ligand it binds LRP in a Ca2+-dependent fashion and its binding is completely inhibited by Using this Fab fragment, we provided evidence for a binding site for pro-urokinase in Cl-II, the binding sites for and t-PA·PAI-1 To ligand binding by Cl-II in we have expressed a series of soluble recombinant cluster II fragments in stably transfected baby hamster kidney cell of the fragments is by a for the fragments the secretory pathway and a to and The epidermal growth factor repeat (E4) that Cl-II is in all fragments to the amino-terminal end of Cl-II, this repeat has been to be important for the binding of to LRP (11Moestrup S.K. Holtet T.L. Etzerodt M. Thøgersen H.C. Nykjær A. Andreasen P.A. Rasmussen H.H. Sottrup-Jensen L. Gliemann J. J. Biol. Chem. 1993; 268: 13691-13696Google Scholar, T.E. Orth K. Herz J. J. Biol. Chem. 1994; 269: 15827-15832Google Scholar). recombinant fragments were analyzed by and were with The of the fragments was than would be on the of the molecular and for and Cl-II, be of of the complement-type repeats of Cl-II G. S. J. Biol. Chem. 1996; Scholar). To that is for the of the we the fragments with and the pattern to that of the As shown in 2 with the of a protein of the molecular results were for the other recombinant fragments and we that the of the fragments during is as a of moieties to the fragments These results the observed 2 which may of the recombinant cluster II of a The the Cl-II fragments as of a and are in In this study we have on the binding of the cluster II ligands RAP, t-PA·PAI-1 complexes, PAI-1, t-PA, and Fab A8. we determined for interactions of these ligands with the receptor. To that end, LRP was on a and the association and dissociation constants were determined by As shown in I, RAP binding to LRP could only be using a the presence of at least two RAP-binding sites on LRP, as by Williams and S.E. Argraves W.S. Strickland D.K. J. Biol. Chem. 1992; 267: Scholar). The two sites of and which are in with by Moestrup et al. and low affinity sites of and S.K. H. Proc. Natl. Acad. Sci. U. S. A. 1996; Scholar). for the determined by binding to be than the may be to the The interactions of t-PA·PAI-1 complexes, PAI-1, t-PA, and Fab A8 could be by a site of and and for the interactions of RAP, t-PA PAI-1 complexes, PAI-1, t-PA, and Fab A8 with the the S.E. of at least four in a the the S.E. of at least four To the binding sites for RAP, t-PA·PAI-1 complexes, PAI-1, and Fab A8 on Cl-II, we these LRP ligands at a low density on and determined the constants for the interactions with the Cl-II As presented in II, RAP binds to a of Cl-II fragments to a site model with of for the Cl-II and for the fragment The fragments and bind to RAP with These results the presence of a RAP-binding site in Cl-II, on a fragment spanning complement-type repeats and and and for the interactions of RAP, t-PA PAI-1 complexes, PAI-1, and Fab A8 with recombinant cluster II cluster II the the S.E. of at least four binding binding in a the the S.E. of at least four binding binding t-PA·PAI-1 complexes bind to recombinant fragments Cl-II and The are (Cl-II) and an site on Cl-II on a fragment spanning epidermal growth factor repeat and complement-type repeats Cl-II and with PAI-1 as with higher and for Cl-II and could binding of Cl-II fragments to that t-PA·PAI-1 complexes as well as PAI-1 bind to a site that is partially overlapping the RAP binding site in Cl-II. The anti-LRP Fab fragment Fab A8, which inhibits the binding of plasminogen activator to LRP, binds to of Cl-II fragments including Cl-II, and are and Cl-II, and a unique binding site for Fab A8 on complement-type repeats and The results of these are in that the recombinant fragments of Cl-II ligand binding sites and Cl-II contains a of at least three binding sites for t-PA·PAI-1 complexes (PAI-1), RAP, and Fab A8. demonstrated that RAP completely prevent the binding of Fab A8 to LRP, whereas Fab A8 is to for the (13Horn I.R. Moestrup S.K. van den Berg B.M.M. Pannekoek H. Nielsen M.S. van Zonneveld A.-J. J. Biol. Chem. 1995; 270: 11770-11775Google Scholar). it has been shown that RAP inhibits t-PA·PAI-1 complex binding to LRP (4Orth K. Madison E.L. Gething M.J. Sambrook J.F. Herz J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7422-7426Google whereas the reverse has been One mechanism to RAP inhibition of ligand binding to LRP is the hindrance to the close spatial association of the and ligand-binding To this we a series of competition to the of t-PA·PAI-1 and Fab to LRP and to Cl-II. we determined the required for binding to the LRP and to Cl-II. As be in of RAP binding to LRP Cl-II is at 0.3 and it be that the is for t-PA·PAI-1 complexes and Fab A8, with at concentrations of and for t-PA·PAI-1 complex and and for Fab A8 binding to LRP and Cl-II, These results that the of the ligand-binding sites of Cl-II is Next, we studied the of t-PA·PAI-1 and Fab to LRP and to Cl-II. As shown in t-PA·PAI-1 complex binding to LRP is inhibited by RAP at an of 0.3 an higher of RAP is required to inhibit t-PA·PAI-1 complex binding to Cl-II (IC50 The inhibition is to be the of hindrance of RAP for t-PA·PAI-1 complex binding as a of the of the binding is by the reverse shown in high concentrations are required to for RAP binding to Cl-II and to LRP. is the in the RAP inhibition of Fab A8 binding to LRP and Cl-II Fab A8 binding to LRP is prevented by RAP at an of RAP inhibition of Fab A8 binding to Cl-II was only and required high RAP concentrations inhibition at the reverse competition experiment, in a of inhibition of RAP binding to LRP Cl-II by Fab A8. these we that RAP is to bind to Cl-II, it is to its ligand To ligand binding by Cl-II of LRP, we have generated a of Cl-II that of soluble Cl-II by baby hamster kidney cell with Ca2+-dependent ligand binding Using these fragments we revealed the presence of an of three binding sites for and PAI-1, for RAP, and for the pseudo-ligand Fab A8. In for the ligand interactions with the fragments are higher than those for the LRP may be by the that for the of the cluster II the ligands were in to the of ligand binding to LRP, the receptor was to the to the Cl-II fragments to the to the of the that is with the The of t-PA binding to the fragments and the that t-PA·PAI-1 complexes and PAI-1 bind to the domain previous that the PAI-1 of the complex is the for binding to Cl-II of LRP. M. M. van den S. K. H. and A. J. van for binding of t-PA to Cl-II could be at we that a t-PA binding site outside Cl-II is for the binding to LRP G. Williams S.E. Strickland D.K. Schwartz A.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7427-7431Google K. Willnow T.E. Herz J. Gething M.J. Sambrook J. J. Biol. Chem. 1994; 269: Scholar, A. L. Gliemann J. Andreasen P.A. Acad. Sci. 1994; Scholar). The of the RAP binding site in Cl-II, in between a partially overlapping t-PA·PAI-1 complex binding site and an independent binding site for Fab A8, the recombinant Cl-II an to study the mechanism of inhibition of ligand binding by even though the binding of RAP to Cl-II and LRP are similar, RAP is a inhibitor of ligand binding to the Cl-II with its inhibition of ligand binding to the it that RAP ligand binding by a mechanism that binding to overlapping binding Our are with a model of ligand inhibition by RAP that assumes RAP-induced allosteric in the receptor, as by Willnow et al. (18Willnow T.E. Goldstein J.L. Orth K. Brown M.S. Herz J. J. Biol. Chem. 1992; 267: 26172-26180Google Scholar). including this the presence of at least two RAP binding sites on LRP (12Willnow T.E. Orth K. Herz J. J. Biol. Chem. 1994; 269: 15827-15832Google Scholar, S.P. Bu G. Maksymovitch E.A. Schwartz A.L. Biochem. J. 1993; 296: 867-875Google Scholar, G. S. J. Biol. Chem. 1996; Scholar, S.E. Argraves W.S. Strickland D.K. J. Biol. Chem. 1992; 267: Scholar, S.K. H. Proc. Natl. Acad. Sci. U. S. A. 1996; Scholar). One site is in Cl-II, the second in the cluster IV A low affinity site is on cluster III, as by Bu and G. S. J. Biol. Chem. 1996; Scholar). the RAP molecule may be for LRP binding on the of LRP binding domains (14Bu G. Geuze H.J. Strous G.J. Schwartz A.L. EMBO J. 1995; 14: 2269-2280Scopus (268) Google Scholar). least two of these at the and carboxyl-terminal have been shown to bind to LRP with high affinity I. Bu G. Schwartz A.L. J. Biol. Chem. 1994; 269: Scholar). The presence of receptor binding sites may a RAP molecule with the to with RAP binding sites on LRP, a conformational change in the receptor that ligand Cl-II may the to the proposed allosteric model for RAP inhibition of ligand The observed of RAP and t-PA·PAI-1 complexes for binding to Cl-II may be by steric with the that sites are partially In a study by et al., the of Cl-II in the was demonstrated A. H. K. A. Proc. Natl. Acad. Sci. U. S. A. 1996; Scholar). contains a involved in the of a highly that may be well for Our on ligand binding to Cl-II of LRP this the may serve as a for ligand binding to other members of the low density lipoprotein receptor gene family as it was demonstrated that RAP as a of chaperone, premature ligand which may in of the receptor in the endoplasmic pathway (14Bu G. Geuze H.J. Strous G.J. Schwartz A.L. EMBO J. 1995; 14: 2269-2280Scopus (268) Google Scholar, 15Willnow T.E. Rohlmann A. Horton J. Otani H. Braun J.R. Hammer R.E. Herz J. EMBO J. 1996; 15: 2632-2639Google Scholar, G. S. J. Biol. Chem. 1996; Scholar). The model may to the of RAP as