Identification of Residues in α-Macroglobulins Important for Binding to the α2-Macroglobulin Receptor/Low Density Lipoprotein Receptor-related Protein

Nielsen, Kåre Lehmann; Holtet, Thor L.; Etzerodt, Michael; Moestrup, Søren K.; Gliemann, Jørgen; Sottrup‐Jensen, Lars; Thøgersen, Henning

doi:10.1074/jbc.271.22.12909

Cited by 85 publications

(82 citation statements)

References 45 publications

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“…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 (27). 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 (28). 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.…”

Section: Discussionmentioning

confidence: 99%

“…The highest resolution studies have been done on the LRP-binding site in activated ␣ 2 -macroglobulin, which identified lysine residues 1370 and 1374 as essential for binding to the LRP (28). The LRPbinding site in thrombospondin 1 has been localized to the same amino-terminal fragment that contains the heparin-binding domain (25), and the carboxyl-terminal folding domain of lipoprotein lipase, which also contains the heparin-binding domain, has been implicated in binding to the LRP (26).…”

Section: Discussionmentioning

confidence: 99%

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Analysis of a Structural Determinant in Thrombin-Protease Nexin 1 Complexes That Mediates Clearance by the Low Density Lipoprotein Receptor-related Protein

Knauer

Crisp

Kridel

et al. 1999

Journal of Biological Chemistry

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We recently identified a synthetic peptide, Pro 47 -Ile 58 , 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 Pro 47 -Ile 58 , 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 His 48 , or two Ala substitutions, one at His 48 and another at Asp 49 , 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 serine protease 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 1 (PN1)1 is a member of the SERPIN superfamily (1, 2), and an important physiological regulator of thrombin (Th) and urinary plasminogen activator (uPA) (2). PN1 forms stoichiometric complexes with both Th and uPA (3) that ultimately results in their removal by cellular endocytosis and degradation (4). The precise biochemical nature of the complexes is still not completely clear, but they are extremely stable and possibly covalent (5-7). 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 (8, 9). 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) (10, 11), heparin cofactor-II (12), antithrombin III (ATIII) (12) and ␣-1-antitrypsin (12). 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 SERPINProtease complexes (12-15). 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 v...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Analysis of a Structural Determinant in Thrombin-Protease Nexin 1 Complexes That Mediates Clearance by the Low Density Lipoprotein Receptor-related Protein

Knauer

Crisp

Kridel

et al. 1999

Journal of Biological Chemistry

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“…Conserved basic amino acid residues within ApoE amino acids 140-150 are crucial for interaction with LDLR, and a site-directed mutagenesis study has demonstrated that Lys-143 and Lys-146 are absolutely necessary (26). Two Lys residues in a receptor-binding domain of ␣2-macroglobulin have also been shown to be involved in binding to the LA module portion of LRP (27,28). Recent structural studies have revealed the ligand-recognition mechanism of LA modules at an atomic level (12,29,30).…”

Section: Discussionmentioning

confidence: 99%

Structure of a receptor-binding fragment of reelin and mutational analysis reveal a recognition mechanism similar to endocytic receptors

Yasui

Nogi

Kitao

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Reelin, a large secreted protein implicated in the cortical development of the mammalian brain, is composed of eight tandem concatenations of ''reelin repeats'' and binds to neuronal receptors belonging to the low-density lipoprotein receptor gene family. We found that both receptor-binding and subsequent Dab1 phosphorylation occur solely in the segment spanning the fifth and sixth reelin repeats (R5-6). Monomeric fragment exhibited a suboptimal level of signaling activity and artificial oligomerization resulted in a 10-fold increase in activity, indicating the critical importance of higher-order multimerization in physiological reelin. A 2.0-Å crystal structure from the R5-6 fragment revealed not only a unique domain arrangement wherein two repeats were aligned side by side with the same orientation, but also the unexpected presence of bound Zn ions. Structure-guided alanine mutagenesis of R5-6 revealed that two Lys residues (Lys-2360 and Lys-2467) constitute a central binding site for the low-density lipoprotein receptor class A module in the receptor, indicating a strong similarity to the ligand recognition mode shared among the endocytic lipoprotein receptors.brain development ͉ x-ray crystallography

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“…In FP6d-AA, Lys residues at aa 1370 and 1374, which are critical for LRP binding (39,40), were mutated to Ala, using the QuikChange system (Stratagene). In FP6d-AR, Lys-1370 was mutated to Ala, and Lys-1374 was mutated to Arg.…”

Section: Methodsmentioning

confidence: 99%

Distinct Binding Sites in the Structure of α2-Macroglobulin Mediate the Interaction with β-Amyloid Peptide and Growth Factors

Mettenburg

Webb

Gonias

2002

Journal of Biological Chemistry

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␣ 2 -Macroglobulin (␣ 2 M) and its receptor, low density lipoprotein receptor-related protein (LRP), function together to facilitate the cellular uptake and degradation of ␤-amyloid peptide (A␤). In this study, we demonstrate that A␤ binds selectively to ␣ 2 M that has been induced to undergo conformational change by reaction with methylamine. Denatured ␣ 2 M subunits, which were immobilized on polyvinylidene difluoride membranes, bound A␤, suggesting that ␣ 2 M tertiary and quaternary structure are not necessary. To determine whether a specific sequence in ␣ 2 M is responsible for A␤ binding, we prepared and analyzed defined ␣ 2 M fragments and glutathione S-transferase-␣ 2 M peptide fusion proteins. A single sequence, centered at amino acids (aa) 1314 -1365, was identified as the only major A␤-binding site. Impor Accumulation of ␤-amyloid peptide (A␤ and A␤ ) 1 in the brain plays a central role in the development and progression of Alzheimer's disease (AD) (1). Mutations in ␤-amyloid precursor protein (APP), which result in increased production of A␤, are associated with autosomal dominant forms of familial AD in humans (2-4). Mutated forms of human APP may also induce changes consistent with AD when expressed as transgenes in mice (5-8). Furthermore, immunization with A␤ (1-42) prevents progression of AD in animal model systems and may reverse symptoms by promoting resorption of A␤-containing plaques (9, 10). These results suggest that A␤ accumulation in the brain is a dynamic and reversible process. Proteins other than antibodies with the capacity to bind A␤ and promote its catabolism may influence disease progression.␣ 2 -Macroglobulin (␣ 2 M) is a 718-kDa homotetrameric glycoprotein, which is well characterized as an extracellular proteinase inhibitor (11) and as a carrier of specific growth factors, including transforming growth factor-␤ (TGF-␤) and nerve growth factor-␤ (NGF-␤) (12, 13). At least two separate polymorphisms in the A2M gene may be associated with increased risk of late-onset AD. The first involves a region within intron 17, at the 5Ј splice acceptor site for exon 18 (14). This exon is important because it encodes part of the bait region, where proteinases initiate reaction with ␣ 2 M by cleaving susceptible peptide bonds (15,16), and a segment of the growth factor binding sequence (17)(18)(19). In the second A2M gene polymorphism, Val-1000 is replaced by Ile (20). The linkage of A2M gene polymorphisms to late-onset AD remains incompletely understood, because the original observations have been confirmed in only a limited number of populations (21-25) and because there is no molecular explanation regarding how A2M gene mutations may affect ␣ 2 M structure, function, and expression.␣ 2 M is expressed by microglia, which accumulate near amyloid plaques (26). Thus, locally synthesized ␣ 2 M may affect AD progression by regulating the activity of various proteinases or by binding important growth factors. The previously demonstrated ability of ␣ 2 M to bind and neutralize the activity of TGF-...

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Identification of Residues in α-Macroglobulins Important for Binding to the α2-Macroglobulin Receptor/Low Density Lipoprotein Receptor-related Protein

Cited by 85 publications

References 45 publications

Analysis of a Structural Determinant in Thrombin-Protease Nexin 1 Complexes That Mediates Clearance by the Low Density Lipoprotein Receptor-related Protein

Analysis of a Structural Determinant in Thrombin-Protease Nexin 1 Complexes That Mediates Clearance by the Low Density Lipoprotein Receptor-related Protein

Structure of a receptor-binding fragment of reelin and mutational analysis reveal a recognition mechanism similar to endocytic receptors

Distinct Binding Sites in the Structure of α2-Macroglobulin Mediate the Interaction with β-Amyloid Peptide and Growth Factors

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