title: Dissection of the domain architecture of the alpha2macroglobulin-receptor-associated protein.
abstract: The alpha2macroglobulin-receptor-associated protein (RAP) binds to the alpha2macroglobulin receptor/low-density lipoprotein receptor-related protein (alpha2MR/LRP), a multi-functional cell surface receptor known to bind and internalize several macromolecular ligands. RAP has been shown to inhibit binding of all known alpha2MR/LRP ligands. Mutational studies have implicated distinct parts of RAP as specifically involved in inhibition of binding of a multitude of ligands. In the present paper we provide experimental evidence allowing assignment of elements of triplicate internal sequence similarity in RAP, noted previously [Warshawsky, I., Bu, G. & Schwartz, A. L. (1995) Sites within the 39-kDa protein important for regulating ligand binding to the low-density lipoprotein receptor-related protein, Biochemistry 34, 3404-3415], to three structural domains, 1, 2 and 3, comprising residues 18-112, 113-218 and 219-323 of RAP, respectively. Structural analysis by 1H-NMR spectroscopy shows that domains 1 and 2 as separate domains have similar secondary structures, consisting almost exclusively of alpha-helices, whereas domain 3 as a separate domain appears only to be marginally stable. Ligand competition titration of recombinant RAP domains 1, 2 and 3 and double domains 1+2 and 2+3 against 125I-RAP and 125I-alpha2M* (methylamine-activated alpha2M) for binding to alpha2MR/LRP demonstrated (a) that functional integrity in single domains is largely preserved, and (b) that important determinants for the inhibition of test ligands reside in the C-terminal regions of domains 1 and 3.
authors.affiliation: Department of Molecular and Structural Biology, University of Aarhus, Denmark.
authors.forename: L
authors.initials: L
authors.lastname: Ellgaard
authors.forename: T L
authors.initials: TL
authors.lastname: Holtet
authors.forename: P R
authors.initials: PR
authors.lastname: Nielsen
authors.forename: M
authors.initials: M
authors.lastname: Etzerodt
authors.forename: J
authors.initials: J
authors.lastname: Gliemann
authors.forename: H C
authors.initials: HC
authors.lastname: Thøgersen
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D026563:LDL-Receptor Related Protein-Associated Protein; D008024:Ligands; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D017433:Protein Structure, Secondary; D011971:Receptors, Immunologic / Q000378:metabolism; D011973:Receptors, LDL / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000511:alpha-Macroglobulins / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D017931:DNA Primers; D006023:Glycoproteins; D026563:LDL-Receptor Related Protein-Associated Protein; D008024:Ligands; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D011971:Receptors, Immunologic; D011973:Receptors, LDL; D011994:Recombinant Proteins; D000511:alpha-Macroglobulins