Dissection of the domain architecture of the α2macroglobulin-receptor-associated protein.

The α₂macroglobulin-receptor-associated protein (RAP) binds to the α₂macroglobulin receptor/low-density lipoprotein receptor-related protein (α₂MR/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 α₂MR/LRP ligands. Mutational studies have implicated distinct parts of RAP as specifically in- volved 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, L., Bu, C. & 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 α-helices, whereas domain 3 as a separate domain appears only to he 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-α₂M* (methylamine-activated α₂2M) for binding to α₂MR/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. [ABSTRACT FROM AUTHOR]