A MINIMAL BINDING DOMAIN OF THE LOW-DENSITY-LIPOPROTEIN RECEPTOR FAMILY

As more relatives of the low density lipoprotein receptor (LDLR) are d iscovered, defining their minimal binding domain(s) becomes a challeng e. Here we have chosen the multifunctional chicken oocyte receptor for yolk deposition (termed LR8), and the pan-receptor ligand, receptor a ssociated protein (RAP), as model systems to characterize a minirecept or using the phage display approach. Displayed fragments derived from the entire 819 residue LR8 molecule, followed by selection via panning on RAP, led to the definition of an 80 residue stretch LR8 minirecept or. It contains 12 cysteines, and represents parts of the second, the entire third, and parts of the fourth, of the eight clustered 'ligand binding repeats' in LR8; only two of the eight stretches of negatively charged residues of LR8, i.e., EDGSDE and DSGEDEE, are present. The l atter sequence is reminiscent of that in the fifth repeat of the human LDLR, thought to be most critical for interaction with positive charg e clusters in ligands. Baculovirus-mediated expression of the soluble minireceptor in insect cells showed it to fold as a monomer, and sulfh ydryl-reduction-sensitive interaction with RAP was demonstrated for im mobilized as well as soluble minireceptor. Furthermore, the LR8-derive d minireceptor provided a RAP-responsive surface when covalently coupl ed to the surface of a gold electrode. In addition to its use in defin ing minimal binding domains, the phage display approach provides power ful tools for dissection, and consequently, manipulation, of the funct ion of receptors so as to direct their binding activity toward ligands of diagnostic and/or therapeutic interest.