A minimal binding domain of the low density lipoprotein receptor family.
As more relatives of the low density lipoprotein receptor (LDLR) are discovered, defining their minimal binding domain(s) becomes a challenge. Here we have chosen the multifunctional chicken oocyte receptor for yolk deposition (termed LR8), and the pan-receptor ligand, receptor associated protein (RAP), as model systems to characterize a minireceptor 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 minireceptor. 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 latter sequence is reminiscent of that in the fifth repeat of the human LDLR, thought to be most critical for interaction with positive charge clusters in ligands. Baculovirus-mediated expression of the soluble minireceptor in insect cells showed it to fold as a monomer, and sulfhydryl-reduction-sensitive interaction with RAP was demonstrated for immobilized as well as soluble minireceptor. Furthermore, the LR8-derived minireceptor provided a RAP-responsive surface when covalently coupled to the surface of a gold electrode. In addition to its use in defining minimal binding domains, the phage display approach provides powerful tools for dissection, and consequently, manipulation, of the function of receptors so as to direct their binding activity toward ligands of diagnostic and/or therapeutic interest.
Bajari
T M
TM
Department of Molecular Genetics, University of Heidelberg, Germany.
Lindstedt
K A
KA
Riepl
M
M
Mirsky
V M
VM
Nimpf
J
J
Wolfbeis
O S
OS
Dresel
H A
HA
Bautz
E K
EK
Schneider
W J
WJ
D000595:Amino Acid Sequence; D000818:Animals; D030161:Avian Proteins; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D006801:Humans; D007313:Insecta; D008024:Ligands; D008074:Lipoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011973:Receptors, LDL / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D030161:Avian Proteins; D017931:DNA Primers; C116062:LR8 protein, chicken; D008024:Ligands; D008074:Lipoproteins; D011973:Receptors, LDL; D011994:Recombinant Proteins