The first propeller domain of LRP6 regulates sensitivity to DKK1.
The Wnt coreceptor LRP6 is required for canonical Wnt signaling. To understand the molecular regulation of LRP6 function, we generated a series of monoclonal antibodies against the extra cellular domain (ECD) of LRP6 and selected a high-affinity mAb (mAb135) that recognizes cell surface expression of endogenous LRP6. mAb135 enhanced Wnt dependent TCF reporter activation and antagonized DKK1 dependent inhibition of Wnt3A signaling, suggesting a role in modulation of LRP6 function. Detailed analysis of LRP6 domain mutants identified Ser 243 in the first propeller domain of LRP6 as a critical residue for mAb135 binding, implicating this domain in regulating the sensitivity of LRP6 to DKK1. In agreement with this notion, mAb135 directly disrupted the interaction of DKK1 with recombinant ECD LRP6 and a truncated form of the LRP6 ECD containing only repeats 1 and 2. Finally, we found that mAb135 completely protected LRP6 from DKK1 dependent internalization. Together, these results identify the first propeller domain as a novel regulatory domain for DKK1 binding to LRP6 and show that mAb against the first propeller domain of LRP6 can be used to modulate this interaction.
Binnerts
Minke E
ME
Arca Biopharma, San Carlos, CA 94070-6211, USA. minkebinnerts@mindspring.com
Tomasevic
Nenad
N
Bright
Jessica M
JM
Leung
John
J
Ahn
Victoria E
VE
Kim
Kyung-Ah
KA
Zhan
Xiaoming
X
Liu
Shouchun
S
Yonkovich
Shirlee
S
Williams
Jason
J
Zhou
Mei
M
Gros
Delphine
D
Dixon
Melissa
M
Korver
Wouter
W
Weis
William I
WI
Abo
Arie
A
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D015153:Blotting, Western; D002460:Cell Line; D004705:Endocytosis / Q000187:drug effects; D005434:Flow Cytometry; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D026502:LDL-Receptor Related Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D060488:Low Density Lipoprotein Receptor-Related Protein-6; D009154:Mutation; D011485:Protein Binding / Q000187:drug effects; D034622:RNA Interference; D015398:Signal Transduction / Q000187:drug effects; D051153:Wnt Proteins / Q000235:genetics / Q000378:metabolism; D060508:Wnt3 Protein; D060509:Wnt3A Protein
D000911:Antibodies, Monoclonal; C501126:DKK1 protein, human; D036341:Intercellular Signaling Peptides and Proteins; D026502:LDL-Receptor Related Proteins; C555475:LRP6 protein, human; D060488:Low Density Lipoprotein Receptor-Related Protein-6; C555510:WNT3A protein, human; D051153:Wnt Proteins; D060508:Wnt3 Protein; D060509:Wnt3A Protein