The LDL receptor is a prototype for a large family of cell surface receptors implicated in biological processes ranging from lipoprotein uptake to Wnt signal transduction. These proteins combine several types of structural units in similar arrangements, such that groups of cysteine-rich LDL-A modules precede regions with clusters of epidermal growth factor-like (EGF) modules and b-propeller domains containing conserved YWTD motifs. Each receptor then terminates with a transmembrane segment and a cytoplasmic tail of variable length.

The VLDLR and ApoER2, which most closely resemble the LDLR in domain structure and amino acid sequence, act as receptors for Reelin, a large modular secreted protein that directs neuronal migration in cortical development. The cytoplasmic tails of these LDL receptor-related proteins bind to a PTB domain at the amino terminus of the cellular adaptor protein Disabled-1 (Dab-1) in order to communicate a Reelin signal. Our recent 1.5 crystal structure of a complex between the Dab1 phosphotyrosine binding domain and a 14-residue peptide from the ApoER2 tail explains the unusual preference of Dab1 for unphosphorylated tyrosine within the NPxY motif of the peptide (Stolt et al., 2003).

Crystals of the complex soaked with the phosphoinositide PI-4,5P2 (PI) show that PI binds to conserved basic residues on the face of the PTB domain opposite the peptide binding groove (Figure 5). This finding explains how the Dab1 PTB domain can simultaneously bind PI and the ApoER2 tail. In two other cytoplamic adaptor proteins that connect lipoprotein receptors to the endocytic machinery, the residues of the Dab-1 PTB domain that constitute the PI binding site are preserved. This conservation suggests that the Dab-1 PTB domain represents a subset of PTB domain proteins that combine a membrane recruitment site with a peptide-binding site. Recruitment of the Dab1 PTB domain to PI-rich regions of the plasma membrane may facilitate association with the Reelin receptor cytoplasmic tails to transduce a critical positional cue to migrating neurons. The next steps of this project are; (i) to determine whether binding of the two ligands at their respective sites are energetically independent, as suggested by the structure, and (ii) to test the functional role of the PIP-binding site of Dab-1 directly in neurons.

A second class of LDL receptor related proteins that contain LDL-A, EGF-like, and YWTD domains in a different arrangement include LRP-5 and LRP-6. LRP-6 from frogs binds in vitro to the developmental signaling protein Wnt, and frog LRP-6 along with its homologues in mice and flies has been implicated as a co-receptor in Wnt signaling. In humans, loss-of-function mutations of human LRP-5 (which shares 70 % identity with LRP-6) lead to the osteoporosis-pseudoglioma syndrome, whereas seven different point mutations located in the first YWTD b-propeller domain all lead to a high-bone mass trait in otherwise asymptomatic individuals.

Several lines of evidence from the literature suggest that the activity of each of these receptors (LRP5 and LRP6) is allosterically regulated. One explanation for how point mutations of LRP-5 can cause an apparent gain of function in the high bone mass patients is that Dkk-1, a known antagonist of Wnt signaling, can no longer prevent the transmission of a Wnt signal in the LRP-5 mutants, even though it appears to bind to the receptor in a site non-overlapping with the putative Wnt binding site. To understand how Dkk-1 acts as a Wnt signaling antagonist by binding to LRP-5 and LRP-6, we plan to combine molecular biology with structural studies. Once solved, the structure of an LRP-5 or -6 complex with Dkk-1 should both reveal the structural basis for Dkk binding by Wnt co-receptors, and provide insights into the mechanism of allosteric regulation.

Bibliography

Rudenko, G., Henry, L., Henderson, K., Ichtchenko, K., Brown, M. S., Goldstein, J. L., and Deisenhofer, J. (2002). Structure of the LDL receptor extracellular domain at endosomal pH. Science 298, 2353-2358.

Stolt, P. C., Jeon, H., Song, H. K., Herz, J., Eck, M. J., and Blacklow, S. C. (2003). Origins of Peptide Selectivity and Phosphoinositide Binding Revealed by Structures of Disabled-1 PTB Domain Complexes. Structure (Camb) 11, 569-579.