Resonance assignment and secondary structure determination of full length human Dickkopf 4 (hDkk4), a secreted, disulphide-rich Wnt inhibitor protein.

A number of proteins have been shown to modulate canonical Wnt signalling at the cell surface, including members of the Dickkopf (Dkk) family (Baron and Rawadi in J Endocrinol 148:2635-2643, 2007; Cruciat and Niehrs in Cold Spring Harb Perspect Biol 5:a015081, 2013). The Dkk family includes four secreted proteins (Dkk1-4), which are characterised by two highly conserved cysteine-rich regions corresponding to C24-C73 and C128-C201 in human Dkk4 (hDkk4). Here we report essentially complete backbone and comprehensive side chain (15)N, (13)C and (1)H NMR assignments for full length mature hDkk4 (M1-L207) containing a short C-terminal hexa-histidine tag (E208-H222).

Characterization of the interaction of sclerostin with the low density lipoprotein receptor-related protein (LRP) family of Wnt co-receptors.

LRP5 and LRP6 are proteins predicted to contain four six-bladed β-propeller domains and both bind the bone-specific Wnt signaling antagonist sclerostin. Here, we report the crystal structure of the amino-terminal region of LRP6 and using NMR show that the ability of sclerostin to bind to this molecule is mediated by the central core of sclerostin and does not involve the amino- and carboxyl-terminal flexible arm regions. We show that this structured core region interacts with LRP5 and LRP6 via an NXI motif (found in the sequence PNAIG) within a flexible loop region (loop 2) within the central core region.

Characterization of the structural features and interactions of sclerostin: molecular insight into a key regulator of Wnt-mediated bone formation.

The secreted glycoprotein sclerostin has recently emerged as a key negative regulator of Wnt signaling in bone and has stimulated considerable interest as a potential target for therapeutics designed to treat conditions associated with low bone mass, such as osteoporosis. We have determined the structure of sclerostin, which resulted in the identification of a previously unknown binding site for heparin, suggestive of a functional role in localizing sclerostin to the surface of target cells. We have also mapped the interaction site for an antibody that blocks the inhibition of Wnt signaling by sclerostin.

Differential involvement of Galpha16 in CC chemokine-induced stimulation of phospholipase Cbeta, ERK, and chemotaxis.

Chemokines are known to regulate the chemotaxis of leukocytes and play an important role in immunological processes. Chemokine receptors are widely distributed in hematopoietic cells and are often co-localized with the hematopoietic-specific G(16) and its close relative, G(14). Yet, many chemokine receptors utilize pertussis toxin-sensitive G(i) proteins for signaling.

Analysis of endogenous S1P and LPA receptor expression in CHO-K1 cells.

The CHO-K1 cell line is commonly used for studies of recombinantly expressed proteins, including proteins of the G protein-coupled receptor (GPCR) family. This laboratory has used CHO-K1 cells for the functional characterization of Edg family GPCRs. However, parental CHO-K1 cells respond to lysophospholipids in in-vitro functional assays, which suggests expression of endogenous Edg family GPCRs.

Differential chemokine activation of CC chemokine receptor 1-regulated pathways: ligand selective activation of Galpha 14-coupled pathways.

Chemokines regulate the chemotaxis, development, and differentiation of many cell types enabling the regulation of routine immunosurveillance and immunological adaptation. CC chemokine receptor 1 (CCR1) is the target of 11 chemokines. This promiscuity of receptor-ligand interactions and the potential for functional redundancy has led us to investigate the selective activation of CCR1-coupled pathways by known CCR1 agonists.