Structural and functional analysis of Dickkopf 4 (Dkk4): New insights into Dkk evolution and regulation of Wnt signaling by Dkk and Kremen proteins.

Dickkopf (Dkk) family proteins are important regulators of Wnt signaling pathways, which play key roles in many essential biological processes. Here, we report the first detailed structural and dynamics study of a full-length mature Dkk protein (Dkk4, residues 19-224), including determination of the first atomic-resolution structure for the N-terminal cysteine-rich domain (CRD1) conserved among Dkk proteins. We discovered that CRD1 has significant structural homology to the Dkk C-terminal cysteine-rich domain (CRD2), pointing to multiple gene duplication events during Dkk family evolution. We also show that Dkk4 consists of two independent folded domains (CRD1 and CRD2) joined by a highly flexible, nonstructured linker. Similarly, the N-terminal region preceding CRD1 and containing a highly conserved NXI(R/K) sequence motif was shown to be dynamic and highly flexible. We demonstrate that Dkk4 CRD2 mediates high-affinity binding to both the E1E2 region of low-density lipoprotein receptor-related protein 6 (LRP6 E1E2) and the Kremen1 (Krm1) extracellular domain. In contrast, the N-terminal region alone bound with only moderate affinity to LRP6 E1E2, consistent with binding via the conserved NXI(R/K) motif, but did not interact with Krm proteins. We also confirmed that Dkk and Krm family proteins function synergistically to inhibit Wnt signaling. Insights provided by our integrated structural, dynamics, interaction, and functional studies have allowed us to refine the model of synergistic regulation of Wnt signaling by Dkk proteins. Our results indicate the potential for the formation of a diverse range of ternary complexes comprising Dkk, Krm, and LRP5/6 proteins, allowing fine-tuning of Wnt-dependent signaling.

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). Analysis of the backbone chemical shift data obtained indicates that there is a very limited amount of regular secondary structure, with only small stretches of ß-strand identified in both cysteine-rich regions. The N-terminal region of hDkk4 (M1-G21) and the relatively long linker between the two cysteine-rich regions (E77-Q123) appear to be unstructured and relatively mobile.

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 ß-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. This sequence is related closely to a previously identified motif in laminin that mediates its interaction with the ß-propeller domain of nidogen. However, the NXI motif is not involved in the interaction of sclerostin with LRP4 (another ß-propeller containing protein in the LRP family). A peptide derived from the loop 2 region of sclerostin blocked the interaction of sclerostin with LRP5/6 and also inhibited Wnt1 but not Wnt3A or Wnt9B signaling. This suggests that these Wnts interact with LRP6 in different ways.

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. This shows minimal overlap with the heparin binding site and highlights a key role for this region of sclerostin in protein interactions associated with the inhibition of Wnt signaling. The conserved N- and C-terminal arms of sclerostin were found to be unstructured, highly flexible, and unaffected by heparin binding, which suggests a role in stabilizing interactions with target proteins.

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. Given that both G(16) and G(14) are capable of linking G(i)-coupled receptors to the stimulation of phospholipase Cbeta, we examined the capacity of six CC chemokine receptors (CCR1, CCR2a, CCR2b, CCR3, CCR5 and CCR7) to interact with G(14) and G(16) in a heterologous expression system. Among the CC chemokine receptors tested, CCR1, CCR2b, and CCR3 were capable of mediating chemokine-induced stimulation of phospholipase Cbeta via either G(14) or G(16). The G(14)/G(16)-mediated responses exhibited CC chemokine dose-dependency and were resistant to pertussis toxin (PTX) treatment. In contrast, CCR2a, CCR5 and CCR7 were unable to interact with G(14) and G(16). Under identical experimental conditions, all six CC chemokine receptors were fully capable of inhibiting adenylyl cyclase via G(i) as well as stimulating phospholipase Cbeta via 16z44, a G(16/z) chimera that possesses increased promiscuity toward G(i)-coupled receptors. Moreover, CCR1-mediated ERK1/2 phosphorylation was largely PTX-insensitive in THP-1 monocytic cells that endogenously express Galpha(16). In addition, CCR1 agonist was less efficacious in mediating chemotaxis of THP-1 cells following the knockdown of Galpha(16) by overexpressing siRNA, indicating the participation of Galpha(16) in CCR1-induced cell migration. These results show that different CC chemokine receptors can discriminate against G(14) and G(16) for signal transduction.

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. To determine the repertoire of Edg family receptor expression in this cell line, alignments of human and rodent sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) receptor sequences were used to design semi-redundant oligonucleotide pairs. A portion of each receptor gene coding sequence was amplified from Chinese hamster genomic DNA and the resultant gene fragments sequenced. Species-specific oligonucleotide pairs were designed using this novel sequence information and used to detect expression of S1P(1,2,4) and LPA(1) transcripts in CHO-K1 cells by RT-PCR.

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. Chemokines leukotactin-1, macrophage inflammatory protein (MIP)-1alpha, monocyte chemotactic peptide (MCP)-3, RANTES, and MIP-1delta all inhibited adenylyl cyclase activity in cells transiently transfected with CCR1. In contrast, only MIP-1delta was unable to signal via G14-, G16- or chimeric 16z44-coupled pathways. In a stable cell line expressing CCR1 and Galpha14, all of these five chemokines along with hemofiltrate CC chemokine (HCC)-1 and myeloid progenitor inhibitory factor (MPIF)-1 were able to stimulate G(i/o)-coupled pathways, but MIP-1delta, HCC-1 and MPIF-1 were unable to activate G14-mediated stimulation of phospholipase Cbeta activity. In addition, MIP-1delta was unable to promote the phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase. This suggests that different chemokines are able to selectively activate CCR1-coupled pathways, probably because of different intrinsic ligand efficacies. CCR1 and Galpha14 or Galpha16 are co-expressed in several cell types and we hypothesize that selective activation of chemokine receptors provides a mechanism by which chemokines are able to fine-tune intracellular signaling pathways.

The enzymatic activity of ADAM8 and ADAM9 is not regulated by TIMPs.

The ADAM family of proteases are type I transmembrane proteins with both metalloproteinase and disintegrin containing extracellular domains. ADAMs are implicated in the proteolytic processing of membrane-bound precursors and involved in modulating cell-cell and cell-matrix interactions. ADAM8 (MS2, CD156) has been identified in myeloid and B cells. In this report we demonstrate that soluble ADAM8 is an active metalloprotease in vitro and is able to hydrolyse myelin basic protein and a variety of peptide substrates based on the cleavage sites of membrane-bound cytokines, growth factors and receptors which are known to be processed by metalloproteinases. Interestingly, although ADAM8 was inhibited by a number of peptide analogue hydroxamate inhibitors, it was not inhibited by the tissue inhibitors of metalloproteinases (TIMPs). We also demonstrate that the activity of recombinant soluble ADAM9 (meltrin-gamma, MDC9) lacks inhibition by the TIMPs, but can be inhibited by hydroxamate inhibitors. The lack of TIMP inhibition of ADAM8 and 9 contrasts with other membrane-associated metalloproteinases characterised to date in this respect (ADAM10, 12, 17, and the membrane-type metalloproteinases) which have been implicated in protein processing at the cell surface.