WNT RECEPTORS AND PATHWAYS

Contents

• Membrane-bount Wnt Receptors
• Extracellular Wnt-Pathway Mediators and Receptors
• References

Wnt receptors and signaling pathways may constitute the most complex relationship between extracellular ligands and receptors identified to date. This mini-review focuses on the structures and relationships of the Wnt receptors, both with themselves, and with the Wnt ligands. Although there is considerable interest in intracellular, downstream Wnt signaling mechanisms, this review will focus on membrane-bound molecules and soluble mediators. Special emphasis will be placed on the mammalian system; however, Xenopus and Drosophila have fundamentally shaped our knowledge of Wnts. Thus, when necessary, references will be made to both non-mammalian vertebrate and invertebrate systems.

The Wnt ligands are a family of 19 molecules that are secreted, vary in length between 350 and 400 amino acids (aa), possess 22 to 24 conserved cysteines, and show 20%-85% aa identity within the family. The name Wnt is derived from Drosophila and mouse Wnt-1 orthologs. Attempts have been made to categorize Wnts as being "canonical" or "non-canonical" and transforming or non-transforming, depending upon the downstream signaling pathways utilized. Although initially useful, it would appear that such characterizations are more likely a function of the receptors used than any intrinsic activity of the Wnt proteins.^1-8

Figure 1. Wnt Canonical Pathway. See text for details.

Figure 2. Wnt may act through two canonical pathways. A primary event may be the engagement and activation/phosphorylation of LRP. This creates an axin docking site that sequesters and removes axin from the beta-Catenin regulating complex.

The complexity of Wnt intracellular signaling pathways parallels the complexity observed in the diversity of Wnt receptors. In general, there are three signaling pathways associated with Wnt-receptor interaction. The first is commonly called the canonical pathway (Figure 1). The heart of this pathway centers on beta-Catenin.⁹ beta-Catenin is the target molecule of a cytosolic complex that is designed to specifically regulate the activity of beta-Catenin. beta-Catenin, in an active form, is a transcriptional activator for the TCF (T cell factor)/LEF-1 (lymphoid enhancer factor 1) family of DNA binding proteins. Examples of TCF-responsive genes include c-myc and cyclin D1. The pentameric regulatory complex is composed of beta-Catenin, axin (axis inhibition),¹⁰ CK-1, APC (adenomatous polyposis coli protein), and GSK-3 beta (glycogen synthase kinase 3 beta). In the absence of Wnt, GSK-3 beta constitutively phosphorylates beta-Catenin, targeting it for degradation. APC acts in this complex by directing phosphorylated beta-Catenin into a ubiquitination-mediated proteosomal degradation pathway. In the presence of Wnt, GSK-3 beta is inactivated, leaving an unphosphorylated, but active, beta-Catenin. In addition to beta-Catenin, axin also seems to be a target of GSK-3 beta. In the absence of Wnt stimulation, GSK-3 beta constitutively phosphorylates axin. In this phosphorylated state, axin contributes to the phosphorylation of beta-Catenin by GSK-3 beta. When Wnts induce GSK-3 beta inactivation, axin is no longer phosphorylated by GSK-3 beta and becomes unstable in beta-Catenin accumulation and gene activation.^11-14

Traditionally, the proximal signaling molecule in the canonical pathway is Dishevelled (Dsh).¹⁵ Dsh has been pictured to lie between membrane-bound receptors and the beta-Catenin complex. Although human and mouse have three Dsh homologs, Drosophila Dsh is the best studied. Drosophila Dsh is a three-domain, 623 aa protein that is activated by Wnt binding to its seven-transmembrane (TM) receptor Frizzled. Following Wnt binding, PAR-1 or casein kinase II is assumed to phosphorylate Dsh on its N-terminus. At this point, it becomes associated with the beta-Catenin complex and blocks GSK-3 beta activity. This results in beta-Catenin accumulation/stabilization and subsequent gene activation.¹⁶ Although intuitively appealing, the actual role of Dsh in beta-Catenin stabilization is now uncertain. For example, while both canonical Wnt-1 and non-canonical Wnt-5a induce phosphorylation of Dsh, the action of Wnt-5a is not associated with beta-Catenin stabilization. In short, Dsh phosphorylation may be a general phenomenon of Wnt activation, with the "true" canonical signal transmitted through the LRP (Figure 2).¹⁷

Two other pathways are associated with Wnt signaling. Both are poorly defined in vertebrate systems. The first is the Wnt/Ca²⁺ pathway.¹⁸ In this pathway, Wnt binding to a seven-TM Frizzled receptor results in the activation of heterotrimeric G-proteins with subsequent mobilization of phospholipase C and phosphodiesterase. This results in a decrease in cGMP, an increase in intracellular Ca²⁺, and activation of protein kinase C. The exact genes or metabolic pathways activated are unknown. But NF-AT seems to be involved. It is also suggested that the Wnt/Ca²⁺ pathway intersects with the beta-Catenin pathway at multiple points.^18-20

The second non-canonical pathway is the planar cell polarity (PCP) pathway. Activation of this pathway defines polarity in select epithelial tissues, particularly along an axis perpendicular to the apical-basal border.²¹ In Drosophila, it is responsible for the orientation of cuticle hairs on wings.^21-23 In vertebrates, it may contribute to the differentiation and orientation of inner ear hair cell stereocilia,²¹ and direct the expansion of mesoderm and neuroectoderm during gastrulation.¹⁷ The PCP pathway is not well understood. In short, Wnt binding to Frizzled activates Dsh, likely on the C-terminus of the molecule.¹⁵ Dsh then recruits RhoA/Rac, which ultimately leads to JNK (c-jun NH₂-terminal kinase) pathway activation. A major target of the JNK pathway is the AP-1 (activator protein-1) transcription factor.²³

Membrane-bound Wnt Receptors

The molecules involved in Wnt signaling are both soluble and membrane-bound. In general, they do not show any structural similarity. Membrane-bound molecule families will be discussed first, followed by soluble mediators. Priority will be given to structure and expression, with function discussed within the framework provided above.

Figure 3. Phylogenetic relationships between Frizzled receptors.

Frizzled Receptors

To date, there are 10 human Frizzled receptors.²⁴ Frizzled receptors vary in length from 537 - 706 aa. All are seven-TM receptors with an extracellular N-terminus and an intracellular C-terminus. The Frizzled N-terminus is characterized by the presence of an alpha-helical, approximately 130 aa, cysteine-rich domain (CRD) that interacts with both Wnts and other Wnt receptors.^25,26 Notably, this CRD pattern is not unique to Frizzled and Frizzled-type receptors. It is also found in tyrosine kinase receptors such as MuSK and ROR2.²⁶ The C-terminus has a membrane-proximal Lys-Thr-x-x-x-Trp motif that is associated with Dsh binding and activation.^27,28 Phylogenetically, the Frizzled receptors fall into four groups. Frizzled-1, 2 and 7, and Frizzled-3 and 6 make up two related groups, while Frizzled-5 and 8 comprise a third group, and Frizzled-4, 9 and 10 generate a distant fourth group (Figure 3).²⁹

Frizzled-1

Human Frizzled-1 is 648 aa in length. It contains a 251 aa extracellular N-terminus, and a 25 aa intracellular C-terminus that contains a 3 aa (Thr-x-Val) PDZ-binding site and a characteristic Lys-Thr-x-x-x-Trp motif. The N-terminus contains two potential N-linked glycosylation sites and five conserved disulfide bonds.^30,31 Frizzled-1 in rodent has also been cloned. In rat, it is 641 aa,³² while in mouse it is 642 aa in length.³³ Notably, in rat TM region five, there is a leucine zipper that may contribute to Frizzled-1 dimer formation. In the extracellular N-terminus, there is 95% aa identity, human to mouse and rat. In the short C-terminus, there is absolute aa identity among all three species. Finally, Xenopus Frizzled-1 has been cloned. It is 559 aa in length and contains a short 204 aa N-terminus.³⁴ Its 25 aa C-terminus is identical to human, while its N-terminus displays only 82% aa identity. Cells and tissues known to express Frizzled-1 include chondrocytes,³³ presomitic, somitic and intermediate mesoderm,^35,36 cells of the developing optic lens and otic placode,³⁵ and vascular smooth muscle, endothelial cells, and fibroblasts following activation.³⁷ Wnts reported to activate Frizzled-1 include Wnt-1,³⁶ Wnt-8,³⁸ Wnt-3a, Wnt-3, and Wnt-2.³¹ Wnt-5a apparently interacts with Frizzled-1 but does not signal.³¹

Frizzled-2

Human Frizzled-2 is a 565 aa, seven-TM receptor. It contains a 224 aa N-terminus with one 120 aa CRD, and two potential N-linked glycosylation sites. Its C-terminus is 25 aa in length and contains a Thr-Thr-Val PDZ-binding motif plus a Lys-Thr-x-x-x-Trp Dsh-association motif.^30,39 Mouse and rat Frizzled-2 have been identified.^32,40 They are 571 and 570 aa in length, respectively. The 224 aa N-terminus in mouse shows complete aa identity to human, while the same segment in rat demonstrates 98% aa identity to human. The rodent C-termini are identical to human. There is a polyglycine sequence in the N-terminus in human, mouse and rat that is recapitulated in Drosophila but not Xenopus.^41,42 The significance of this is unknown. Frizzled-2 is found in activated fibroblasts, endothelial cells and vascular smooth muscle cells,³⁷ thyroid epithelium,³⁴ myofibroblasts,⁴³ and pharyngeal mesenchyme.⁴³ The only reported Wnt to interact with Frizzled-2 is Wnt-5a.^44,45

Frizzled-3

Human Frizzled-3 is a 666 aa, seven-TM, G protein-coupled receptor that contains a 17 aa signal sequence, a 188 aa N-terminal region, and a lengthy 168 aa C-terminal tail.^46-48 The N-terminal region contains one potential N-linked glycosylation site with a typical CRD domain. The C-terminus demonstrates the standard Frizzled Lys-Thr-x-x-x-Trp motif. It does not, however, have a Thr-Thr-Val tripeptide. Interestingly, an alternate splice variant has been identified that shows a premature truncation of the protein at the junction of the second TM segment and the second extracellular loop.⁴⁷ This is predicted to create a non-signaling, membrane-bound antagonist of Wnt signaling. Mouse^48,49 and rat (GenBank Accession # AAN14442) Frizzled-3 have also been cloned and found to be virtually identical to human Frizzled-3. At the N-terminus, mouse and rat possess 99% aa identity relative to human; at the C-terminus, mouse and rat are 99% and 98% identical to human Frizzled-3, respectively. Cells known to express Frizzled-3 include keratinocytes,⁴⁸ neurons in the dentate gyrus,⁵⁰ mitral and glomerular cells of the olfactory bulb,⁵⁰ and somite cells at the junction of the dermatome and myotome.³⁵ Wnt-1 and Wnt-8 are reported to bind to Frizzled-3.^8,51

Frizzled-3 is somewhat unusual in that it will activate the beta-Catenin pathway without ligand participation.²⁷ This may be attributable to the fact that the receptor will form a homodimer, either through disulfide bonding or CRD interaction.⁵² It is possible that a dimerized state is used in beta-Catenin signaling, while a monomeric state is conducive to PCP pathway activation.⁵¹

Frizzled-4

Human Frizzled-4 is 537 aa in length, with a 36 aa signal sequence, a 186 aa N-terminus, and a 38 aa cytoplasmic tail.^53,54 The N-terminus contains the expected CRD, while the tail exhibits a Thr-x-Val PDZ-binding motif and Lys-Thr-x-x-x-Trp Dsh association site. There is also a soluble variant found in kidney that is 86 aa in size. It contains three of the 13 cysteines that normally contribute to a full-sized Frizzled-4 CRD. The first 59 aa are consonant with the full-length receptor, while the C-terminal 27 aa are unique to the splice event. In Xenopus bioassays using XWnt-8, this soluble Frizzled-4 variant potentiates Wnt-8 activity, suggesting this molecule serves as a positive regulator of Wnt signaling.⁵⁴ Similar to Frizzled-3, Frizzled-4 forms homodimers. Indeed, it has been suggested that all Frizzled receptors are capable of forming homo-oligomers that appear at the cell surface, and that these may form independently of Wnts.⁵⁵ Mouse⁴⁹ and rat (Genbank Accession # Q9QZH0) Frizzled-4 have been cloned and are equal in size to human. The mouse N-terminus shows 97% aa identity to human, while the rat N-terminus displays 98% aa identity to human. At the C-terminus, both mouse and rat are 94% identical to human at the aa level, differing by only two aa. Cells known to express Frizzled-4 include cells lining the telencephalic vesicles³⁵ and skeletal muscle cells.⁵³ Wnts believed to bind to Frizzled-4 include Xenopus Wnt-1,⁴² Wnt-8,^8,56 and possibly, Wnt-5a.²⁷

Frizzled-5

Frizzled-5 in human is 585 aa in size. There is a 26 aa signal sequence, a 212 aa N-terminus, and a 64 aa C-terminus. The N-terminus has two potential N-linked glycosylation sites and a 123 aa CRD. The C-terminus contains the typical Lys-Thr-x-x-x-Trp and Thr-Thr-Val motifs.^30,49,57 In the fifth TM segment there is a 9 aa leucine zipper. While rat Frizzled-5⁵⁸ is equal in length to human Frizzled-5, mouse Frizzled-5 is 8 aa shorter at 577 aa.^49,59 Unlike Frizzled-1 through 4, there is modest divergence in aa sequence between rodent and human. At the N-terminus, there is 92% and 95% aa identity, human to mouse and rat, respectively. At the C-terminus, mouse Frizzled-5 shows only 42% aa identity to human, while rat shows 92% aa identity to human. Cells reported to express Frizzled-5 include intestinal columnar epithelium and goblet cells, and trophoblasts of the placenta. Frizzled-5 knockout mice die because of a lack of blood vessel formation.⁵⁹ Thus, it might be expected to appear in vascular-associated cells. Wnts known to act through Frizzled-5 include Wnt-7a,⁵⁸ Wnt-5a,^8,59 Wnt-10b, Wnt-2,⁵⁹ Wnt-8, and Wnt-11.⁸

Frizzled-6

Human Frizzled-6 is 706 aa in length.⁶⁰ It contains a 183 aa extracellular N-terminus, and a 212 aa intracellular C-terminus. The C-terminus is lacking a Thr-x-Val PDZ-binding site but does possess a membrane-proximal Lys-Thr-x-x-x-Trp motif. The length of the C-terminus is interesting in light of the fact that Frizzled-6 is an inhibitor of beta-Catenin signaling. Frizzled-6 signaling activates NEMO-like kinase (NLK). NLK, in turn, phosphorylates TCF transcription factors and inhibits their DNA-binding activity.⁶¹ The fifth TM segment demonstrates an 8 aa leucine zipper. Mouse Frizzled-6 has also been reported.⁴⁹ It is 709 aa long and differs significantly from human. Overall, there is 83% aa identity. Within the N-terminus there is 85% aa identity, and within the C-terminus, there is 71% aa identity. Canine Frizzled-6 has also been cloned (Genbank Accession # AAL12245). At its N-terminus, there is 93% aa identity to human Frizzled-6, while at its C-terminus there is 87% aa identity. Frizzled-6 is found in thyroid epithelium,⁶² on cells of the first branchial arch, the metanephric duct, notochord, and in ectoderm overlying somites.³⁵ Not a great deal of information exists regarding Frizzled-6 ligands. Notably, Frizzled-6 may be a primary target of the Wnt-modulating, secreted Frizzled-related proteins (sFRPs), adding more complexity to the Frizzled system.⁷

Frizzled-7

Human Frizzled-7 is a 574 aa precursor with a 32 aa signal sequence, a 224 aa extracellular N-terminus, seven TM segments, and a 25 aa C-terminus that contains a Lys-Thr-x-x-x-Trp motif with a Thr-x-Val PDZ-binding tripeptide.^30,63 Mouse Frizzled-7 is of approximately equal length (572 aa) and shows striking orthology to human. At the N-terminus, it is 97% identical, and at the C-terminus it is 100% identical at the aa level.^44,49 Frizzled-7 is described as being a monomer.⁵² It is also potentially able to heterodimerize with other Frizzled receptors.⁵⁵ The molecule is reportedly found on neurons,⁵⁰ limb bud mesenchyme,³⁵ and on the lateral aspect of developing somites.³⁶ Wnts reported to bind to Frizzled-7 include Wnt-5a,²⁷ Wnt-8,⁸ and Wnt-11.⁶⁴

Frizzled-8

Human Frizzled-8 is quite long at 694 aa in length.⁶⁵ It possesses a 27 aa signal sequence, a 248 aa extracellular N-terminus, and an 89 aa C-terminus. The N-terminus is somewhat unusual in that there are two potential N-linked glycosylation sites plus a polyproline segment and a polyglycine segment. The usual CRD also appears with ten cysteines embedded in a 120 aa segment. The C-terminus has a Thr-x-Val tripeptide, a Lys-Thr-x-x-x-Trp motif, and a polyglycine repeat of 25 aa that is essentially iterated in mouse Frizzled-8⁴⁹ but not Xenopus Frizzled-8.⁶⁶ Human to mouse, the N-terminus is 97% identical, while the C-terminus is 92% identical at the aa level. The Frizzled-8 CRDs are reported to form homodimers.²⁵ Frizzled-8 is reportedly found in early somites and later myotomes.³⁵ Wnt-8 is believed to be a ligand for Frizzled-8.⁵⁶

Frizzled-9

Frizzled-9 is a 569 aa, G protein-coupled receptor that contains a 207 aa N-terminus, seven TM segments, and a 62 aa C-terminus. The N-terminus has two potential glycosylation sites and a 122 aa CRD. The C-terminus has a Lys-Thr-x-x-x-Trp motif, but no Thr-x-Val tripeptide.^67,68 The fifth TM segment contains a leucine zipper. Both rat⁶⁹ and mouse⁷⁰ Frizzled-9 have been cloned. Each are 95% identical to human at the N-terminus and 95% identical at the C-terminus. Frizzled-9 is found on neuroepithelial precursor cells,⁶⁹ skeletal muscle,⁷⁰ all spermatogenic cell types in the adult,⁷⁰ and myotomes.^35,70 Wnt-2 is reportedly a ligand for Frizzled-9.⁶⁹

Frizzled-10

Human Frizzled-10 is expressed as a 581 aa precursor with a 20 aa signal sequence, a 205 aa N-terminus, and a 58 aa C-terminus.^71,72 The N-terminus contains a CRD and two potential N-linked glycosylation sites. The C-terminus contains both a Lys-Thr-x-x-x-Trp motif and a terminal Thr-x-Val tripeptide. There is a short leucine zipper in the fifth TM segment. Mouse Frizzled-10 shows 94% and 83% aa identity to the human Frizzled-10 N-terminus and C-terminus, respectively.⁷³ Notably, mouse Frizzled-10 does not contain a Thr-x-Val terminal tripeptide. It may be that this motif simply serves as a binding motif for scaffold proteins such as PSD-95 and ZO-1.⁷¹ Even the significance of the Lys-Thr-x-x-x-Trp motif has been questioned. Although highly conserved, it may only be needed to maintain receptor conformation.⁶⁹ Frizzled-10 is found in Müllerian duct and limb bud ectoderm and mesenchyme.⁷³ Xenopus Wnt-8 binds to Frizzled-10.⁷²

LRPs (Low Density Lipoprotein Receptor-related Proteins)

The two principal Wnt-related receptors in this group belong to the low density lipoprotein receptor (LDLR) gene family. In mammals, there are at least 10 members, five of which bind ApoE (Apolipoprotein E; LDL-R, VLDL-R, LRP-8, LRP-1, and LRP-2), and five that do not (LRP-4, LRP-5, LRP-6, LRP-3, and LR11).^74,75 ApoE is part of the protein coat of chylomicrons, chylomicron remnants, VLDL (very low density lipoprotein) particles, and IDL (intermediate density lipoprotein) particles. Chylomicrons transport dietary triglyceride (TG) and cholesterol from the gut to fat and muscle. These non-polar constituents lie in the center of the chylomicron complex, covered by polar phospholipids and (apo)proteins, such as ApoE. Upon delivery to fat/muscle, the chylomicrons are immobilized on endothelium, and lipoprotein lipase acts on the TGs to release free fatty acids. These freely diffuse into tissue. The remaining constituents of the complex (cholesterol, ApoE, etc.) make up chylomicron remnants and travel to the liver where ApoE receptors (ApoE-R) bind them and remove the cholesterol. If a diet contains high carbohydrate, this excess sugar is converted into fatty acid in the liver. This fatty acid is attached to glycerol to form TG, and the TG is released into the blood. Instead of being transported by chylomicrons, it is packaged as VLDL, and again travels to fat/muscle where lipoprotein lipase breaks down the newly formed TG into fatty acids. The fatty acids again enter the tissues, and the TG-depleted VLDL again starts to circulate. At this point, the ApoE:B-100:TG-poor complex is called IDL and either binds to LDL receptors on liver membranes (via ApoE), or is "transformed" in the blood into true LDL which contains only cholesterol and ApoB-100.

The two LRP-family, Wnt-associated receptors are LRP-5 and LRP-6. They are the orthologs of the Drosophila receptor Arrow. Although they are often described as being only structural relatives of the LDL receptor, there is evidence that they, too, may play a role in lipid metabolism. First, LRP-5 is reported to bind ApoE.^76,77 Second, LRP-5 is also reported to be essential for normal cholesterol and glucose metabolism. LRP-5^-/- mice develop increased plasma cholesterol due to decreased hepatic clearance of remnant chylomicrons.⁷⁸ In addition, low ApoE levels induce LRP-5 to participate in the clearance of dietary TGs. This effect may be indirect, however. Dickkopf-1 (Dkk-1) is a soluble modulator of Wnt-LRP activity. Structurally, Dkk-1 has two characteristic CRDs, and it is known to bind to LRP-5. One of the CRDs is reminiscent of colipase, which binds liver lipase and/or lipoprotein lipase. This binding results in lipoprotein lipase activation and subsequent lipid hydrolysis. Thus, it is possible that a LRP-5 complexed to Dkk-1 may contribute to lipase activity and TG hydrolysis.^78,79

As mediators of Wnt activity, LRP-5 and LRP-6 are presumed to function as coreceptors for Wnt signaling.⁸⁰ Select Frizzled receptors, as well as LRP-5 and 6, are associated with signaling through the beta-Catenin pathway. beta-Catenin accumulation/activation/stabilization may be accomplished either via axin or Dsh.⁸¹ Activation of Frizzled results in Dsh activation, while activation of LRP results in axin destabilization.^81,82 Both events impact the status of beta-Catenin. On the cell surface, LRP-5 and 6 are normally inactive and exist as heterodimers, homodimers, or oligomers via interactions between their extracellular EGF/Tyr-Trp-Thr-Asp (YWTD) repeats.⁸³ When Frizzled and LRP are brought together, Frizzled appears to separate the "associating" intracellular domains of multimer LRP, resulting in the exposure of a cytoplasmic [Pro]-Pro-Pro-Ser-Pro (PPSP) signaling motif.^82-84 The interaction of LRP and Frizzled is complex and is not fully understood. Wnt, in this complex, perhaps serves as a bridge for Frizzled and LRP or induces some type of conformational change in LRP.^80,83 In any event, an exposed LRP cytoplasmic PPSP site is phosphorylated, creating a docking site for axin that leads to axin destabilization.

LRP-5

Human LRP-5 is a 175-180 kDa, type I, single-pass TM glycoprotein that is synthesized as a 1615 aa precursor.^76,85,86 The precursor contains a 24 aa signal sequence, a 1361 aa extracellular region, a 23 aa TM domain and a 207 aa cytoplasmic tail. The extracellular region is complex. There are four, 40-50 aa EGF-repeats. Each EGF repeat contains six cysteines and an Arg-Gly-Gly-(Cys) motif at its N-terminus. An approximately 250 aa "spacer" separates each EGF repeat, and in each of these spacers lies a propeller-like structure composed of six blades. Five of the propeller blades contain an unusual and variable Tyr/Phe-Trp-Ile/Gly/Thr-Asp/Cys/Asn (YWTD) tetrapeptide in its structure.^86,87 Distal to the EGF region lie three LDL-R repeats.^75,86 A typical LDL-R repeat is approximately 40 aa long. In LRP-5, the three LDL-R repeats are 35-50 aa in length. There are six cysteines with a Ser-Asp-Glu tripeptide at the C-terminus of each repeat. It is thought that EGF repeats direct ligand dissociation, while LDL-R repeats are necessary for proper ligand binding. In general, the number of LDL-R repeats correlates with the number of different ligands that can be bound by a receptor.⁸⁶ Although LRP-5 is a member of the LDL-R gene family, its EGF and LDL-R modules appear in reverse order relative to those on the receptor that normally binds LDL.

The LRP-5 molecules for mouse and Xenopus have also been cloned. Mouse LRP-5 shows 94% overall aa identity to human LRP-5, with 95% identity in the extracellular region.^79,86 Xenopus LRP-5 shows 80% overall aa identity to human, with 82% aa identity in the extracellular region.⁸⁸ LRP-5 has been found in osteoblasts, beta-cells, macrophages, neurons, retinal pigment epithelium, proximal and distal renal epithelium, hepatocytes, Ito cells, and Kupffer cells.⁸⁵

LRP-6

Human LRP-6 is synthesized as a 1613 aa precursor that contains a 19 aa signal sequence, a 1353 aa extracellular domain, a 23 aa TM segment, and a 218 aa cytoplasmic tail.⁸⁹ As with LRP-5, there are four EGF repeats with intervening propeller structures that are followed by three LDL-R repeats. Studies reveal that EGF repeats 1 and 2 interact with Frizzled receptors, while repeats 3 and 4 interact with Dkk proteins.⁹⁰ The cytoplasmic region is known to contain multiple PPSP motifs, the most membrane-proximal of which is absolutely required for Wnt signaling.^82,84 As noted earlier, multimers of LRP-6 are inactive, while monomers and mutants missing the extracellular domain are constitutively active.⁸³ LRP-6 is ubiquitously expressed.⁹¹ This allows it to serve as a beta-Catenin pathway signal transducing receptor for a variety of Wnt/Frizzled pairs. Those included are Wnt-8/Frizzled-5, Wnt-11/Frizzled-5, Wnt-5a/Frizzled-5, Wnt-8/Frizzled-4, and Wnt-8/Frizzled-7.⁸

Mouse and Xenopus LRP-6 have also been cloned. Mouse LRP-6 is 98% identical to human at the aa level, both overall and within the extracellular region.⁸⁹ Xenopus LRP-6 also shows the same pattern of orthology. It is 85% identical to human LRP-6 in both the extracellular region and across the entire molecule.⁸⁸ Human LRP-5 is 72% identical at the aa level to human LRP-6 in the extracellular domain. In both LRP-5 and LRP-6, the cytoplasmic region contains multiple PPSP motifs.

LRP-1

LRP-1 is a 4500 aa member of the 600 kDa, ApoE-binding subfamily of the LDL receptor family. It not only binds ApoE and lipoprotein lipase, but also binds growth factors and matrix proteins, participates in the activation of lysosomal enzymes, and serves as a receptor for viruses and alpha₂-Macroglobulin.^92-94 Like LRP-5 and 6, LRP-1 has EGF and propeller motifs in its extracellular region. Notably, the entire extracellular region of LRP-6 represents only one of a pattern of repeats that occur repeatedly in the LRP-1 molecule. Unlike LRP-5 and 6, mature LRP-1 is the product of a Furin-processed precursor that generates a 515 kDa, extracellular (alpha-)subunit that is non-covalently linked to a 90 kDa, TM and cytoplasmic (beta-)subunit.

Although LRP-1 has documented roles in coagulation, inflammation, lipid metabolism, and viral disease, it only recently has become associated with Wnt signaling. In this context, LRP-1 is a negative regulator of Wnt activity. Full-length LRP-1 blocks Wnt-3a canonical signaling by sequestering Frizzled-1 via its CRD. Remarkably, this can even occur in a transcellular manner.⁹⁵ Thus, there appears to be an inhibitory class of LRPs that are counterparts to the activating class of LRP Wnt receptors.

Kremens

As noted with LRP-1, not all membrane-bound molecules promote Wnt signaling. Two molecules that contribute to down-modulation of LRP-5/6 activity are Kremen-1 and 2. Kremen-1 was discovered in mice during a search for kringle-containing motifs.⁹⁶ Although the cloning of Kremen-1 provided no clue as to its function, subsequent studies searching for Dkk-binding proteins revealed a structurally homologous protein, Kremen-2,⁹⁷ that interacted with Dkk to inhibit LRP-6 mediated beta-Catenin signaling.⁹⁷ The Kremens are now known to be high-affinity receptors that bind Dkk.^97,98

Kremen-1

Human Kremen-1 is synthesized as a 52 kDa, 475 aa precursor that contains a 19 aa signal sequence, a 375 aa extracellular region, a 19 aa TM segment, and a 60-aa cytoplasmic tail (GenBank Accession # Q96MU8).⁹⁹ The extracellular domain contains six potential N-linked glycosylation sites, an 84 aa kringle domain, an 82 aa WSC (cell wall integrity and stress response component) domain, and a 108 aa CUB (C1q-Uegf-BMP-1) domain. Three disulfide bonds maintain its two dimensional structure. The WSC sequence contains eight cysteines and a Ser/Thr-rich domain. This may be involved in carbohydrate binding. The CUB domain demonstrates an antiparallel beta-barrel structure that is often found in developmental proteins. There is no unique motif in the C-terminus. There is, however, an alternate splice form that deletes the final 3 aa in the cytoplasmic tail and replaces it with a 20 aa extension. Its significance is not known. Mouse⁹⁶ and rat (GenBank Accession # Q924S4) Kremen-1 have been cloned and found to be 92% identical to human Kremen-1 over the mature aa segment (mouse and rat are 95% identical).

Kremen-2

Human Kremen-2 is synthesized as a 462 aa precursor protein that contains a 48 kDa, 437 aa mature segment (GenBank Accession # Q8NCW0). There is a 339 aa extracellular region that contains a kringle segment, a WSC region, and a CUB domain, a 23 aa TM segment, and a 75 aa cytoplasmic tail. Three alternate splice forms are reported, none involving the extracellular region. Two encode truncated molecules with 54 and 33 aa substitutions beginning in the TM segment, which may represent soluble forms. The third splice form retains the TM but shows a 31 aa substitution in the cytoplasmic tail. Its significance is unknown (Genbank Accession # Q8NCW0). Human Kremen-1 and Kremen-2 show only 40% aa identity in the mature segment. In the mature region, human Kremen-2 is 89% identical to mouse Kremen-2.

The canonical beta-Catenin pathway is normally activated when Wnts interact with Frizzled (activating Dsh) and LRP (recruiting axin). The LRP component is blocked when Dkk interacts with Kremen. Together, they tether to LRP and induce a rapid endocytosis of a LRP/Dkk/Kremen complex. An intact cytoplasmic tail is necessary for Kremen action.^97,99 Thus, the alternate splice versions of Kremen may be important. In the extracellular region, deletion of any of the three specific domains abolishes Kremen binding to Dkk.⁹⁷ All Kremens and all Dkks do not interact equally. Dkk-1 prefers Kremen-2, while Dkk-2 prefers Kremen-1. Neither Kremen-1 nor 2 interact with Dkk-3. Kremen-2 is reported to interact with Dkk-4. Dkks use their C-terminal CRDs to bind to Kremens.¹⁰⁰

ROR2 (Receptor Tyrosine Kinase-like Orphan Receptor 2)

Not all membrane-bound Wnt receptors impact the beta-Catenin, or canonical, pathway. Two classes of receptors that potentially affect the PCP pathway are ROR and Strabismus (or Van Gogh-like Protein). ROR2 is the second of two orphan receptors identified during a search for receptor tyrosine kinase-like molecules in a neuroblastoma cell line, SH-SY5Y.¹⁰¹ Native human ROR2 is a 125 kDa, 910 aa glycoprotein that contains a 370 aa extracellular region, a 21 aa TM domain, and a 519 aa cytoplasmic tail.^101,102 In the extracellular region, and beginning at the N-terminus, there is a 91 aa, immunoglobulin-like, C2 segment, a 135 aa CRD, and a 79 aa kringle domain that is likely involved in protein-protein interaction. In the C-terminus, human ROR2 demonstrates a 274 aa kinase domain, two 8 aa ATP-binding sites, and one extended, potential phosphorylation site that contains a 30 aa Ser/Thr-rich area, a 74 aa Pro-rich area, and another 24 aa Ser/Thr-rich area. It also exhibits a Tyr-Ala-Leu-Met (YALM) motif that can potentially interact with Shc, Csk and p85 of PI-3 kinase.^101,103 ROR2 has tyrosine kinase activity.¹⁰¹ It also utilizes its Ser/Thr and Pro-rich areas. These areas associate with a MAGE family protein called Dlxin that regulates homeodomain proteins.¹⁰⁴ When these areas are disrupted, brachydactyly type B occurs.¹⁰⁴ Mouse and rat ROR2 have also been identified (GenBank Accession # XP225181).¹⁰³ In the extracellular region, mouse and rat ROR2 are 91% and 94% aa identical to human ROR2, respectively. Mouse and rat ROR2 are 95% aa identical over the entire open reading frame. There is a human ROR1 and it shows 57% overall aa identity to ROR2.¹⁰¹

A ligand for this receptor has been identified. ROR2 has a CRD that resembles the Wnt binding site on Frizzled receptors,^105,106 and Wnt-5a is now known to bind to ROR2. Perhaps as importantly, ROR2 is also known to bind to Frizzled-2 and Frizzled-5 via their CRDs. Wnt-5a binding of ROR2 results in activation of the PCP non-canonical pathway.¹⁰⁶ Although the physiological significance of a Wnt-ROR interaction is not yet known, Wnt-5a^-/- mice develop short limbs and tails.

Strabismus/Van Gogh-like Protein

The uncertainty and complexity of the Wnt system is underscored by the role Strabismus may play in Wnt signaling. Wnt binding to Frizzled recruits/activates Dsh. Activated Dsh interferes with GSK-3 beta kinase, an enzyme that normally destabilizes beta-Catenin. Stabilized beta-Catenin interacts with TCF molecules to initiate gene transcription.¹⁰⁷ Dsh is also suggested to interact with small GTPases that ultimately turn on the JNK cascade.¹⁰⁸ This implies a dual function for Dsh.^23,107 In vertebrates, Strabismus is a four-TM molecule that recruits Dsh to a PDZ domain on its cytoplasmic tail.¹⁰⁹ This suggests that Strabismus can compete with Frizzled for recruitment of Dsh. Dsh interaction with Frizzled leads to beta-Catenin signaling. Dsh interaction with Strabismus interferes with beta-Catenin signaling and activates the PCP pathway. This does not appear to be a random event. Evidence suggests that it is Wnt itself that determines the pathway involved. Canonical Wnts such as Wnt-1 and Wnt-3a block Strabismus binding of Dsh, and encourage its association with Frizzled. Wnt-5a and Wnt-11, however, promote Dsh interaction with Strabismus and block its association with Frizzled.¹⁰⁸

Strabismus was initially identified in Drosophila,¹¹⁰ but has since been cloned in both human and mouse. Remarkably, both the N-terminus and C-terminus are cytosolic. There are two Strabismus genes in mouse and human. Human Strabismus-1 (also known as Van Gogh-like Protein-2) is 60 kDa and 521 aa in length. It contains a 110-aa N-terminus and a 280 aa C-terminus. Human Strabismus-2 (also known as Van Gogh-like Protein-1) is 524 aa in length and almost identical to Strabismus-1 in orientation. Strabismus-1 shows 73% aa identity to Strabismus-2.^111-114 Mouse Strabismus-1 and 2 have also been cloned (GenBank Accession # AAH52195).¹¹⁵ Mouse and human Strabismus-1 display 99% aa identity while mouse and human Strabismus-2 show 95% aa identity.

Flamingo/CELSR

In Drosophila, wing hair formation requires Flamingo. Flamingo orientation is key in determining hair "polarity," and the proper orientation requires the participation of Frizzled and Dsh. Dsh is known to co-localize with Flamingo, and Frizzled directs Dsh to the area of cell boundaries. When Frizzled or Flamingo are absent, intracellular Dsh cannot attach to the cell membrane. Although the relationship between Frizzled, Dsh and Flamingo are poorly understood, Flamingo would seem to be a target of Dsh binding.^116,117 In summary, Flamingo would seem to participate in the orienting of fly hair via a PCP pathway.

In vertebrates, ear hair cells have microtubule cilia that extend from one particular side of each cell. All cilia point in the same direction. In mice, when putative mouse orthologs of Drosophila Flamingo and Strabismus are mutated, these cilia, and their cells, lose their directional orientation.^21,118 Thus, it would appear that vertebrates have some counterpart to Drosophila Flamingo and that it may play a role in Wnt receptor signaling.

Drosophila Flamingo is a 3575 aa, seven-TM protein that is best described as a non-classic, Cadherin subfamily molecule. The extended, N-terminal, extracellular region has nine Cadherin repeats, three CRDs, and two Laminin A globular segments. This is consistent with a classic Cadherin family molecule. What makes this molecule different is the absence of a typical cytoplasmic Catenin-binding sequence, which precludes its binding to cytosolic Catenins.¹¹⁹

Both human and mouse have three somewhat distant orthologs named CELSRs [Cadherin, EGF-like, Laminin A globular-like (LAG), and seven-pass receptors].¹²⁰ Human CELSR1, 2, and 3 all show the same structural features and have been referred to as long N-termini, seven-TM (LN) receptors. CELSR1 is synthesized as a 3014 aa precursor with a 20 aa signal sequence, a 2449 aa extracellular region, seven TM segments, and a 310 aa cytoplasmic tail.¹²¹ The extracellular region contains multiple N-linked glycosylation sites plus two potential hydroxylation sites. Beginning with the N-terminus, there are nine 100-200 aa Cadherin domains, three 35-60 aa, EGF-like segments, one LAG region, five 35 aa, EGF-like segments and one membrane proximal GPS (G protein-coupled receptor proteolytic site) domain.¹²¹ GPS domains are associated with proteolytic activity that results in two subunits, which remain non-covalently associated.¹²² Relative to CELSR1, human CELSR2, the ortholog to Flamingo, is 100 aa shorter than CELSR1 resulting from differences in the extracellular region.¹²³ Human CELSR3 is the longest of the three receptors at 3300 aa and contains two additional extracellular Laminin EGF-like domains, plus an extended 532 aa cytoplasmic tail.¹²⁴ Considering the size of the three CELSR molecules, aa identity is low, at 56% between CELSR1 and 2. Mouse CELSR1, 2, and 3 have also been cloned.^119,120,125 Their lengths and structural domains are virtually identical to their human counterparts. Mouse relative to human, CELSR1, 2, and 3 display 81%, 95%, and 92% aa identity, respectively.

CELSR1 is expressed in the primitive streak and adult ependyma, choroid plexus, and area postrema.¹²¹ CELSR2 is expressed in the anterior neural ectoderm,¹²⁵ and hippocampal pyramidal cells and dentate granule cells.¹²⁶ CELSR3 is found in the neural tube anterior to the forelimb buds, and postnatal neurons of the cerebrum and olfactory system.^125,126

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