The frizzled genes were first identified in Drosophila in a screen for mutations that disrupt the polarity of epidermal cells in the adult fly 1. Subsequently, frizzleds have been found in diverse metazoans 2, including at least ten in vertebrates, four in Drosophila, and three in Caenorhabditis elegans. Frizzleds have also been identified in primitive metazoans, including the sponge Suberites domuncula 3 and in Hydra vulgaris 4, but they have not been described in protozoans. They have been shown to encode receptors for Wnt proteins 5. The smoothened (smo) gene, which functions in the Hedgehog signaling pathway in various developmental processes, is distantly related to frizzled genes. Additional information on the Wnt pathway can be found on the Wnt gene homepage 6 and in various comprehensive reviews 1789.

Sequence analysis suggests that the ten human frizzled (FZD) genes fall into four main clusters 10. FZD1, FZD2, and FZD7 share approximately 75% identity; FZD5 and FZD8 share 70% identity; FZD4, FZD9, and FZD10 share 65% identity; and FZD3 and FZD6 share 50% amino acid identity 10. Frizzled genes from different clusters share between 20% and 40% sequence similarity. A dendrogram of human and selected invertebrate frizzled genes is shown in Figure 1. The overall genomic organization of frizzled genes does not appear to be highly conserved across this broad species diversity. Several frizzled genes appear to lack introns, however, including vertebrate orthologs of human FZD1, FZD2, and FZD7 to FZD10 (this is also a feature of many G-protein-coupled receptor (GPCR) genes); other frizzled genes, such as human FZD5 and Drosophila frizzled2 (Dfz2), contain one intron but the entire open reading frame is encoded by a single exon. Interestingly, the intron-deficient frizzled genes appear to be derived from a common ancestor, as they cluster into a subfamily that includes Dfz2 (Figure 1).

Frizzled proteins range in length from about 500 to 700 amino acids (Figure 2). The amino terminus is predicted to be extracellular and contains a cysteine-rich domain (CRD) followed by a hydrophilic linker region of 40-100 amino acids. The proteins also contain seven hydrophobic domains that are predicted to form transmembrane α-helices. The intracellular carboxy-terminal domain has a variable length and is not well conserved among different family members 2.

The CRD, which is necessary and sufficient for binding to Wnt molecules, consists of 120-125 residues with ten conserved cysteines, all of which form disulphide bonds 511. The crystal structures of the CRDs from mouse Frizzled 8 (mFz8) and mouse secreted Frizzled-related protein 3 (sFRP-3) reveal that CRDs are predominantly α-helical and form a previously unknown protein fold 11. A ligand-binding interface, involving a single region of the CRD surface, was predicted from analysis of the crystal structure integrated with comprehensive mutagenesis. Within the crystal, the CRDs form a conserved dimer interface, although in solution they appear to exist as monomers. Whether dimerization of the CRD has a role in ligand binding in vivo is not yet known 11.

The presence of seven hydrophobic domains has raised speculation that these receptors are related to the GPCR super-family. The sequence similarity to GPCRs is low, however, and is limited to the hydrophobic domains, which might be expected to have some similarity because of the shared higher frequency of hydrophobic residues. An intriguing sequence similarity, potentially derived from evolutionary conservation, has been described between Frizzleds and members of the Taste2 subfamily of taste receptors (which are GPCRs) 10.

A motif (KTXXXW) located two amino acids after the seventh hydrophobic domain is highly conserved in Frizzleds and is essential for activation of the Wnt/β-catenin pathway 12. Point mutations affecting any of the three conserved residues are defective in Wnt/β-catenin signaling (see below for more details on this pathway). A peptide derived from this conserved motif interacts in vitro with a peptide from the PDZ domain of mouse Dishevelled 1 - an intracellular signal-transduction protein - suggesting that this motif might mediate interaction between Frizzled proteins and Dishevelled proteins, although an interaction between the full-length proteins has not yet been demonstrated 13. Apart from the KTXXXW motif, the carboxy-terminal tail is not well conserved among Frizzleds. The carboxy-terminal S/T-X-V motif found in some Frizzleds is apparently not required for Frizzled function 14. The distantly related protein Smo also contains an amino-terminal CRD and seven hydrophobic domains, but it lacks the KTXXXW motif and does not bind Wnts 515.

Frizzled proteins are found exclusively at the plasma membrane. They are located at the surface of Wnt-responsive cells, although recent evidence has suggested that they may be internalized as part of a mechanism for regulating the extracellular level of Wnt protein and/or the cellular response to Wnts 1617. The tissue-specific expression of frizzled genes is complex, given that numerous frizzleds have been described in metazoans. In general, frizzleds are widely and dynamically expressed and, indeed, it is rare to find a cell that does not express one or more frizzleds. Specific expression patterns of frizzleds in model organisms have been described 261418.

Frizzleds function in three distinct signaling pathways, known as the planar cell polarity (PCP) pathway, the canonical Wnt/β-catenin pathway, and the Wnt/calcium pathway. The PCP pathway is defined by the set of genes that, when mutated, result in defects in the polarity of cells in a planar tissue, as described below; the canonical Wnt/β-catenin pathway is characterized by stabilization of β-catenin protein in response to ligand binding; and the Wnt/calcium pathway is defined by the ability of overexpressed Wnts and Frizzleds to cause increases in intracellular calcium. As discussed above, the frizzled gene (fz) was first identified genetically from mutations that cause a PCP phenotype in Drosophila 1. Asymmetric subcellular distribution of Frizzled has a central role in establishing cell polarity in flies, and most likely in other organisms as well. The dorsal epidermis of the adult fly shows a highly polarized pattern referred to as planar cell polarity, in which a single hair extends from the posterior end of each cell and points from anterior to posterior. The PCP pathway also regulates the organization of photoreceptor cells in the Drosophila eye. Frizzled and Dishevelled proteins become asymmetrically localized at the distal boundary of each pupal wing cell during the generation of polarity 78. Furthermore, polarization of sensory organ precursor (pI) cells in developing bristles requires fz, and Frizzled protein is localized to the posterior apical cortex of the pI cell prior to mitosis. The C. elegans frizzled genes lin-17 and mom-5 are also required for asymmetric cell divisions (Table 1) 14. A role for frizzleds in vertebrate gastrulation movements was first suggested by the observation that expression of a truncated form of Xenopus fz8 that encodes just the CRD, which inhibits full-length Fz8 function, blocks convergent-extension movements in Xenopus gastrulae 19, in a similar way to overexpression of Wnt-5a 20 and a dominant-negative form of Dishevelled 21. Subsequent work in zebrafish and Xenopus suggested this convergent-extension phenotype arises through disruption of a PCP pathway that orients cell movements during gastrulation 7.

The first evidence that Frizzled proteins can function as receptors for canonical Wnt signaling was the observations that Drosophila frizzled-2 (Dfz2) can make Drosophila S2 cells responsive to the Wnt protein Wingless (Wg); these cells normally do not respond to Wg 5. Although fz interacts genetically with dishevelled in the PCP pathway, a fz loss-of-function mutant does not disrupt canonical Wnt signaling in the fly, as fz and Dfz2 are functionally redundant for canonical signaling 22. Evidence that frizzleds are required for Wnt signaling therefore required removing both fz and Dfz2, which was accomplished by RNA interference against Dfz2 in an fz mutant background, by analysis of chromosomal deficiencies that delete Dfz2 (see Table 1), and by identifying mutations in Dfz2 and crossing these mutants to fz flies 22. In vertebrates, overexpression studies suggest that different Frizzleds function in either the canonical or the noncanonical pathways 23, but at least some vertebrate frizzleds appear to function in multiple pathways, including the PCP, Wnt/calcium, and canonical Wnt/β-catenin pathways 12.

Description of the Wnt/calcium pathway derives originally from the observations that overexpression of Wnt5a or rat frizzled2 can cause an increase in intracellular calcium in zebrafish and can activate protein kinase C and calcium/calmodulin-dependent protein kinase (CaM kinase) in Xenopus 7. This pathway appears to require G proteins and Dishevelled, although a distinct Wnt/calcium pathway has also been proposed to regulate protein kinase C independently of Dishevelled in a frizzled7 pathway that maintains the separation of mesoderm and ectoderm during gastrulation in Xenopus 24.

The specific functions of Frizzled proteins are as varied as the number of cell types that express them. In addition to Drosophila and C. elegans, frizzled mutants have also been described in mouse and humans, and interference with frizzled function using antisense or dominant-interfering constructs has been described in Xenopus and zebrafish. Some of the phenotypes associated with loss of function of Frizzleds in various organisms are listed in Table 1. Of particular note is the fact that mutations in human FZD4 are found in familial exudative vitreoretinopathy (FEVR), an inherited form of retinal degeneration with associated progressive hearing loss 25; investigation into the related Nome's disease, which arises from mutations in a novel, secreted protein called Norrin, led to the exciting recent discovery that Norrin is a ligand for Fz4 that can activate canonical Wnt signaling and yet is distinct from the Wnt proteins 26.

An important remaining question is how Frizzleds transduce a signal upon binding of the ligand. For the canonical Wnt pathway, as discussed above, ligand binding may initiate interaction with Arrow/LRPs, but the nature of the interaction is not known. Arrow/LRP does not appear to be involved in the PCP pathway, and other potential coreceptors have not been identified for this pathway. Whether Frizzleds are regulated by a secreted ligand in the PCP pathway also remains an open question, at least in Drosophila. The mechanism of signal transduction in the Wnt/calcium pathway is also an area of intense research, and the exciting possibility that Frizzleds couple directly to G proteins is still a controversial area, perhaps in part because of the lack of genetic data to support the idea of this interaction.

Information on the specificity of ligand-receptor interaction is also limited. Direct binding assays have been performed for a limited number of ligands, although this is likely to change now that a purification protocol has been established for Wnt proteins 6. A classification of Wnt proteins has suggested that some ligands, such as Wg, Wnt1, and Wnt3a, function as ligands that activate the canonical pathway, whereas others, such as Wnt5a, Wnt11, and Dwnt4, function in noncanonical pathways. Whether this distinction applies to Frizzleds remains to be resolved. In Drosophila, Fz functions in both pathways but Dfz2 functions only in canonical signaling; in vertebrates, this distinction is less clear (compare 23 with 12).

Frizzled proteins are asymmetrically distributed in tissues that exhibit planar polarity in the fly, and PCP signaling has been proposed to regulate oriented cell movements in vertebrate gastrulation; so far, however, an asymmetric subcellular distribution of vertebrate Frizzled proteins has not been demonstrated, largely because of the difficulty in generating antibodies sensitive enough to detect the endogenous protein. In addition, the biochemistry of PCP signaling is in its early stages, mainly because a biochemical readout for this pathway has not been clearly established, and it remains unclear whether PCP is regulated by a ligand-receptor interaction.

Finally, Wnt/Frizzled signaling clearly plays important roles in adult tissues as well as embryonic development. The limited number of human diseases found so far to be linked to mutations in frizzled genes is likely to expand in the near future.