four-jointed: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - four-jointed

Synonyms -

Cytological map position - 55C1--3

Function - a type II transmembrane kinase

Keywords - leg, eye, brain, tissue polarity, phosphorylates of the atypical cadherins Fat and Dachsous, regulates growth via the Hippo/Warts pathway

Symbol - fj

FlyBase ID: FBgn0000658

Genetic map position - 2-81.5

Classification - type 2 transmembrane protein

Cellular location - surface



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Li, C., Li, B., Ma, S., Lu, P. and Chen, K. (2017). Dusky works upstream of Four-jointed and Forked in wing morphogenesis in Tribolium castaneum. Insect Mol Biol. PubMed ID: 28677915
Summary:
Dusky (dy) is required for cytoskeletal reorganization during wing morphogenesis in Drosophila melanogaster, but which genes participate together with dy for wing morphogenesis has remained unclear. In Tribolium castaneum, dy is highly expressed at the late embryonic stage. Tissue-specific expression analysis indicated high expression levels of dy in the epidermis, head and fat body of late-stage larvae. RNA interference (RNAi) targeting dy significantly decreased adult wing size and caused improper folding of the elytra. Meanwhile, dy knockdown reduced the transcription of four-jointed (fj) and forked (f). These results show that fj RNAi reduces adult wing size and that silencing f results in abnormal wing folding in T. castaneum. Interestingly, knocking down fj and f simultaneously phenocopies dy RNAi, suggesting that dy probably acts upstream of fj and f to regulate wing morphogenesis in T. castaneum. cvvv
BIOLOGICAL OVERVIEW

four-jointed is involved in conveying positional information in the leg and eye, perhaps acting downstream of Notch. fj mutants show reduced growth and altered differentiation only within restricted sectors of the proximal-distal (PD) axis in the leg and wing, thus fj is a candidate for a gene with a coordination function. Consistent with a position-sensitive role, fj is expressed in a regional pattern in the developing leg, wing, eye and optic lobe. The fj gene encodes a novel type II membrane glycoprotein. It is proposed that fj encodes a secreted signal that functions as a positive regulator of regional growth and differentiation along the PD axis of imaginal discs (Villano,1995 and Brodsky, 1996).

Four-jointed was identified in a search for P-element insertions in genes expressed in patterns that might convey positional information in imaginal discs. The beta-galactosidase markers expressed by these P-elements were examined at the late third instar and prepupal stages, a period when axis specification is complete but overt differentiation of each disc has not yet occurred. Based on its provocative expression pattern in the leg disc, eye disc and optic lobes, a single insertion line was chosen for further study (Villano, 1995). Similarly, in the Brodsky (1996) study, an enhancer trap screen was carried out for patterned expression patterns in the eye.

In the third instar larval leg disc, beta-gal expression marking transcription of fj is expressed in a pattern of concentric circles, similar to a subset of the concentric restrictions that mark the future segment boundaries of the leg. Non-uniform expression is observed in fj eye discs, with strongest expression in the central portion of the disc just anterior to the morphogenetic furrow, but with no expression in the lateral regions of the disc. An apparent gradient of expression is seen centrally declining toward the posterior tip of the disc. This triangular zone of expression does not correspond to any known developmental or physiological compartment boundaries. In the larval brain, expression is concentrated in the outer optic anlage (ooa), which forms a circumferential band around the optic lobe. The ooa is a proliferation zone that contributes cells to the lamina and distal medulla regions of the optic lobe, that form the postsynaptic targets for the photoreceptor axons. Staining in the eye and optic lobe are transient and no staining is detectable in adults. However, some beta-gal expression persists in the wings, legs and antennae of adult flies (Villano, 1995).

In the leg, segments occupying intermediate positions in the PD axis are specifically affected in the strongest mutations, fj2 and fj3. The adult leg can be divided into five regions. The most distal portion of the leg is called the tarsus, followed proximally by the tibia, femur, trochanter and coxa. The tarsus is composed of five segments, T5-T1 (distal to proximal), which are separated from each other by joints and are distinguishable by their lengths, patterns of bristles and cuticular specializations. The tarsus is reduced to four segments in these mutants. The middle tarsal segments, T2 and T3, are replaced by a single segment that appears to be a fusion of these two, while T1, the tibia and the femur are truncated. The more proximal segments of the leg (the coxa and trochanter) and the most distal (T5 and T4) are unaltered in fj mutants. Even within the affected segments, anterioposterior and dorsal-ventral patterning appears to be normal, based on cuticular patterns (Villano, 1995).

Loss of growth in the PD axis of the leg and wing might arise from an initial failure in proliferation and differentiation, or might result from a late degeneration event subsequent to proliferation. To distinguish between non-proliferation and degeneration, the emergence of the tarsal segments in prepupal discs that had just begun to evert was examined. By 4 hours APF in wild-type discs, all five of the prospective tarsal segment boundaries can be observed as indentations of the leg epithelium. Only four such boundaries are seen in fj2 discs and the sizes of the presumptive T2/T3 and T1 segments are already abnormally small (also see Waddington, 1943). Presence of cell death was examined by acridine orange staining during the early or late third instar, between the time of active cellular proliferation in the leg disc and the time of leg eversion. No cell death was found. These results suggest that the fj leg phenotype arises from an initial failure in the proliferation of a subset of tarsal segments, as well as a failure to initiate the T2/T3 segment boundary (Villano, 1995).

Previous work on the fj gene suggested that it functions as a regional signaling molecule regulating growth and differentiation in specific portions of the PD axis in the leg and wing. Analysis of the phenotype, expression pattern, molecular identity and biochemistry of fj strongly supports this hypothesis. A role in intercellular communication may be inferred from the nonautonomous behavior of wild-type cells juxtaposed with mutant cells in mosaic patches at the T2/T3 boundary in the leg (Tokunaga, 1976). The heterozygous cells immediately adjacent to the mutant patch fail to form a joint. This would be expected if a defective signal from the mutant cells was unable to stimulate a receptor on the immediately adjacent wild-type cells. However, the majority of fj clones crossing the T2/T3 segment boundary display autonomous failure to form a joint (the nonautonomous group were attributed to probable mitotic recombination between the marker and the fj gene), suggesting fj may be required in both cells. A nonautonomous signal or ligand would presumably be in the form of a cell surface or secreted molecule, although the molecule defective in the mosaics could also be involved in the generation of the signal. Analysis of the fj gene product is consistent with the fj protein representing the signal itself. Conceptual translation of fj cDNA predicts a type II trans-membrane glycoprotein with a potential internal signal peptidase cleavage site. In vitro translation of fj mRNA in the presence of membranes results in cleavage of a portion of the nascent polypeptides at the predicted site. However, the microsomal cleavage of fj is incomplete and a significant portion of the protein remains transmembrane and full length (Villano, 1995).

It is thought that fj is required for local proliferation in imaginal tissue. (1) Loss of function results in a loss of tissue in both the leg and the wing. In the leg, a loss of growth is apparent as early as the prepupa. (2) fj is strongly expressed in known proliferation zones, including the region of proliferation just anterior to the morphogenetic furrow of the eye disc and the outer proliferation center of the optic lobe. Although only mild phenotypes in the eye were found in the Villano (1995) study, Waddington (1943) observed that the eye is greatly diminished or lost in double mutants of fj and dachsous (ds), a gene with homology to cadherins that by itself has no phenotype in the eye. A role for fj in controlling growth of the eye may therefore be masked by the activity of other genes. (3) Two distinct tumor-suppressor genes, Gull, an allele of fat (Bryant, 1988 and Mahoney, 1991) and expanded (Boedigheimer, 1993), are able to suppress the fj leg phenotype in double mutants (Villano and Katz, unpublished observations cited in Villano, 1995), suggesting that loss of fj can be compensated for by deregulation of proliferative activity. Whether fj is sufficient for growth can be tested by ectopic expression. However, if fj is indeed a signal then the effect of ectopic expression will depend on the distribution of its receptor and downstream intracellular signaling pathways, which may not be ubiquitous (Villano, 1995).

Cleavage and secretion is not required for Four-jointed function in Drosophila patterning: fj interacts genetically with ft and ds in planar polarity and proximodistal patterning

four-jointed (fj) is required for proximodistal growth and planar polarity in Drosophila tissues. It encodes a predicted type II transmembrane protein with putative signal peptidase sites in its transmembrane domain, and its C terminus is secreted. Fj has therefore been proposed to act as a secreted signalling molecule. Fj protein has a graded distribution in eye and wing imaginal discs, and is largely localized to the Golgi in vivo and in transfected cells. Forms of Fj that are constitutively secreted or anchored in the Golgi were assayed for function in vivo. Cleavage and secretion of Fj is shown to not be necessary for activity, and Golgi-anchored Fj has increased activity over wild type. fj has similar phenotypes to those caused by mutations in the cadherin-encoding genes fat (ft) and dachsous (ds). fj is shown to interact genetically with ft and ds in planar polarity and proximodistal patterning. It is proposed that Fj may act in the Golgi to regulate the activity of Ft and Ds (Stutt, 2004).

Fj has been proposed to act as a secreted signalling molecule, based on the fact that its C-terminal region can be cleaved and secreted, and that it exhibits non-autonomous functions in mosaic clones. The functional significance of this cleavage was tested by making modified forms of Fj that are either poorly cleaved, constitutively secreted or anchored in the Golgi apparatus. Several different assays support the conclusion that cleavage and secretion of Fj is not essential for its activity in either planar polarity or PD patterning. In both overexpression experiments and rescue assays, Golgi-tethered Fj has significantly more activity than wild-type Fj, while secreted Fj is less active. Furthermore, even though Golgi-tethered Fj is not secreted, it still produces non-autonomous polarity phenotypes similar to or stronger than those of wild-type Fj. Therefore, it is proposed that secreted Fj is not the active form, and that Fj acts intracellularly. Fj most likely acts by modulating the activity of other molecules involved in intercellular signalling (Stutt, 2004).

Thus, the results show that rather than acting in an analogous manner to the cleaved type II transmembrane protein Hedgehog, a better model for Fj function may be the type II transmembrane protein Fringe (Fng). Fng is Golgi-localized and acts as a glycosyltransferase enzyme to post-translationally modify the receptor Notch (N). This renders N more sensitive to its ligand Delta, and less sensitive to the ligand Serrate. In the case of Fj, there are no molecular homologies that give any clues as to a possible enzymatic activity. Consequently, the precise location of its function is uncertain. However, since the results show that if Fj is tethered in the Golgi, it has higher than normal activity, the Golgi seems most likely to be its preferred site of action (Stutt, 2004).

An important question is whether Fj cleavage has any functional significance. Since forced retention of Fj in the Golgi causes hyperactivity, it is possible that cleavage and secretion could be a mechanism to downregulate Fj activity during normal development. However, further experiments will be required to determine if the cleavage step is temporally or spatially regulated (Stutt, 2004).

The mouse homolog of Fj (Fjx1) has also been proposed to act as a secreted molecule, on the basis of a hydrophobic stretch at the N terminus that might represent a signal peptide and the presence of predicted signal peptidase cleavage sites. However, the hydophobic region is not at the extreme N terminus and is sufficiently long to be predicted to be a transmembrane domain. This structure suggests that Fjx1 may also be a type II transmembrane protein. Consistent with this, in tissue culture experiments, no secretion of the C-terminal region of Fjx1 into the medium is observed. However, this failure to detect any activity of Fjx1 when overexpressed in flies suggests that there may be a divergence of function between the fly and vertebrate proteins (Stutt, 2004).

In Drosophila, the atypical cadherins Ft and Ds are good candidates for being the ultimate targets of fj activity. They are required for both planar polarity and PD patterning, and have similar mutant phenotypes to fj. In addition, fj interacts genetically with ds and ft in both planar polarity and PD patterning. Interestingly, ds fj double mutants have surprisingly strong phenotypes, which were qualitatively different to those of the single mutants, including duplications or transformations of limb structures. However, no such phenotypes are seen in any of the double mutant combinations, suggesting that the duplications/transformations may be specific to the combination of chromosomes used in classical experiments. The current results instead show that mutations in fj enhance the phenotypes of both ft and ds hypomorphic mutations, suggesting that these genes act in a common pathway (Stutt, 2004).

Epistasis experiments further demonstrate that ds is required to mediate fj function, and therefore ds acts downstream of fj; this is in agreement with data based on clonal analysis of ds and fj. Interestingly, recent experiments have also revealed a role for fj in regulating the intracellular distribution of Ds and Ft. In wild-type tissue, Ds and Ft colocalize at apicolateral membranes, and their localization is mutually dependent. Inside fj mutant clones, Ds and Ft localization is largely unaltered. However, in the row of mutant cells immediately adjacent to wild-type tissue, Ft and Ds preferentially accumulate on the boundary between fj+/fj- cells. In addition, cells inside the fj clones appear to be 'rounded-up', suggesting that they prefer to adhere to each other rather than to non-mutant cells. Thus, it is thought that fj modulates the activity and intermolecular binding properties of Ft and Ds (Stutt, 2004).

An interesting point to note is that both ds and ft show planar polarity phenotypes as homozygotes, whereas fj only shows polarity phenotypes on the boundaries of mutant clones. The fj phenotypes have been explained by models in which fj acts redundantly to regulate the production of a gradient, the direction of which determines polarity. Thus, in homozygotes the direction of the gradient is unchanged, and animals show no major defects; but at clone boundaries there is a discontinuity in the direction of the gradient, leading to inversions of polarity. This model can be extended to suppose that Fj may modulate Ds/Ft activity, but that it does not act as a simple on-off switch; rather Ds/Ft retain some activity even when Fj is not present (Stutt, 2004).

In the absence of a known enzymatic function for Fj, the mechanism by which it might modulate Ft and Ds activity remains uncertain. It is speculated that since Fj acts intracellularly, it is possible that it promotes or mediates the post-translational modification of Ds and/or Ft proteins, and that these molecules mediate the non-autonomous signalling functions of Fj. However, the large size of the Ft and Ds gene products (5147 and 3380 amino acids, respectively) renders the analysis of their post-translational modification highly challenging (Stutt, 2004).

Four-jointed is a Golgi kinase that phosphorylates a subset of cadherin domains

The atypical cadherin Fat acts as a receptor for a signaling pathway that regulates growth, gene expression, and planar cell polarity. Genetic studies in Drosophila identified the four-jointed gene as a regulator of Fat signaling. This study shows that four-jointed encodes a protein kinase that phosphorylates serine or threonine residues within extracellular cadherin domains of Fat and its transmembrane ligand, Dachsous. Four-jointed functions in the Golgi and is the first molecularly defined kinase that phosphorylates protein domains destined to be extracellular. An acidic sequence motif (Asp-Asn-Glu) within Four-jointed is essential for its kinase activity in vitro and for its biological activity in vivo. These results indicate that Four-jointed regulates Fat signaling by phosphorylating cadherin domains of Fat and Dachsous as they transit through the Golgi (Ishikawa, 2008).

The Fat and Hippo signaling pathways intersect at multiple points and influence growth and gene expression through regulation of the transcriptional coactivator Yorkie. Fat signaling also influences planar cell polarity (PCP). Fat acts as a transmembrane receptor, and is a large (5147 amino acids) atypical cadherin protein, with 34 extracellular cadherin domains. Dachsous (Ds) is also a large (3503 amino acids) transmembrane protein with multiple cadherin domains and is a candidate Fat ligand because it appears to bind Fat in a cultured cell assay, acts non-cell autonomously to influence Fat pathway gene expression, and acts genetically upstream of fat in the regulation of PCP. A second protein, Four-jointed (Fj), also acts non-cell autonomously to influence Fat pathway gene expression and acts genetically upstream of fat in the regulation of PCP. However, Fj is a type II transmembrane protein that functions in the Golgi. Thus, Fj might influence Fat signaling by posttranslationally modifying a component of the Fat pathway (Ishikawa, 2008).

To investigate the possibility of modification of Fat or Ds, FLAG epitope-tagged fragments of their extracellular domains together were coexpressed with Fj in cultured Drosophila S2 cells. When the first 10 cadherin domains of Ds (Ds1-10) were coexpressed with Fj, a shift in mobility was observed. A common posttranslational modification of secreted and transmembrane proteins as they pass through the Golgi is glycosylation. Most glycosyltransferases contain a conserved sequence motif, Asp-X-Asp (DXD; X, any amino acid), which is essential for their activity. Because a related sequence motif [Asp-Asn-Glu (DNE) at amino acids 490 to 492] is present in Fj and its vertebrate homologs, a mutant form of Fj was created in which DNE was changed to GGG (FjGGG; G, glycine). The expression levels and Golgi localization of FjGGG appear normal, but FjGGG expression did not shift Ds1-10 mobility (Ishikawa, 2008).

To identify modified cadherin domains, smaller fragments of Ds1-10 were expressed. The smallest fragments whose mobility was shifted in cells expressing Fj were two-cadherin-domain polypeptides: Ds2-3, Ds5-6, and Ds8-9. Ds2-3 and Ds5-6 appeared to be stoichiometrically modified in cells expressing Fj, whereas Ds8-9 was only partially modified. Fat4-5 was also partially shifted by Fj coexpression. The mobility shifts of these two-cadherin-domain polypeptides were not observed with FjGGG. To identify potential sites of modification, their sequences were aligned. This identified four sites at which a Ser or Thr residue was conserved, whose hydroxyl groups could potentially be sites of posttranslational modification. To evaluate their influence, each was mutated in turn to Ala within the Ds2-3 polypeptide. Three of the four mutants had no effect; however, one, Ds2-3S236A (mutation of Ser236 to Ala), completely eliminated the Fj-dependent mobility shift. Introduction of an analogous mutation into Ds8-9 also eliminated its mobility shift. Thus, a Ser reside at a specific location within the second of the two cadherin domains was essential for the Fj-dependent mobility shift. This amino acid was a Ser in each of these dicadherin domains, but Thr was also compatible with the Fj-dependent modification. In a structurally solved cadherin domain, this Ser is the seventh amino acid and predicted to be located on the surface near the middle of the cadherin domain (Ishikawa, 2008).

To identify posttranslational modifications associated with this mobility shift, Ds2-3 was purified from S2 cells expressing or not expressing Fj, the proteins were digested with trypsin, andthe resulting peptides were analyzed by mass spectrometry. One peptide from Fj-expressing cells was stoichiometrically shifted by 80 daltons relative to the same peptide from cells not expressing Fj, and it also eluted earlier on high-performance liquid chromatography (HPLC). Mass and tandem mass spectrometry (MS/MS) fragmentation patterns identified this peptide as amino acids 215 to 237 of Ds and refined the site of modification to within amino acids 232 to 237. The mass of the equivalent peptide from Ds2-3S236A was not altered by Fj expression. Most of the peptides corresponding to Ds2-3 cadherin domains were identified, and none of the others were detectably modified in cells expressing Fj. Thus, the Fj-dependent modification of Ds2-3 comprises an addition of 80 daltons, which is attached to Ser236. An 80-dalton mass does not correspond to that of any known glycans, but does correspond to the mass associated with addition of a phosphate group. Incubation of Fj-modified Ds fragments with either calf intestinal alkaline phosphatase (CIP) or Antarctic phosphatase (AnP) reversed the Fj-dependent mobility shifts of Ds2-3, Ds8-9, and Fat 4-5. Thus, Ds and Fat cadherin domains are subject to Fj-dependent phosphorylation at a specific Ser residue (Ishikawa, 2008).

To investigate whether Fj itself has kinase activity, a secreted, epitope-tagged Fj (sFj:V5) was purified from the medium of cultured S2 cells. Purified sFj:V5 was then incubated with affinity-purified Ds2-3 and [γATP (adenosine 5'-triphosphate)] in buffer. Transfer of 32P onto Ds2-3 was observed in the presence of sFj, but not in its absence, and not when sFjGGG was used as the enzyme. Moreover, Ds2-3S236A was not detectably phosphorylated by sFj. The activity of Fj expressed in a heterologous system was also characterized by expressing a glutathione S-transferase:Fj (GST:Fj) fusion protein in Escherichi coli and partially purifying it on glutathione beads. GST:Fj, but not GST:FjGGG, catalyzed the transfer of 32P onto Ds2-3. Thus, Fj is a protein kinase (Ishikawa, 2008).

The generic kinase substrates myelin basic protein and casein were not detectably phosphorylated by sFj. Thus, Fj appears to have a limited substrate specificity. Only a few proteins have been identified as being phosphorylated in the secretory pathway, and none of the responsible kinase(s) have been molecularly identified. A Golgi kinase activity, referred to as Golgi casein kinase, preferentially phosphorylates Ser or Thr residues within a S/T-X-E/D/S(Phos) consensus sequence. Because Fj does not phosphorylate casein, and the Ser residues within cadherin domains targeted by Fj do not conform to Golgi casein kinase sites, Fj is not Golgi casein kinase. Fj autophosphorylation was detected, but this reaction was weak compared to phosphorylation of Ds2-3. The autophosphorylation reaction is apparently unimolecular, because GST:Fj and sFj:V5 did not phosphorylate each other and the fraction of Fj phosphorylated was independent of concentration (Ishikawa, 2008).

Some cadherin domain polypeptides that include a Ser as the seventh amino acid were not detectably shifted, but the mobility shift on Ds2-3 might reflect a conformational effect. To examine the ability of Fj to phosphorylate other cadherin domains, in vitro kinase reactions were performed with [γ-32P]ATP. This identified phosphorylation sites on polypeptides that were not gel shifted, including Fat2-3, Fat10-11, and Fat12-13. The in vitro kinase reactions also identified differences in the efficiency with which different cadherin domains were phosphorylated by Fj, with Ft3, Ds3, and Ds6 being the best substrates (Ishikawa, 2008).

If the presence of a Ser or Thr at the seventh amino acid of a cadherin domain is taken as the minimal requirement for Fj-mediated phosphorylation, there are nine potential sites in Ds and 11 in Fat. However, Fat10, Ds2, Ds11, Ds13, and Ds18 were not detectably phosphorylated, despite the presence of Ser or Thr at this position. Presumably, there are other structural features important for recognition by Fj. This was also emphasized by the detection of phosphorylation of this Ds2-3 polypeptide, but not the Ds3-4 polypeptide, even though both contain Ser236. The dicadherin constructs were based on published annotations, but in comparing Ds cadherin domains to structurally solved cadherin domains, it was realized that these misposition the intercadherin domain boundary, and consequently these constructs lacked three amino acids of the first cadherin domain. Addition of these amino acids, together with the intercadherin domain linker sequence, enabled phosphorylation of a Ds3 single-cadherin domain construct (Ishikawa, 2008).

A weak similarity between Fj and the bacterial kinase HipA, and between Fj and the mammalian lipid kinase phosphatidylinositol 4-kinase II (PI4KII), has been suggested previously on the basis of bioinformatic analyses in which HipA or PI4KII were used as the starting point for PSI-BLAST searches. Asp residues play critical roles in catalysis and in the coordination of Mg2+ in these and other kinases, and the loss of Fj kinase activity associated with mutation of the conserved DNE motif is thus consistent with the inference that Fj is related to other kinases. A single Fj ortholog, Fjx1, is present in a range of vertebrate species, including humans (Ishikawa, 2008).

To investigate the biological requirement for Fj kinase activity, the catalytically inactive fjGGG mutant was assayed in vivo. A V5 epitope-tagged form of this gene was expressed in transgenic Drosophila. At the same time, V5-tagged wild-type fj was contructed. To ensure that both forms were expressed in similar amounts, site-specific integration was used to insert transgenes at the same chromosomal location. Immunostaining confirmed that FjGGG:V5 and Fj:V5 both exhibited normal Golgi localization and were expressed in similar amounts. Uniform overexpression of fj reduces the growth of legs and wings and interferes with normal PCP. Fj:V5 exhibited phenotypes consistent with previous studies, but FjGGG:V5 was completely inactive. Thus, mutation of the DNE motif in Fj abolishes its biological activity (Ishikawa, 2008).

The identification of Fj's cadherin domain kinase activity provides a biochemical explanation for the influence of Fj on Fat signaling and supports a model in which Fj directly phosphorylates Fat and Ds as they transit through the Golgi to influence their activity, presumably by modulating interactions between their cadherin domains. Because there was a substantial difference in the efficiency with which individual cadherin domains could be modified by Fj, both in cell-based and in vitro assays, it is also possible that differences in the extent of Fat and Ds phosphorylation normally occur in vivo and might differentially modify their binding or activity (Ishikawa, 2008).


GENE STRUCTURE

Transcript size - 3.7 kb

Bases in 5' UTR - 1345

Bases in 3' UTR - 456


PROTEIN STRUCTURE

Amino Acids - 583

Structural Domains

Although the predicted ORF has no similarity to any known gene in the available data bases, several structural motifs are present. The Kyte-Doolittle Hydropathy test detects a potential membrane spanning region at amino acids 77-101 but no comparable region at the N terminus that would correspond to a signal sequence. The charge distribution surrounding this potential transmembrane region strongly predicts that the polypeptide should be oriented with its N terminus inside the cell and its C-terminal domain external, as in a type II membrane protein. Two sites within the hydrophobic region predict cleaved signal sequences. Although the algorithm was modeled on N-terminal signal sequences and has not been tested extensively with internal signal sequences of type II membrane proteins, it was successful in predicting a cleavage site in the Drosophila Hedgehog (Hh) protein, which has a similar structure to fj. Cleavage at either of the predicted sites in the fj protein would allow the mature C-terminal polypeptide chain to be released into the lumen of the rough endoplasmic reticulum and eventually secreted. The sequence also contains 14 pairs of adjacent basic residues, which may serve as sites for post-translational proteolytic processing; a consensus site for alpha-amidation near the C terminus (a common modification in proteolytically processed neuropeptides) and a PEST sequence, associated with rapid protein degradation. Finally, there are four consensus sites for asparagine-linked glycosylation, three in the C-terminal domain (Villano, 1995 and Brodsky, 1996).

To test the predicted size, topology, glycosylation and potential cleavage of the fj protein, an in vitro transcription-translation-coupled translocation assay was performed using rabbit reticulocytes and canine pancreatic microsomes. Together, these results indicate that fj encodes a type II membrane glycoprotein and strongly suggest that, at least in this in vitro system, fj is synthesized in both a membrane-bound and a secreted form (Villano, 1995).


EVOLUTIONARY HOMOLOGS

The cloning of the mouse four-jointed1 gene is reported and its pattern of expression in the brain during embryogenesis and in the adult. In the neural plate, fjx1 is expressed in the presumptive forebrain and midbrain, and in rhombomere 4, however a small rostral/medial area of the forebrain primordium is devoid of expression. Expression of fjx1 in the neural tube can be divided into three phases. (1) In the embryonic brain fjx1 is expressed in two patches of neuroepithelium: the midbrain tectum and the telencephalic vesicles. (2) In fetal and early postnatal brain, fjx1 is expressed mainly by the primordia of layered telencephalic structures: cortex (ventricular layer and cortical plate), and olfactory bulb (subependymal layer and in the mitral cell layer). In addition, expression is observed in the superior colliculus. (3) In the adult, fjx1 is expressed by neurones evenly distributed in the telencephalon (isocortex, striatum, hippocampus, olfactory bulb, piriform cortex), in the Purkinje cell layer of the cerebellum, and numerous medullary nuclei. In the embryo, strong expression can further be seen in the apical ectodermal ridge of fore- and hind-limbs, and in the ectoderm of the branchial arches (Ashery-Padan, 1999).

The murine sequence has an open reading frame encoding a polypeptide of 437 amino acids. The calculated relative molecular mass of the conceptual translation product is 48,912 Da, and its isoelectric point would be 10.55. Comparison of the predicted amino acids sequence with the currently available database entries reveal significant homology to the Drosophila Fj protein (50% similarity, 42% identity). In addition to sequence similarity, Fj and Fjx1 share structural motives; a putative transmembrane domain, and a signal sequence cleavage site are located in the N-terminal end of both proteins. In the C-terminal region of both proteins, consensus sites for asparagine linked glycosylation are present. In addition, a consensus sequence for alpha amidation, a common modification in proteolytically processed neuropeptides, is predicted in Fj and Fjx1. Finally, the predicted topology of both proteins implies that the C-terminal region is processed in the endoplasmic reticulum and secreted. This is in agreement with in vitro experimental evidence showing that the Drosophila Fj is glycosylated and secreted (Ashery-Padan, 1999).


four-jointed:
Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 10 August 2004

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