BIOLOGICAL OVERVIEW

Steroid hormones are key regulators of numerous physiological and developmental processes, including metastasis of breast and ovarian cancer. The Drosophila gene taiman encodes a steroid hormone receptor coactivator related to AIB1. Mutations in tai cause defects in the migration of specific follicle cells (the border cells) in the Drosophila ovary. Mutant cells exhibit abnormal accumulation of E-cadherin, ß-catenin, and focal adhesion kinase. Tai protein colocalizes with the ecdysone receptor in vivo and augments transcriptional activation by the ecdysone receptor in cultured cells. The finding of this type of coactivator required for cell motility suggests a novel role for steroid hormones, in stimulating invasive cell behavior, independent of effects on proliferation (Bai, 2000).

A small group of follicle cells in the Drosophila ovary, the border cells, has been studied as a model system for a forward genetic approach to the study of cell motility. The border cells originate within an epithelium of approximately 1100 follicle cells that surrounds a cluster of 16 germline cells to form an egg chamber. Early in oogenesis, a pair of specialized follicle cells, called polar cells, differentiates at each end of the egg chamber. The anterior polar cells recruit an additional four to eight cells, and this cluster then detaches from the follicle cell epithelium and invades the neighboring group of fifteen nurse cells (Bai, 2000).

A number of genes are known to be required to convert the border cells from stationary, epithelial cells to invasive, migratory cells. The first gene found to be required for border cell migration was slow border cells: slbo encodes a basic region/leucine zipper transcription factor related to the mammalian CCAAT enhancer binding protein (C/EBP) family. It has been proposed that expression of C/EBP is one factor controlling the timing of border cell migration during oogenesis (Bai, 2000).

A screen was carried out for mutations on the left arm of the second chromosome (2L) that cause border cell migration defects in mosaic clones. Border cell position was monitored using beta-galactosidase expression from an enhancer trap line, PZ6356. Of 2885 mutant lines screened for migration defects, one mutant named taiman^61G1 (tai^61G1, meaning 'too slow') was selected for further study. In egg chambers containing tai^61G1 mosaic clones, PZ6356 expression is unaffected. In some egg chambers containing tai^61G1 clones, migration is completely inhibited, and border cells remain at the anterior tip of the egg chamber. In other egg chambers, the border cells migrate partway. Border cell clusters that undergo partial migration are typically composed of a mixture of heterozygous and homozygous mutant cells (Bai, 2000).

Border cells mutant for tai express wild-type levels of the Slbo protein, indicating that the tai mutant phenotype is not due to reduced expression of Slbo. For example, in a border cell cluster composed of a mixture of wild-type cells and cells homozygous mutant for tai, Slbo protein is expressed similarly in all of the cells (Bai, 2000).

A second protein known to be required for border cell migration is Drosophila E-cadherin (Shotgun). To determine whether the tai migration defect might be due to reduction in Shotgun expression, egg chambers containing tai mutant clones were stained with antibodies against Shotgun. In all wild-type stages examined, Shotgun accumulates in the central, nonmigratory polar cells, as well as in the junctions between individual border cells. Shotgun colocalizes with cortical F-actin in these locations. Prior to migration, when the border cells are still part of the follicular epithelium, Shotgun also accumulates at the junctions between border cells and nurse cells. However, once the border cells leave the follicular epithelium and invade the neighboring germline cell cluster, much less Shotgun staining is evident at the junctions between the nurse cells and border cells, relative to the level between border cells or in the polar cells. When migration is complete, Shotgun accumulates again in the junctions between the border cells and the oocyte (Bai, 2000).

In tai mutant clusters, Shotgun staining is abnormally elevated at the border cell/nurse cell junctions. In contrast, in slbo mutants, Shotgun expression fails to rise at the time of migration and Shotgun immunoreactivity is only detected at high levels within the polar cells. Armadillo (Arm) colocalizes with Shotgun in wild-type and mutant border cells. The abnormal accumulation of Shotgun and Arm in tai mutants does not appear to result from increased transcription of Shotgun because overexpression of Shotgun in border cells causes neither a migration defect nor specific accumulation of cadherin staining at the border cell/nurse cell junctions. Nor does the abnormal accumulation of Shotgun and Arm appear to be simply a consequence of the migration failure. In addition to slbo, Shotgun and Arm expression were examined in border cells that fail to migrate due to mutations in the jing locus: no defect in either expression or localization of adhesion complexes was observed. Nor are defects in either Shotgun or Arm expression or localization found in border cells that fail to migrate due to expression of dominant-negative Rac (Bai, 2000).

The accumulation of Shotgun at the border cell/nurse cell boundary suggests that the role of tai in border cell migration might be to stimulate turnover of adhesion complexes during migration in order to allow forward movement. One protein believed to play a role in turnover of adhesion complexes is Focal adhesion kinase. Drosophila FAK (Fak56D) is highly enriched in the border cells during their migration, but not in the polar cells (Bai, 2000).

To determine whether Fak56D expression or localization is affected by mutations that disrupt border cell migration, wild-type and slbo mutant egg chambers were stained and the staining was compared to that of egg chambers containing tai mosaic clones. Fak56D expression is significantly reduced in slbo mutant border cells. Furthermore, the level of reduction correlates with the degree of inhibition of migration. That is, in some slbo egg chambers, border cell migration fails completely and the cells remain at the anterior tip. In such egg chambers, Fak56D expression is undetectable. In a minority of slbo mutant chambers, the cells migrate a little. In these egg chambers, Fak56D expression is reduced compared to wild type, but is detectable. In tai mutant border cells, Fak56D expression is present; however, its distribution is altered relative to wild type. Rather than being evenly distributed throughout the cytoplasm, Fak56D appears to accumulate at the would-be leading edge of the cluster. Some border cell clusters that are mutant for tai exhibit partial migration and in these clusters, the abnormal distribution of Fak56D is only slightly affected such that little Fak56D accumulation can be detected at the most posterior position within the cluster. Thus, the severity of the migration defect in tai mutants correlates with the severity of the defect in Fak56D localization (Bai, 2000).

The similarity of Taiman to steroid hormone receptor coactivators suggests that Tai might interact with one or more steroid hormone receptors. The only known steroid hormone in Drosophila is ecdysone, and the ovary is a major site of ecdysone synthesis, which peaks at stage 9. The functional ecdysone receptor is a heterodimer composed of Ultraspiracle (Usp), which is the fly retinoid X receptor (RXR) homolog, and the Ecdysone receptor. To determine whether the ecdysone receptor complex would be a good candidate for interaction with Tai, expression of ecdysone receptor subunits in egg chambers was examined using antibodies against Usp, EcR-A, and EcR-B. EcR-A and EcR-B are distinct isoforms of the EcR subunit, which are generated by alternative splicing. Usp, EcR-A, and EcR-B colocalize with Tai protein in migrating border cells; Usp and EcR-A are expressed generally, in both follicle cells and nurse cells (Bai, 2000).

These observations raise the possibility that the timing of border cell migration might be controlled by ecdysone. To test whether border cell migration is responsive to hormone, the effects of injecting hormone into female flies were examined. It was not expected that increasing the hormone concentration alone would be sufficient to cause precocious border cell migration because expression of the slbo gene and its targets are independently required for migration. Therefore, slbo was precociously expressed using transgenic flies carrying a heat-inducible slbo transgene, followed by injection of hormone. Border cell migration was assayed in stage 8 egg chambers dissected from flies treated with heat shock and hormone, and compared to control flies treated with heat shock and ethanol, or with hormone in the absence of heat shock. Precocious border cell migration was observed in 20% of egg chambers that were treated with both heat shock and hormone but not in controls. The observed effects are consistent with a role for ecdysone in regulating the timing of border cell migration (Bai, 2000).

If the rising ecdysone level at stage 9 is required to stimulate border cell migration, then reducing the ecdysone level should cause a delay in border cell migration. The ecdysoneless mutant ecd¹ is temperature sensitive for production of ecdysone. Females homozygous for ecd¹ are sterile when held at the nonpermissive temperature for 5 days, and egg chambers in these flies arrest development at stage 8 and subsequently degenerate. Border cells fail to develop in these arrested egg chambers. However, when ecd¹ mutants are held at the nonpermissive temperature for 2 days, some stage 10 egg chambers develop, in which border cells differentiate and express Slbo protein. Greater than 50% of these egg chambers exhibit delayed border cell migration (Bai, 2000).

Since the effects on border cell migration of increasing or decreasing ecdysone levels could have been indirect, whether there is a cell autonomous requirement for the ecdysone receptor in border cells was tested. The EcR locus is proximal to available FRT insertion sites, preventing mosaic analysis. Therefore, the analysis was carried out using mutations in usp. Border cells that were homozygous mutant for a null allele of usp exhibit inhibition of border cell migration, but no obvious defects in other follicle cells (Bai, 2000).

To assess whether Tai and the ecdysone receptor are likely to associate in a complex in vivo, Tai expression was examined in third instar larvae. Antibodies against Tai react specifically with the salivary gland nuclei, as well as other larval tissues. Polytene chromosome spreads were stained with antibodies against Tai and Usp proteins in a double labeling experiment. Anti-Tai antibody labels specific loci on the polytene chromosomes. Moreover, Usp and Tai proteins colocalize precisely. Since previous experiments have shown that Usp and EcR colocalize as a complex on polytene chromosomes, these results indicated that Tai colocalizes with the functional Ecdysone receptor complex at specific target sites (Bai, 2000).

Whether expression of Tai can enhance ecdysone receptor-dependent transcriptional activation in EcR-293 mammalian cells was tested. These cells respond to hormone, either ecdysone or an analog known as ponasterone, with a substantial increase in transcriptional activation of genes placed under the control of a cis-acting sequence known as an E/GRE. Transcriptional activation was tested in cells expressing varying amounts of Tai in transient transfection assays. Tai expression increases transcriptional activation up to 5-fold, in a dose-dependent manner, specifically in the presence of hormone (Bai, 2000).

Furthermore, a GST-fusion protein containing the region of Tai protein containing the LXXLL motifs predicted to interact with EcR (residues 1028 to 1235 of Tai) associates with in vitro translated EcR in a ligand-dependent manner. The same fusion protein does not associate detectably with Usp alone. However, in the presence of EcR and ligand, the Tai-GST fusion protein is able to coprecipitate Usp. Taken together, these results suggest that Tai is a bona fide ecdysone receptor coactivator (Bai, 2000).

Thus, Tai appears to be a coactivator of the p160 class based not only on amino acid sequence similarity and overall domain structure, but based also on its in vivo colocalization with EcR, its direct, ligand-dependent binding to EcR, and its ability to potentiate hormone-dependent transcription in cultured cells. The homology of Tai to SRC proteins suggests that Tai might interact with a steroid hormone receptor. Although there are more than 20 genes in Drosophila that code for proteins related to nuclear hormone receptors, ecdysone is the only known steroid hormone. Since SRC proteins require the presence of a ligand in order to interact with receptors, the ecdysone receptor seems like the best candidate partner for Tai. The colocalization of Tai protein with the ecdysone receptor complex at specific chromosomal loci in third instar larva, the direct and ligand-dependent binding of Tai to EcR in vitro, and the ability of Tai to potentiate the ecdysone response in cell culture lend substantial support to this proposal (Bai, 2000).

The ligand-dependent interaction of Tai with the ecdysone receptor suggests that ecdysone regulates border cell migration. The strongest evidence in support of this is that border cells lacking Usp are unable to migrate. Consistent with this observation, numerous unfertilized eggs were produced from females lacking usp function. Moreover usp is required specifically in somatic cells for production of a fertilizable egg. Defects in border cell migration are known to lead to the production of unfertilized eggs. Whether EcR loss of function mutations affect border cell migration could not be examined. This is because the EcR locus, at 42A, is proximal to available FRT insertions, making it impossible to make FLP-mediated mosaic clones. The frequency of X-ray induced mitotic clones is too low to be useful, and marking such clones is problematic. A temperature-sensitive allele of EcR exists and flies at the nonpermissive temperature exhibit a variety of defects in oogenesis, including arrest prior to border cell migration. Even though it was not possible to assess the effect of EcR mutations specifically in the border cells, the observations that hormone injections can lead to precocious border cell migration and that reduced ecdysone levels can lead to delayed migration provide additional support for the hormonal control of migration (Bai, 2000).

The rise in ecdysone after eclosion, specifically in females, occurs in response to adequate nutrition. In the absence of a rich diet, yolk protein synthesis is inhibited and oogenesis does not progress. Yolk protein synthesis can be restored in the absence of a rich diet by applying ecdysone or juvenile hormone (JH) to cultured ovaries. Recent studies indicate that functional ecdysone receptors are required in the germline for progression of oogenesis through vitellogenesis, the stages during which yolk is taken up by the oocyte. In summary, then, adequate nutrition appears to lead to elevated hormone levels, which in turn stimulate yolk protein synthesis and uptake, and progression of oogenesis beyond stage 8. Together with the results reported here, these findings suggest that a rising ecdysone titer coordinates a variety of events that occur in early vitellogenic egg chambers, including border cell migration (Bai, 2000).

These studies indicate a role for steroid hormones in cell motility that is independent of any role in cell proliferation or cell fate determination. If tai function were required for follicle cell proliferation, it would not be possible to generate homozygous mutant clones since the homozygous mutant cells would fail to proliferate. Many homozygous mutant tai clones are found in the follicular epithelium, some of which are quite large; therefore, there does not appear to be a requirement for tai function in follicle cell proliferation. In addition, tai mutant border cells clearly differentiate from the neighboring follicle cells, based on their morphology, and they continue to express all of the border cell markers tested. Therefore, there does not appear to be any detectable change of cell fate or differentiation state in these cells. Rather, they appear to have a relatively specific defect in their ability to migrate through the neighboring nurse cell cluster (Bai, 2000).

These findings may have significance for steroid hormone-dependent human cancers since hormones are known to promote progression of breast, ovarian, and prostate cancers, which includes acquisition of highly invasive characteristics. The prevailing view is that the hormones act to stimulate proliferation of the cancer cells, leading to an increased likelihood of mutation and appearance of an invasive phenotype. However, the data presented in this paper suggest that steroid hormones can also stimulate invasive behavior independently of any discernible effect on proliferation. Thus, steroid hormones, like many peptide growth factors, may possess both mitogenic and motogenic properties. This notion is supported by studies that show effects of an anti-estrogen on metastasis of prostate cancer cells in the rat. Raloxifene, an anti-estrogen, inhibits metastasis of PAIII adenocarcinoma cells to the lymph nodes and lungs, in vivo, without effects on growth of the primary tumor, or proliferation of the PAIII cells in vitro. The treatment also extends the survival of the animals (Bai, 2000 and references therein)

A number of genes that have been described, slbo, jing, breathless, shotgun, and PZ6356 define a slbo-dependent pathway required for border cell migration. Experiments reported in this study indicate that expression of Fak56D also depends upon the slbo pathway. In contrast, tai function appears to be independent of slbo, based on the lack of effect of slbo mutations on tai expression and the lack of effect of tai mutations on slbo expression. In addition, overexpression of tai fails to rescue even mild slbo migration defects and overexpression of slbo fails to rescue tai migration defects. Mutations in either slbo or tai affect cadherin and Fak56D. Whereas slbo function is required for expression of these two proteins, tai function is required for proper subcellular localization of both proteins (Bai, 2000).

The finding that Shotgun is required both in the border cells and in the nurse cells for normal border cell migration is surprising since the prevailing view has been that E-cadherin promotes formation of stable cell–cell adhesion belts and inhibits motility. However, there are numerous exceptions to the general correlation of decreased E-cadherin expression with increasing motility. One particularly interesting exception is the human ovarian epithelium. Normal cells within the human ovarian surface epithelium express relatively low levels of E-cadherin. However, carcinomas derived from this epithelium express high levels of E-cadherin, and overexpression of E-cadherin in T antigen transformed ovarian surface epithelium cells, causing them to become invasive and to form distant metastases in nude mice. Thus, in both human and Drosophila ovaries, E-cadherin seems to promote rather than inhibit motility (Bai, 2000).

Why then do some cells respond to increased cadherin expression by increasing invasiveness whereas other cells respond by decreasing invasiveness? It is proposed that the difference is that some cell types are capable of rapidly turning over E-cadherin-containing adhesion complexes whereas other cells are not. If the complexes can be turned over efficiently, the cells behave like wild-type border cells and become invasive. If the complexes cannot be turned over efficiently, the cells behave as tai mutant border cells, accumulate stable cell–cell adhesion complexes and do not migrate. It will be important to identify the critical downstream targets of Tai because one or more of these may be a protein important for turnover of adhesion complexes (Bai, 2000).

Steroid hormones may stimulate formation and turnover of cadherin-containing cell adhesion complexes in human breast cancer as well. In support of this, MCF7 human breast cancer cells have been found to respond to beta-estradiol treatment by extending motile lamellipodia, which make small, transient, E-cadherin-containing contacts with underlying cells. This behavior is prevented by treatment of the cells with anti-estrogens. Taken together with the proven effectiveness of anti-estrogens in preventing and reversing metastasis of hormone-dependent cancers, these findings suggest that steroid hormones may stimulate invasive cell behavior by facilitating rapid turnover of E-cadherin containing cell adhesion complexes. This could be one mechanism by which amplification of AIB1 contributes to the progression of breast and ovarian cancer (Bai, 2000).

Receptor tyrosine kinases CAD96CA and FGFR1 function as the cell membrane receptors of insect juvenile hormone

Juvenile hormone (JH) is important to maintain insect larval status; however, its cell membrane receptor has not been identified. Using the lepidopteran insect Helicoverpa armigera (cotton bollworm), a serious agricultural pest, as a model, this study determined that receptor tyrosine kinases (RTKs) cadherin 96ca (Cad96Ca), a protein with one n-terminal extracellular cadherin domain, and fibroblast growth factor receptor homologue (FGFR1) function as JH cell membrane receptors by their roles in JH-regulated gene expression, larval status maintaining, rapid intracellular calcium increase, phosphorylation of JH intracellular receptor MET1 and cofactor Taiman, and high affinity to JH III (see Juvenile Hormone III). Gene knockout of Cad96Ca and Fgfr1 by CRISPR/Cas9 in embryo and knockdown in various insect cells, and overexpression of CAD96CA and FGFR1 in mammalian HEK-293T cells all supported CAD96CA and FGFR1 transmitting JH signal as JH cell membrane receptors (Li, 2025).

Juvenile hormone (JH) plays a vital role in insect development and maintaining insect larval status. JH is an acyclic sesquiterpenoid known to enter cells freely via diffusion because of its lipid-soluble character. JH binds its intracellular receptor methoprene-tolerant protein (MET), a basic helix-loop-helix/Per-ARNT-SIM (bHLH-PAS) family protein. MET forms a transcription complex with the transcription factor Taiman (TAI, also known as FISC, p160/SRC, and is a steroid receptor coactivator) to initiate gene transcription. An important gene in the JH pathway is Kruppel homologue 1 (Kr-h1), which encodes the zinc-finger transcription factor Kr-h1. Kr-h1 acts downstream of MET and is induced rapidly by JH to regulate larval growth and development. Other genes, for example, the early trypsin gene of Aedes aegypti (AaEt), JH-inducible 21 kDa protein (Jhp21), JH esterase (Jhe), vitellogenin (Vg), Drosophila JH-inducible gene 1 (Jhi-1), and JH-inducible gene 26 (Jhi-26) are regulated by JH (Li, 2025).

However, some studies suggest that cell membrane receptors also play essential roles in JH signaling. For example, in A. aegypti, receptor tyrosine kinases (RTKs) are involved in JH-induced rapid increases in inositol 1,4,5-trisphosphate, diacylglycerol, and intracellular calcium, leading to activation of calcium/calmodulin-dependent protein kinase II (CaMKII) to phosphorylate MET and TAI, resulting in Kr-h1 gene transcription in response to JH. JH III, also via RTKs, leads to rapid calcium release and influx in Helicoverpa armigera epidermal cells. JH induces MET phosphorylation to increase MET interacting with TAI, which enhances Kr-h1 transcription in H. armigera. In Drosophila melanogaster, JH through RTK and PKC protein kinase C (PKC) induces phosphorylation of Ultraspiracle (USP). The phenomenon that RTK transmits JH signal has long been predicted; however, the RTKs critical for JH signaling have yet to be identified from numerous RTKs in vivo (Li, 2025).

RTKs constitute a class of cell surface transmembrane proteins that play important roles in mediating extracellular to intracellular signaling. Humans carry approximately 60 RTKs, the Drosophila genome encodes 21 RTK genes, Bombyx mori has 20 RTKs, and the German cockroach genome identifies 16 RTKs. H. armigera has 20 RTK candidates with gene codes in the H. armigera genome. The cotton bollworm is a well-known and worldwide distributed agricultural pest in Lepidoptera, which threatens cotton and many other vegetable crops by rapidly producing resistance to various chemical insecticides and Bt-transgenic cotton. Using H. armigera as a model, this study focuses on identifying the RTKs functioning as the JH receptors and demonstrating the mechanism. The RTKs were screened sequentially, including examining the roles of 20 RTKs identified in the H. armigera genome in JH-regulated gene expression to obtain primary candidates, followed by screening of the candidates by their roles in maintaining larval status, JH-induced rapid increase of intracellular calcium levels, JH-induced phosphorylation of MET and TAI, and affinity to JH. The cadherin 96ca (CAD96CA) and fibroblast growth factor receptor 1 (FGFR1) were finally determined as JH cell membrane receptors by their roles in JH-regulated gene expression, maintaining larval status, JH-induced rapid increase of intracellular calcium levels, JH-induced phosphorylation of MET and TAI, and their JH-binding affinity. Their roles as JH cell membrane receptors were further determined by knockdown and knockout of them in vivo and cell lines, and overexpression of them in mammal HEK-293T heterogeneously. These data not only improve knowledge of JH signaling and open the door to studying insect development but also present new targets to explore the new growth regulators to control the pest (Li, 2025).

JH regulates insect development through intracellular and membrane signaling; however, the cell membrane receptors and the mechanism are unclear. In this study, CAD96CA and FGFR1 were screened out from the total 20 RTKs in the H. armigera genome and identified as JH III cell membrane receptors, which transmit JH signal for gene expression to prevent pupation and have a high affinity to JH III (Li, 2025).

JH induces a set of gene expression, such as Kr-h1, Vg, Jhi-1, and Jhi-26 , a rapid intracellular calcium increase, phosphorylation of MET and TAI, and prevents pupation. Several RTKs are involved in JH III-induced gene expression and calcium increase; however, only Cad96ca, Nrk, Fgfr1, and Wsck are involved in the JH III-induced pupation delay, in which, only CAD96CA, NRK, and FGFR1 are involved in the JH-induced phosphorylation of MET1 and TAI, and only CAD96CA and FGFR1 can bind JH III. Therefore, CAD96CA and FGFR1 are finally determined as JH III receptors (Li, 2025).

CAD96CA (also known as Stitcher, Ret-like receptor tyrosine kinase) activates upon epidermal wounding in Drosophila and promotes growth and suppresses autophagy in the Drosophila epithelial imaginal wing discs. There is a CAD96CA in the genome of the H. armigera, which is without function study. This study reports that CAD96CA prevents pupation by transmitting JH signal as a JH cell membrane receptor. CAD96CA of other insects has a universal function of transmitting JH signal to trigger Ca2+ mobilization, as demonstrated by the study in Sf9 cell lines of S. frugiperda and S2 cell lines of D. melanogaster (Li, 2025).

FGFRs control cell migration and differentiation in the developing embryo of D. melanogaster. The ligand of FGFR is FGF in D. melanogaster. FGF binds FGFR and triggers cell proliferation, differentiation, migration, and survival. Three FGF ligands and two FGF receptors (FGFRs) are identified in Drosophila. The Drosophila FGF-FGFR interaction is specific. Different ligands have different functions. The activation of FGFRs by specific ligands can affect specific biological processes. The FGFR in the membrane of Sf9 cells can bind to Vip3Aa toxin. One FGF and one FGFR are in the H. armigera genome, which have yet to be studied functionally. The study found that FGFR prevents insect pupation by transmitting JH signal as a JH cell membrane receptor. Exploring the molecular mechanism and output by which multiple ligands transmit signals through the same receptor is exciting and challenging in future work (Li, 2025).

CAD96CA and FGFR1 have similar functions in JH signaling, including transmitting JH signal for Kr-h1 expression, larval status maintaining, rapid intracellular calcium increase, phosphorylation of transcription factors MET1 and TAI, and high affinity to JH III. CAD96CA and FGFR1 are essential in the JH signal pathway, and the loss-of-function of each is sufficient to trigger strong effects on pupation, suggesting they can transmit JH signal individually. The difference is that CAD96CA expression has no tissue specificity, and the Fgfr1 gene is highly expressed in the midgut. A possibility is that CAD96CA and FGFR1 play roles by forming homodimer or heterodimer with each other or with other RTKs in tissues, which needs to be addressed in future studies. CAD96CA and FGFR1 transmit JH III signals in three different insect cell lines, suggesting their conserved roles in other insects (Li, 2025).

Homozygous Cad96ca null Drosophila die at late pupal stagee, suggesting that CAD96CA is critical to insect pupation. This study further revealed that Cad96ca mosaic mutation by CRISPR/Cas9 caused precocious pupation in H. armigera, suggesting that CAD96CA plays roles to prevent pupation. Similarly, null mutant of Fgfr1 or Fgfr2 in mouse is embryonic lethal. Htl (Fgfr) homozygous mutant in D. melanogaster die during late embryogenesis, too, suggesting FGFR1 is important to embryogenesis. However, in H. armigera, Fgfr1 mosaic mutation mainly caused precocious pupation, suggesting FGFR1 is necessary to prevent pupation. The double mutation of Cad96ca and Fgfr1 caused earlier pupation and death compared to the single mutation of Cad96ca or Fgfr1. These data suggested that both CAD96CA and FGFR1 can transmit JH signal to prevent pupation independently and cooperatively. MET1 is the intracellular receptor of JH. Knockout of Met1 in the B. mori leads to precocious metamorphosis and death in the penultimate instar. In the Spodoptera exigua, knockout of Met1 also results in precocious metamorphosis and death in the penultimate and final instars. In D. melanogaster, both Met and Gce null mutants die during the larval-pupal transition. In the H. armigera, most of the Met1 mutants died during the transformation from the final instar larva to the pupa, too. These data suggest that JH via MET1 prevents pupation. In the triple mutants of Met1, Cad96ca, and Fgfr1, most larvae died at the 5th and 6th instar larval stages, which is much more serious than the Met1 mutation, or Cad96ca and Fgfr1 double mutation, because of the mutation both intracellular receptor MET1 and two membrane receptors CAD96CA and FGFR1 of JH. These data suggest that JH exerts a complete regulatory role through cell membrane receptors and intracellular receptor, because the cell membrane receptors regulate the phosphorylation of the intracellular receptor MET1 and the interacting transcription factor TAI. The results from different insects suggest that JH via MET1, CAD96CA, and FGFR1 play roles in preventing metamorphosis. In B. mori, after the knockout of JH acid methyltransferase (Jhamt) using TALENs, the larvae died during L1 or L2 larval stages. The knockdown of Jhamt in Drosophila by RNAi does not exhibit a visible effect on development. Knockdown of Jhamt by RNAi in the Tribolium larval stage results in precocious larval-pupal metamorphosis. Homozygous Jhamt -/- larvae in mosquitoes hatch normally and live to the final-instar larvae (L4), and die prior to pupation. The results from different insects suggest that the insect development in the early larval instars is less dependent on JH. The phenotypes from H. armigera CAD96CA and FGFR1 editing are consistent with the general knowledge that the primary role of JH is to maintain larval status and antagonize 20E-induced metamorphosis. The insulin/insulin-like growth factor signaling (IIS) plays a major role in promoting cell proliferation and determining the larval growth rate, and 20E promotes metamorphosis. These hormones cross talk to regulate insect growth and metamorphic development (Li, 2025).

The phenotypes of gene mutation in H. armigera are somehow different from those obtained by homozygous mutation in other animals, due to the mosaic mutation by CRISPR/Cas9. In addition, RNAi of Cad96ca and Fgfr1 was observed precocious pupation as was the case in CRISPR/Cas9, suggesting the RNAi can be used for the study of gene function in insects, especially when the gene editing is embryonic lethal (Li, 2025).

The knockdown of Cad96ca, Nrk, Fgfr1, and Wsck showed phenotypes resistant to JH III induction and the decrease of Kr-h1 and increase of Br-z7 expression, but knockdown of Vegfr and Drl only decreased Kr-h1, without increase of Br-z7. Br-z7 is involved in 20E-induced metamorphosis in H. armigera, whereas, Kr-h1 is a JH early response gene that mediates JH action and represses Br expression. The high expression of Br-z7 is possible due to the down-regulation of Kr-h1 in Cad96ca, Nrk, Fgfr1 and Wsck knockdown larvae. The different expression profiles of Br-z7 in Vegfr and Drl knockdown larvae suggest other roles of Vegfr and Drl in JH signaling, which need further study (Li, 2025).

This study found six RTKs that respond to JH induction by participating in JH-induced gene expression and intracellular calcium increase; however, they exert different functions in JH signaling, and finally CAD96CA and FGFR1 are determined as JH cell membrane receptors by their roles in JH-induced phosphorylation of MET1 and TAI and binding to JH III. The RTKs transmitting JH signal were screened primarily by examining some of JH-induced gene expression. By examining other genes or by other strategies to screen the RTKs might find new RTKs functioning as JH cell membrane receptors; however, the key evaluation indicators, such as the binding affinity of the RTKs to JH and the function in transmitting JH signal to maintain larval status are essential (Li, 2025).

In addition, GPCRs also play a role in JH signaling. JH triggers GPCR, RTK, PLC, IP3R, and PKC to phosphorylate Na+/K+-ATPase-subunit, consequently activating Na+/K+-ATPase for the induction of patency in L. migratoria vitellogenin follicular epithelium; JH activates a signaling cascade including GPCR, PLC, extracellular Ca2+, and PKC, which induces vitellogenin receptor (VgR) phosphorylation and promotes vitellogenin (Vg) endocytosis in Locusta migratoria. JH activates a signaling cascade including GPCR, Cdc42, Par6, and aPKC, leading to an enlarged opening of patency for Vg transport. In Tribolium castaneum, the dopamine D2-like receptor-mediated JH signaling promotes the accumulation of vitellogenin and increases the level of cAMP in oocytes. In H. armigera, GPCRs are involved in JH III-induced broad isoform 7 (Br-Z7) phosphorylation. In summary, these published results indicate that RTKs and GPCRs contribute to JH signaling on the cell membrane; however, the GPCR functions as JH receptor needs to be addressed in the future study. The RNAi of RTKs does not affect JH-induced Jhi-1 expression, which implies that other receptors exist, presenting a target for future study of the new JH III receptor (Li, 2025).

RTKs are high-affinity cell surface receptors for many cytokines, polypeptide growth factors, and peptide hormones. Up to now, there is no report that RTK binds lipid hormone. This study determined that CAD96CA and FGFR1 have a high affinity to JH III by MST and ITC methods after they were isolated from the cell membrane (Li, 2025).

The [3H]JH III detection method is used to determine Drosophila MET in vitro translation product binding JH III, and Tribolium MET binding JH III. However, the commercial production of [3H]JH III has ceased, whereas the microscale thermophoresis (MST) method is a widely used method to detect protein binding of small molecules. Therefore, MST was used in this study as the alternative method to measure the binding strengths of RTKs with JH III. Using the MST method, this study determined that the saturable specific binding of Helicoverpa MET1 to JH III is Kd of 6.38 nM, which is comparable to that report for Drosophila MET and Tribolium MET determined using [3H]JH III, confirming MST method can be used to detect protein binding JH III. The CAD96CA exhibited saturable specific binding to JH III with a Kd of 11.96 nM, and FGFR1 showed a Kd of 23.61 nM, which is higher than that of MET1 for JH III, suggesting lower binding affinity of RTKs than the intracellular receptor MET1 for JH III. A similar phenomenon is reported in another study, the binding affinities of steroid membrane receptors are orders of magnitude lower than those of nuclear receptors. NRK did not bind JH III. One possible explanation is that NRK has a low affinity to JH III and thus transmits JH signal without binding JH, or NRK requires association with other proteins to play roles. This study provides new evidence for the binding of lipid hormones by RTK and a new method to study the binding of ligands to receptors (Li, 2025).

This study also verified the affinity of CAD96CA and FGFR1 with JH III through the ITC method, determining their respective Kd values as 79.6 and 88.5 nanomolar. ITC is a versatile analytical method for the character of molecular interactions. ITC is applied in the membrane protein family, containing GPCRs, ion channels, and transporters. The ITC method requires relatively high ligand and receptor concentrations for better saturation curves. However, when a protein solution of 1000 nM, protein aggregation occurred, thus a protein solution was used with a concentration of 700 nM. The Kd value detected by ITC is slightly higher than the result of the MST method; the results are sufficient to confirm the high affinity of CAD96CA and FGFR1 binding to JH III (Li, 2025).

Although JH I and JH II are natural hormones for lepidopteran larvae, H. armigera and B. mori also respond to JH III. In B. mori Bm-aff3 cells, the effective concentrations (EC50) of JHs (JH I, JH II, JH III, JHA, or methyl farnesoate) to induce Kr-h1 transcription are 1.6x¹⁰10, 1.2x¹⁰10, 2.6x¹⁰10, 6.0x¹⁰8, and 1.1x¹⁰7 M, respectively. In cultures of wing imaginal discs from B. mori, 1-2 μM JH III promotes cuticle protein 4 gene expression (Deng et al., 2011). The effective concentration of JH III to induce rapid calcium increase in HaEpi cells is ≥1 &mi;M and 500 ng of 6th instar larva. JH III is a commercially available reagent; therefore, JH III was used to carry out the experiments in this study, and the results hypothesize the possibility of CAD96CA and FGFR1 binding other JHs in addition to JH III, which should be addressed in future study (Li, 2025).

MET is determined as JH’s intracellular receptor by its characters binding to JH and regulating Kr-h1 expression. In this study, cell membrane receptors CAD96CA and FGFR1 are also able to bind JH III and transmit JH III signal to regulate a set of JH III-induced gene expression including Kr-h1. Obviously, both intracellular receptor MET and cell membrane receptor CAD96CA and FGFR1 are involved in JH III signaling as receptors. JH III transmits signal by cell membrane receptor and intracellular receptor at different signaling stages, with cell membrane receptor CAD96CA and FGFR1 inducing rapid Ca2+ signaling, which regulates the phosphorylation of MET and TAI to enhance the function of MET for gene transcription, and the intracellular receptor MET regulates gene transcription by diffusion into cells based on its lipid characteristics (Li, 2025).

The study in human cell line HEK293 shows that overexpression of B. mori JH intracellular receptor MET2 and its cofactor SRC together in HEK293 cells may induce JH-dependent luciferase reporter expression through a plasmid that contains the JH specific kJHRE (JH response element containing the E-box core sequence for JH binding), suggesting JH can diffuse into cells to initiate a gene expression when the insect MET2 and SRC and kJHRE exist. This study showed that overexpressing CAD96CA or FGFR1 in HEK-293T cells elicits Ca2+ elevation under JH III induction, suggesting CAD96CA or FGFR1 transmit a rapid signal of JH III in HEK-293T cells, which might trigger further cellular responses of HEK-293T to JH III. These data suggest that both cell membrane receptors CAD96CA and FGFR1 and intracellular receptor MET of JH can respond to JH. These proteins might be used as switches to induce a gene expression or regulate cell fate in heterogeneous cells by JH induction when the side effects are determined (Li, 2025).

CAD96CA and FGFR1 were involved in JH III signaling, including maintaining larval status, JH III-induced rapid intracellular calcium increase, gene expression, and phosphorylation of MET and TAI. CAD96CA and FGFR1 had high affinity to JH III and were possible cell membrane receptors of JH III and other JHs. CAD96CA and FGFR1 had a general role in transmitting the JH III signal for gene expression in various insect cells, suggesting their conserved roles in other insects. JH III transmits the signal by either cell membrane receptor or intracellular receptor at different stages in the signaling, with JH III transmitting the signal by cell membrane receptor CAD96CA and FGFR1 to induce rapid Ca2+ signaling, which regulates the phosphorylation of MET and TAI to enhance the function of MET for gene transcription, and intracellular receptor MET regulates gene transcription by diffusion into cells based on its lipid characteristics. CAD96CA and FGFR1 can transmit JH signal to prevent pupation independently and cooperatively. This study presents a platform to identify the agonist or inhibitor of JH cell membrane receptors to develop an environmental-friendly insect growth regulator (Li, 2025).

GENE STRUCTURE

cDNA clone length - 7535

Bases in 5' UTR - 813

Bases in 3' UTR - 614

PROTEIN STRUCTURE

Amino Acids - 2035

Structural Domains

The tai locus encodes a protein with amino acid sequence similarity to steroid hormone receptor coactivator proteins of the p160 family (SRCs). SRCs bind to steroid hormone receptor (SHR) complexes in a ligand-dependent manner and potentiate hormone-induced transcriptional activation (Leo, 2000). The most related protein is AIB1, a steroid hormone receptor coactivator that is amplified in breast and ovarian cancer (Anzick, 1997). SRCs typically contain a basic helix-loop-helix (bHLH) domain near the N terminus, a PAS domain, LXXLL motifs, which are responsible for binding to the hormone bound receptor (McInerney, 1998), and one or more polyglutamine stretches that mediate transcriptional activation. The predicted Tai protein contained all of the domains featured in the p160 class of SRCs, and the top 19 BLAST scores are from members of this family. The highest level of amino acid sequence identity is found in the bHLH domain, which is 48% identical and 71% similar between AIB1 and Tai (Bai, 2000).

date revised: 15 April 2001

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