The larval fat body of newly eclosed adults of Drosophila was found to contain a single major binding protein specific for juvenile hormone (JH). Binding to this protein was saturable, of high affinity, and specific for JH III. The protein has a subunit molecular weight (Mr) of 85,000, as determined by photoaffinity labeling. The same or similar JH-binding protein was found in larval fat body and cuticle of third instar larvae and in male accessory glands and heads of newly eclosed adults. It was not found in several other tissues in adults. Male accessory gland cytosol from wild-type flies was found to contain a single binder with a dissociation constant (KD) of 6.7 nM for JH III; a binder in similar preparations from the methoprene-tolerant (Met) mutant had a KD value 6-fold higher. JH III stimulated protein synthesis in glands cultured in vitro, but this effect was reduced in Met flies as compared to wild-type flies, establishing a correlation between JH binding and biological activity of the hormone. In addition, glandular protein accumulation during the first 2 days of adult development was less in Met flies than in wild-type flies. These results strongly suggest that the binding protein identified here mediates this JH effect in male accessory glands and thus is acting as a JH receptor (Shemshedini, 1990a).
Juvenile hormones (JHs) of insects are sesquiterpenoids that regulate a great diversity of processes in development and reproduction. As yet the molecular modes of action of JH are poorly understood. The Methoprene-tolerant (Met) gene of Drosophila melanogaster has been found to be responsible for resistance to a JH analogue (JHA) insecticide, methoprene. Previous studies on Met have implicated its involvement in JH signaling, although direct evidence is lacking. This study examined the product of Met (MET) in terms of its binding to JH and ligand-dependent gene regulation. In vitro synthesized MET directly binds to JH III with high affinity, consistent with the physiological JH concentration. In transient transfection assays using Drosophila S2 cells the yeast GAL4-DNA binding domain fused to MET exerted JH- or JHA-dependent activation of a reporter gene. Activation of the reporter gene was highly JH- or JHA-specific with the order of effectiveness: JH III JH II > JH I > methoprene; compounds which are only structurally related to JH or JHA did not induce any activation. Localization of MET in the S2 cells is nuclear irrespective of the presence or absence of JH. These results suggest that MET may function as a JH-dependent transcription factor (Miura, 2005).
The MET protein was obtained by using coupled in vitro transcription/translation (TNT) reaction. The production of the full-length polypeptide was confirmed by analysing the product of reaction in the presence of 35S-methionine by SDS/PAGE and autoradiography. The principal product had the expected full-length molecular mass of 79 kDa. The programmed lysate was used as a protein source for binding assay by the dextran-coated charcoal (DCC) method. Specific binding of 3H-labeled JH III to the TNT protein showed saturable profile. By these experiments, it was demonstrated that MET binds to JH III directly with a nanomolar Kd value although the possibility that factors in the rabbit reticulocyte lysate may influence binding cannot be ruled out. The specific binding was competed away by 100-fold molar excess of cold JH III (Miura, 2005).
The Met gene product was examined for its transactivation ability. The binding sequence motif of MET is presently uncertain. In addition, it is unknown whether MET functions as a homo- or heterodimer. So, a heterologous approach was used with the yeast GAL4–DBD (DNA binding domain) fusion/UAS (upstream activating sequence) system. The GAL4–DBD possesses a zinc finger that directs homodimerization and binding to UAS elements, and a potent nuclear localization sequence. By using this system, it was possible to assess the transactivation potential of MET independently of its dimerization properties or nuclear localization signals. The MET protein was expressed in S2 cells as a fusion with GAL4–DBD, and a luciferase reporter construct possessing five tandem copies of the UAS in its regulatory region was used. In this system, the effect of JH on transcription from the reporter gene was tested (Miura, 2005).
When an empty expression vector was transfected, the addition of 5 µm JH III to the culture medium caused no elevation of reporter activity over that of controls given the vehicle ethanol. Expression of native MET lacking GAL4–DBD slightly elevated the reporter activity, but did not show any JH dependency. Next, only the GAL4–DBD was expressed. In this case, the reporter activity was elevated about twofold above the empty vector control in either the presence or absence of JH III, indicating that the GAL4–DBD translocates into the nucleus and functions as a moderate, constitutive activator of transcription in a JH-independent manner. The GAL4–DBD–MET fusion in the presence of ethanol did not bring about any enhanced reporter activity relative to the empty vector control, but when JH III was added, the reporter activity was elevated about fivefold over the case of the empty vector control or the case of the GAL4–DBD–MET with ethanol. This activation by JH III can also be described as about twofold when compared to the case of GAL4–DBD with JH III. This indicates that MET has transactivation domain(s), and its transactivation function is JH dependent. It is noteworthy that in the absence of JH III the MET moiety of the fusion protein repressed the moderate transactivation produced by GAL4–DBD. This suggests that unliganded MET may function as a transcriptional repressor (Miura, 2005).
If MET represents a JH-dependent transcription factor, it should show stringent ligand specificity. To test this, several compounds that are structurally related to JH or JHA but show no JH activity were examined in the GAL4-MET fusion/UAS system. The effects of these potential ligands on the reporter activity are shown as fold induction by dividing the activity obtained with the pAcGAL4–DBD–Met by that in negative controls using empty pAc vectors. Addition of squalene, farnesol, farnesyl acetate and geraniol at a final concentration of 5 µm did not result in any activation of the reporter. JH III, however, again brought about enhanced reporter activity. Interestingly, a JHA (methoprene) showed weaker ligand activity than JH III. Thus, the transactivation exerted by MET shows stringent ligand specificity apparently related to JH activity, ruling out nonspecific transactivation by lipid-soluble compounds (Miura, 2005).
The binding assay showed that MET has a nanomolar level Kd for JH III and several potential ligands weere used at 5 µm in the experiments described above. If MET functions as a JH-dependent transcription factor, it should respond to nanomolar levels of ligand, consistent with its high affinity for JH III. Three natural JHs, JH I, JH II, and JH III, and the JHA methoprene were tested in varying concentrations using the GAL4-MET fusion/UAS transfection assay. The effects on the reporter activity were recored as fold induction. Every compound tested showed ligand activity on transactivation, nearing saturation at 500 nm while showing only marginal increase at 5 µm. Among these, JH III, which is one of the native JHs of Drosophila, was found to be the most effective over the range of concentrations tested. Of note is that JH III was conspicuously active in the range of 5–50 nm, whereas the other JHs or JHA showed only slight effects. The other native JH of Drosophila, JH-bisepoxide was not tested. The induction activities are in the following order: JH III JH II > JH I > methoprene. The most effective transcriptional activation produced by Drosophila MET with its native JH species further supports the putative role of MET as a JH-dependent transcription factor. These data, thus, indicate that the threshold activity concentration of JHs is reasonably low in this transient transfection system (Miura, 2005).
In these transfection assays, MET was fused to GAL4–DBD, which has a nuclear localization sequence. To test the subcellular localization of MET, a fusion to enhanced green fluorescent protein (EGFP), which does not have a nuclear localization sequence, was used. S2 cells were transfected with the expression plasmid pAcMET–EGFP together with the reporter and coreporter constructs used above. After transfection, cells were incubated for 24 h in the presence or absence of JH III, then observed by Nomarski DIC (differential-interference contrast) or fluorescence microscopy. In both cases, the fluorescence of the fusion proteins was seen in the nucleus. In these experiments the use of cultured cells allows for complete depletion of JH. These observations are consistent with the previous report in vivo (Pursley, 2000) and rule out the ligand-dependent nuclear translocation reported for the aryl hydrocarbon receptors (Ahrs) of vertebrates. Then, how is JH transported to the nucleus? A process such as vertebrate retinoid transport including cellular retinol-binding protein may be involved (Miura, 2005).
The principal new contributions of this study are: (1) demonstration of direct, reversible binding of JH III to MET; (2) demonstration of its JH-dependent transactivation potential. The former was enabled by the use of coupled in vitro transcription and translation, since difficulty was experienced in obtaining soluble preparations of full-length bHLH-PAS proteins from mosquitoes using prokaryotic expression systems. The binding of JH III by MET showed high affinity with a nanomolar Kd value, and was competed away by an excess of cold JH III (Miura, 2005).
In the present study, MET was tethered to a promoter by using the GAL4–DBD fusion/UAS reporter system. In this heterologous system, the MET fusion exhibited specific ligand-dependent activation of a reporter gene placed downstream of the UAS. The MET fusion responded only to JH or the JHA methoprene while compounds that are structurally related but hormonally inactive elicited no response. Among compounds tested, JH III was the most effective ligand, even at nanomolar concentrations, which is in accordance with its nature as one of Drosophila's native JHs. The typical range of concentration for JH In insect haemolymph is 0.3–180 nm. Further, the maximal JH titre in the Drosophila life cycle is 5–7 pmol·g-1 wet weight, which would correspond to 25–35 nm in the haemolymph, assuming that haemolymph occupies one-fifth of the body weight. In view of these physiological JH titres, it is thus notable that in this study JH III was found to be overwhelmingly active in the physiological range of 5–50 nm over the other JHs or JHA. The ligand-dependent transactivation profile exhibited by MET clearly rules out the possibility that it is simply a JH binder, like cytosolic JH binding proteins, and suggests that it might play a role in JH signaling in vivo (Miura, 2005).
Conformational changes of recombinant Drosophila USP exposed to several different farnesoid compounds including natural JHs have been reported. JH III and JH I at 100 µm elicit the conformational changes to a similar degree whereas JH II is the least effective. Furthermore, by using Drosophila white puparial bioassay it has been have demonstrated that the biochemical differences in the three JHs mentioned above parallel the respective biological activity. For example, in prevention of adult emergence the 50% effective doses (ED50s) for JH I–III are 153, 678 and 143 pmol·puparium-1, respectively. The ED50 of methoprene has been reported as 5 pmol·puparium Another point is that JH I has been shown to be more active than JH II in the white puparial assay whereas JH II is more active in the current transfection assay. The difference between these two studies is the concentrations of JH used. In the current assay JH I and JH II were similarly much less effective than JH III in the physiological range while these differences were somewhat obscured at higher doses, although JH II was still more effective than JH I. At present this discrepancy is not explained. Possibly, there might be more than one pathway of JH signaling underlying in the white puparial bioassay, one mediated by USP and another by MET (Miura, 2005).
In the reporter assays, it was noted that unliganded MET repressed the intrinsic activation function possessed by GAL4–DBD. Although GAL4–DBD is believed to lack transactivation domains, it showed moderate transactivation potential in Drosophila S2 cells in this study. The nuclear localization of MET was confirmed by the finding that the MET–EGFP fusion is concentrated in the nuclei of transfected S2 cells. In addition, GAL4–DBD has a nuclear localization sequence. Therefore, it is reasonable to consider that the GAL4–DBD fusion of MET sits on the UAS of the reporter construct even in the absence of ligand, and that the MET moiety is responsible for the observed repression. In the case of vertebrate Ahr, a multimeric complex including hsp90 anchors the unliganded Ahr in the cytoplasm, thereby preventing its transactivation function. Upon ligand binding, Ahr translocates to the nucleus and forms a transcription factor complex with Arnt. In fact, the C-terminal portion of Ahr fused to GAL4–DBD has been shown to act as a constitutive activator of gene regulation. Contrary to this, MET exists in the nucleus even in the absence of ligand. Upon ligand binding, it becomes a transcriptional activator. This resembles the ligand-dependent activation that has been shown in the activation function of many nuclear hormone receptors, rather than the case of the vertebrate Ahr whose activation function is regulated by its subcellular localization (Miura, 2005).
Two questions arise here as to whether MET functions as a homo- or heterodimer, and as to what DNA sequences are responsible for the binding of this transcriptional regulator complex. These questions are related since DNA-binding specificities of bHLH-PAS proteins are determined by their dimerization properties. For example, the dioxin receptor complex Ahr/Arnt heterodimer binds to TNGCGTG. Ahr recognizes the 5'-half-site TNGC, while Arnt recognizes the 3'-half-site GTG. Arnt is also capable of forming a homodimer that recognizes a consensus palindromic E-box sequence, CACGTG. Drosophila Sim protein forms a heterodimer with Tango (a Drosophila Arnt-like protein) and binds to ACGTG core sequence. Thus, DNA binding specificities of bHLH-PAS dimers are dependent upon the dimer configuration while Arnt or Tango always recognize the GTG motif. In the present study, the GAL4–DBD fusion of MET was used in transfection assays. Under these conditions MET is likely to behave as a homodimer because of its overexpression and because of dimerization interfaces provided by the GAL4–DBD moiety. Therefore, the natural dimerization partner and binding sequence of MET are unknown at present. Since the bHLH domain of MET shows relatively high similarity to vertebrate Arnts, the use of the consensus sequence CACGTG may be a good starting point to answer these questions (Miura, 2005).
Based on the framework by Wilson and coworkers, these results have further supported the notion that MET may function as a JH-dependent transcription factor. In further studies, identification of its target genes will help elucidate its in vivo function. Molecular dissection of MET and structural studies may lead to the development of new biologically active JHA and new strategies for pest management (Miura, 2005).
Metamorphosis of holometabolous insects, an elaborate change of form
between larval, pupal and adult stages, offers an ideal system to study the
regulation of morphogenetic processes by hormonal signals. Metamorphosis
involves growth and differentiation, tissue remodeling and death, all of which
are orchestrated by the morphogenesis-promoting ecdysteroids and the
antagonistically acting juvenile hormone (JH), whose presence precludes the
metamorphic changes. How target tissues interpret this combinatorial effect of
the two hormonal cues is poorly understood, mainly because JH does not prevent
larval-pupal transformation in the derived Drosophila model, and
because the JH receptor is unknown. The red flour beetle
Tribolium castaneum has been used to show that JH controls entry to metamorphosis
via its putative receptor Methoprene-tolerant (Met). This study demonstrates that
Met mediates JH effects on the expression of the ecdysteroid-response gene
Broad-Complex (BR-C). Using RNAi and a classical mutant, it has been
show that Tribolium BR-C is necessary for differentiation of pupal
characters. Furthermore, heterochronic combinations of retarded and
accelerated phenotypes caused by impaired BR-C function suggest that
besides specifying the pupal fate, BR-C operates as a temporal
coordinator of hormonally regulated morphogenetic events across epidermal
tissues. Similar results were also obtained when using the lacewing
Chrysopa perla (Neuroptera), a member of another holometabolous group
with a primitive type of metamorphosis. The tissue coordination role of BR-C
may therefore be a part of the Holometabola groundplan (Konopova, 2008).
In both Tribolium and Chrysopa, BR-C RNAi compromises the
larval-pupal transition without affecting earlier development, regardless of
the time of dsRNA injection. The TcBR-CKS342 homozygotes
die at the same stage. These data suggest that the moderate levels of
BR-C mRNAs, detectable during premetamorphic stages in both species,
has no essential role. This scenario would agree with the fact that zygotic
BR-C function is not required in Drosophila BR-C null
nonpupariating mutants until the onset of metamorphosis. However, as neither RNAi nor the likely hypomorphic TcBR-CKS342 allele present a complete loss-of-function situation, a possibility that BR-C plays some
additional role, not visualized by the phenotypes cannot be excluded. Importantly, the lethal phase correlates with a strong upregulation of BR-C expression. At
least in beetles, this stage coincides with a peak of ecdysteroid titer that
causes larvae to initiate prepupal development (Konopova, 2008).
In contrast to Drosophila npr1 mutants, metamorphosis was not
completely blocked by BR-C deficiency in Tribolium or
Chrysopa. Instead the arrested prepupae showed a blend of larval,
pupal, and partially even adult features. Based on the absence of the
pupal-specific gin traps in Tribolium and on the surface
microsculpture, the cuticle was apparently larval in both species, thus
confirming the requirement of BR-C for the pupal commitment of the
epidermis. Interestingly, although the thorny cuticle in Chrysopa BR-C(RNAi)
animals was distinctly larval, similar to in Tribolium, the body
pigmentation resembled that of pupae. It is not certain whether this mixed
character of the epidermis might be due to persisting CpBR-C function, or might be because CpBR-C is not necessary for the pupal pigmentation (Konopova, 2008).
Pupal characters in BR-C(RNAi) animals included rudimentary wings.
In particular, the weak phenotypes in Tribolium (produced with either
isoform-specific or diluted common-core dsRNAs) revealed that wing elongation
was highly sensitive to BR-C depletion. A similar effect of BR-C RNAi
was described for pupal appendages in Bombyx mori.
BR-C silencing prevented the gradual wing enlargement even in larvae
of the hemimetabolous milkweed bug Oncopeltus fasciatus.
Imaginal discs fail to elongate in Drosophila br mutants with
disrupted BR-C Z2 function. The short legs and wings are not due to insufficient
proliferation of the disc cells but are due to their inability to change shape
in response to the ecdysteroid. This cell shape change requires cytoskeletal
components whose mutations enhance the effect of br. The
rudimentary wings, present even in animals most severely affected by
TcBR-CKS342 mutation or by RNAi, suggest that cell shape
changes, rather than cell proliferation may be disrupted by the loss of BR-C
in Tribolium as well. Growing wings marked by EGFP in arrested beetle
prepupae support this idea. The legs in Tribolium BR-C(RNAi) animals were short also but were distally specified as pupal with two tarsal claws. By contrast, the arrested Chrysopa prepupae retained pretarsi with the larval-specific
elongated arolium, thus suggesting a stronger requirement for BR-C
function in the Chrysopa leg (Konopova, 2008).
Except for small deviations, gross morphology of Tribolium genital
segments with the pupal genital papillae was pupal in BR-C(RNAi)
animals. In addition, the larval-pupal transformation of the visual system was initiated,
as larval stemmata were replaced with ommatidia of the compound eyes. However,
as in Drosophila, TcBR-C was important for compound eye
differentiation. These observations suggest that not all aspects of pupal
development are completely blocked by BR-C depletion (Konopova, 2008).
While the above described structures are retarded in their development in
BR-C(RNAi) animals, others appeared accelerated in their development
towards the adult state, although none could be unambiguously defined as
adult. For instance, the antennae in Tribolium or the compound eyes
in Chrysopa resembled their adult counterparts, but in fact were
intermediates between pupal and adult organs. These heterochronic phenotypes
suggest that BR-C may not only be a pupal specifier,
but rather a temporal coordinator of the extensive morphogenesis in diverse
tissues during metamorphosis (Konopova, 2008).
Drosophila organs require a temporally regulated balance between
both inductive and repressive BR-C functions, represented by the individual
isoforms. Two alternative explanations are seen for the heterochronically advanced
phenotypes. First, these structures may require BR-C to repress precocious adult morphogenesis in them, but the inductive BR-C function is dispensable for development beyond larval state. Consequently, loss of BR-C accelerates their development. Second, if both functions are required but the repressive one is more sensitive to reduced BR-C dose, then the inductive function will prevail under an incomplete BR-C knockdown. The first alternative alternative is favored, because progression beyond the pupal stage seems to depend on BR-C downregulation (Konopova, 2008).
Periods of JH absence are required first in larvae to initiate the pupal
program, and later in pupae to exit it. BR-C in both cases promotes the pupal
fate, and therefore JH must regulate BR-C differently in
larvae and in pupae. In lepidopteran, as well as in Tribolium larvae,
JH prevents BR-C expression until the onset of metamorphosis, and
presumably that is how JH prevents pupal differentiation. Conversely, removal
of the JH source (allatectomy) causes both BR-C misexpression and
precocious pupal development. In pupae, ectopic JH induces BR-C, and in many
insects, including Tribolium, such JH application causes reiteration of the pupal stage. In Drosophila, BR-C misexpression alone is sufficient to inhibit adult cuticle formation. BR-C is therefore a prime target of JH signaling, but how JH
regulates BR-C expression is unknown (Konopova, 2008).
Precocious pupation, triggered by interference with the
putative JH receptor Met, coincided with precocious TcBR-C mRNA
increase in the sixth instar. Thus, disrupted JH signaling induced
TcBR-C similarly to allatectomy in lepidopteran larvae. As
expected, TcBR-C not only marked but also was necessary for the
untimely pupation, as TcMet; TcBR-C double-RNAi resulted in
a phenotype similar to TcBR-C RNAi alone, i.e. entry to a lethal
prepupal stage, except one or two instars too early.
Therefore, although the metamorphic program could be prematurely induced by
silencing of TcMet, it could not be completed without TcBR-C. However, loss of Met has been shown to worsen the effect of BR-C mutations in Drosophila, without altering BR-C expression. This again might reflect the different response to JH in the fly (Konopova, 2008).
The evidence that TcMet is required for regulation of
TcBR-C came from pupae, where the JH mimic methoprene induced
TcBR-C mRNA, but not after TcMet knockdown. This result
places TcBR-C downstream of TcMet in JH signaling.
Importantly, the averting of ectopic TcBR-C expression by
TcMet RNAi also rescued the methoprene-treated animals from repeating
the pupal stage and allowed them to become adult. Together, these findings suggest that, similar to in Drosophila, downregulation of BR-C is required to exit the pupal state in Tribolium (Konopova, 2008).
The following model for BR-C function in holometabolan metamorphosis. In larvae, JH acts through Met to prevent BR-C induction until the final
instar, when JH decline relieves the repression, and BR-C coordinates pupal
morphogenesis. Loss of BR-C function causes both retardation and acceleration
of development in diverse epidermal tissues, thus producing a mix of larval-,
pupal- and adult-like features. In early pupae, low JH titer normally allows
BR-C expression to drop, which is necessary for proper adult
differentiation. Exogenous JH, again acting via Met, causes BR-C misexpression, which in turn promotes another round of pupal, instead of adult, development. Whether Met regulates BR-C expression directly, and what determines whether BR-C will be repressed or activated requires further work (Konopova, 2008).
Drosophila S2 cells were screened for the presence of endogenous MET, but neither Western blotting with MET polyclonal antibody nor Met mRNA amplification by RT-PCR performed with S2 cell-derived RNA detected MET protein or mRNA. Therefore, the proteins of interest were introduced into S2 cells by expression of the respective cDNAs following transfection.
The interaction between MET and GCE proteins co-expressed in S2 cells was examined as GST-MET and GCE-V5 fusion proteins. Using a GSH pull-down technique, the GCE-V5 fusion protein was readily detected with V5 antibody as an immunoprecipitation product, demonstrating interaction between the two proteins (Godlewski, 2006).
Since MET is capable of binding JH in nanomolar concentration (Miura, 2005), the influence of JH treatment on MET-GCE interaction was examined. In the initial experiment, the transfected cell cultures were treated with JH III in final concentrations ranging from 10 μM to 1 nM for 1 h prior to homogenization and pull-down. JH III had no influence on the appearance of the MET-GCE protein complex. Conversely, analogous experiments with the JH agonists methoprene and pyriproxyfen revealed a disruptive effect of each of these juvenile hormone antagonists (JHAs) on the MET-GCE interaction. The strongest effect was observed at the highest concentrations examined. At the lowest concentration of 1 nM, pyriproxyfen showed no effect on the MET-GCE interaction, but methoprene showed a slight effect at 10 nM (Godlewski, 2006).
Both of the JH agonists are thought to be more resistant to catabolism by endogenous enzymes than is JH III. Therefore, the JH III treatment experiment was repeated using a series of time periods shorter than the previous 1-h incubation. At shorter incubation times, especially 15 min, JH III also disrupted MET-GCE interaction as did methoprene. However, the effect of JH III is reversible after 1 h, whereas methoprene requires 4 h for complete reversibility. At this shorter time period of 15 min for JH III, a dose–response effect is seen. The lowest detectable effect on MET-GCE interaction was 10 nM, an amount that is within the physiological concentration range for hormone action (Godlewski, 2006).
To determine if the hormone effect is specific to JH analogs, analogous experiments were carried out using farnesol and geraniol. Both of these compounds share overall chemical similarity with JH, but neither has JH activity in cell culture (Miura, 2005). Even when present in high concentration (10 μM) for the most effective time of JH III treatment (15 min), neither had an effect on MET-GCE interaction. Therefore, the effect on the interaction appears to be specific for JH III and JH agonists (Godlewski, 2006).
To determine if MET can form homodimers, S2 cells were co-transfected with a plasmid expressing GST-MET and one expressing MET-V5. Following incubation and V5 antibody immunoprecipitation, a strong band of MET was detected. To ensure that the MET-MET interaction was specific, a plasmid expressing MET was substituted for GST-MET and subjected to MET antibody detection following incubation and GSH pull-down. Since the MET antibody also recognizes the GST-MET fusion protein, two bands were seen on the Western blot, and the larger GST-MET band was far more intense, as expected. However, a band of the appropriate size for MET (79 kDa) was also evident. Therefore, MET-MET homodimers can form from different fusion proteins for MET and detected with different immunoprecipitation techniques (Godlewski, 2006).
When JH III was included for a 15-min preincubation, far less MET-MET was detected, similar to its effect on MET-GCE heterodimer formation. Therefore, MET homodimers can form in S2 cells, and they are also sensitive to the presence of JH III (Godlewski, 2006).
It was of interest to examine the effect of mutations of Met on the MET-GCE interaction. A variety of types of mutants were constructed, including severe truncations of Met, 10-amino acid deletions in conserved domains, and finally point mutations in each of the conserved domains. S2 cells were transfected with each mutant together with gce. Following incubation, GSH pull-down and V5 antibody detection of GCE were carried out to detect interaction (Godlewski, 2006).
Neither the truncated halves of MET nor the mutants with deletions in the bHLH and PAS-A domains interacted with GCE. The presence of JH III had no effect on the failure of interaction, as expected. However, Met mutants having a point mutation in either the bHLH (Met3) or PAS-A (Met1) domain expressed MET proteins capable of heterodimerization with GCE, and the presence of JH III greatly diminished the interaction. The Met128 mutant having an R433G point mutation in the PAS-B region showed a slight, almost undetectable interaction. So, it appears that severe mutations in Met as well as the R433G mutation in the PAS-B domain can block heterodimer formation or stability, but certain point mutations in the bHLH or PAS-A domains do not affect it (Godlewski, 2006).
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