MET goes to the membrane—A new place for the “old” juvenile hormone receptor

The insect juvenile hormone (JH) is a unique lipophilic signaling molecule. While insects play indispensable ecosystem roles, some species destroy crops and still others spread infectious diseases. The latter applies to mosquitoes, the subject of current PNAS study from the Jinsong Zhu’s team (1). PNAS has historically covered chief advances in JH research, and this time is no exception. Zhao et al. (1) disclose a mechanism that engages the intracellular JH receptor methoprene-tolerant (MET) as a sensor of JH while additionally operating at the plasma membrane. Their work identifies the ligand-activated transcription factor MET as a hot contender for the elusive cell-membrane receptor of this vital insect hormone.

JH is essential in the yellow fever mosquito (Aedes aegypti) for completion of development (2) and to promote female reproduction (1–3). Although these roles of JH have been recognized for almost a century, the mode of JH action was not revealed until the basic helix–loop–helix-PER-ARNT-SIM (bHLH-PAS) protein MET (4) was causally linked with developmental effects of JH (5) and finally established as a JH receptor (JHR) 15 y ago (6). Work by the Zhu laboratory (7) and others (6, 8) has further shown that upon binding JH, MET interacts with another bHLH-PAS protein taiman (TAI) and the DNA-bound complex activates genes furnished with JH-response elements (Fig. 1), such as Krüppel-homolog 1 (Kr-h1). This gene-regulatory action of the JHR resembles that of the vertebrate aryl hydrocarbon receptor (AHR) (9, 10) and executes many—but not all—functions of the pleiotropic hormone.

Fig. 1.

A tentative model for the established nuclear action of the JHR and the novel membrane-initiated JH signaling pathway involving MET, PVR, and second messenger signaling. PIP2, phosphatidylinositol-4,5-bisphosphate; DAG, diacylglycerol; IP3, inositol-1,4,5-trisphosphate; SRSF, serine/arginine-rich splicing factors. It is unknown whether or how MET circulates between the cytoplasmic chaperone complex and the membrane-associated pool (broken line). See text for further details.

JH has long been known to trigger rapid cellular responses, particularly important during oogenesis (11), which cannot be ascribed to the gene-regulatory mechanism. These short-term effects are mediated via second messenger signaling and protein kinase cascades initiated at the cell membrane. It was again the Zhu team who have uncovered a branch of JH signaling through phospholipase C (PLC) acting downstream of a hypothetical JH receptor tyrosine kinase (RTK) and upstream of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) (12). Somewhat paradoxically, this membrane-initiated JH signaling converged back at the gene-regulatory branch, stimulating the transcriptional activity of the nuclear MET–TAI complex (12). Nonetheless, the study has reignited the ongoing search for a cell-membrane JH receptor. Work on oogenesis in the migratory locust (Locusta migratoria) has suggested an unidentified G protein–coupled receptor (GPCR) for the role (11). In the cotton bollworm, Helicoverpa armigera, two candidates from the RTK family have even been proposed to bind JH directly (13). However, no definitive answers were available as no compelling mechanism of JH interaction with cell-membrane receptors of either type have been revealed, until now.

Zhao et al. (1) disclose a mechanism of JH action that engages the intracellular JH receptor methoprene-tolerant (MET) as a sensor of JH while additionally operating at the plasma membrane.

The long search for the cell-membrane JH receptor wound up identifying the missing hormone-binding entity as MET itself. Zhao et al. (1) firmly link the membrane-initiated JH/MET signaling with an RTK termed PVR, the insect counterpart of the mammalian platelet-derived growth factor and vascular endothelial growth factor receptors. While the exact mechanisms remain to be defined, a tentative model can be drawn from their work (Fig. 1). A subpopulation of MET residing at the plasma membrane of mosquito cells associates with the intracellular domain of PVR and, upon JH addition, mediates its phosphorylation. The ensuing PVR signaling requires the JH-binding capacity of the MET PAS-B domain; the DNA-binding function and dimerization with TAI are dispensable. Likewise, the extracellular portion of PVR is not required. This JH- and MET-dependent PVR phosphorylation in turn activates PLC and further downstream signaling through the CaMKII and previously identified targets of the phosphoinositide 3-kinase (PI3K). Those encompass the MET and TAI proteins (12) and serine/arginine-rich splicing factors that modulate the function of TAI through selective expression of its alternatively spliced isoforms (14).

The MET–PVR signaling eventually translates into gene-expression signatures, wherein large sets of transcripts affected by MET or PVR knockdown in the mosquitoes only partially overlap. Most of the differentially expressed genes have metabolic and proteosynthetic roles, corresponding with the mosquito reproductive physiology. Consistently, the observed interaction between PVR and the JH-sensing MET has a common phenotypic outcome. Loss of either PVR or MET compromises the reproductive fitness of female mosquitoes, markedly reducing their ovary size. PVR becomes the first membrane receptor conclusively identified as a direct partner of MET in the JH regulatory network within the physiological context of mosquito oogenesis.

The rapid signaling events triggered by JH at the cell membrane may either cooperate with the nuclear MET–TAI complex or act separately to regulate other processes. Apparently, both options apply. First, as the MET–PVR activity essentially contributes to Kr-h1 induction, both branches of JH signaling synergistically converge at the transcriptional level (1, 12). Thus, in this case, the nongenomic pathway reinforces the gene-regulatory JH activity. Second, the partially overlapping but distinct transcriptome signatures of MET and PVR indicate that the membrane-initiated signaling branch expands the diversity of cellular responses to JH beyond the “canonical” nuclear pathway. Integration of the diverse signals may enable various insect tissues in different developmental and physiological settings to interpret the same endocrine cue in distinct ways.

In its dual action at the cell membrane and at the gene-regulatory level, JH is no exception among small signaling molecules. Classic examples are provided by steroid hormones (15). Upon binding an agonist, steroid receptors of the nuclear receptor (NR) family dimerize to directly control transcription of target genes. However, monomers of the same NRs localized at the cell membrane associate with kinases such as Src and PI3K to engage in other signaling modes that often complement their own gene-regulatory function. Adding further complexity, hormones such as estradiol or progesterone additionally bind to transmembrane receptors, typically GPCRs.

The already mentioned ligand-dependent bHLH-PAS protein AHR closely parallels JHR signaling (9, 10). Both AHR and MET reside in cytoplasmic chaperone complexes with HSP90 (insect HSP83). Ligand binding to the PAS-B domain of either receptor leads to nuclear import, dissociation from HSP90/83 (16, 17), and concomitant assembly of the transcriptionally active heterodimer with the respective bHLH-PAS partner (Fig. 1). Besides this “canonical” nuclear action, AHR signals through a nongenomic branch involving the Src kinase, which is also a component of the AHR-HSP90 chaperone complex that dissociates once AHR becomes liganded, leading to the kinase activation. The Src kinase activity then feeds into RTK signaling via the epidermal growth factor receptor (18). It remains to be seen whether similar regulatory paths might lead from the membrane-associated MET to JH-dependent activation of PVR. Given the analogous action of the related AHR, the nongenomic JH signaling through MET interacting with PVR is perhaps not as unexpected as it might have seemed.

While the present study of Zhao et al. (1) advances the field to the next level, it naturally raises many questions. Just by looking at the suggested model drawn here on behalf of the authors (Fig. 1), one can spot several question marks. First, the mechanism of MET–PVR interaction and PVR phosphorylation is unclear. While the JH-binding capacity of MET and JH itself is required for PVR phosphorylation, the PAS-B of MET appears to associate with the intracellular tail of PVR independently of JH. Does MET cause PVR to autophosphorylate or does it recruit a protein kinase, and which one? Could it be Src as in the case of AHR?

Second, is it the PVR monomer or dimer that interacts with MET? The fact that the extracellular ligand-binding domain of PVR is dispensable for the JH-induced MET–PVR signaling would argue that a PVR monomer might act independently of its cognate Pvf1/2/3 ligands in this setting. If so, one would expect the JH-MET–PVR branch to exert effects distinct from those of Pvf-dependent PVR signaling. This might be reflected by the largely different transcriptome profiles obtained from MET and PVR knockdown mosquitoes. Yet, the nuclear JHR and the PVR pathways do intercept, at least by regulating common target genes including Kr-h1.

Third, the authors postulate a subpopulation of MET constitutively residing at the cell membrane. While JH induces nuclear import of MET, part of the pool remains associated with the membrane, where it stimulates PVR phosphorylation (Fig. 1). It will be important to know what distinguishes the membrane-associated MET from its cytoplasmic and nuclear pools, and how the trafficking dynamics induced by JH is regulated. At this point, it is unclear whether MET localization is determined by its posttranslational modifications (such as phosphorylation or palmitoylation) or interactions with other proteins. The role of chaperones associated with MET, primarily HSP83, may be critical. Follow-up studies should establish whether the membrane (PVR)-associated MET originates from the cytoplasmic MET–HSP83 complex and to what extent the chaperones impact the MET–PVR branch of JH signaling.

As master Yoda would observe, “always more questions than answers there are.” The present study by Zhao et al. (1) uncovers an intricate interplay between MET and PVR, suggesting even more complex regulation of JH signaling than previously suspected. Their work points out mechanisms that may underlie the manifold roles of the pleiotropic insect hormone and injects a healthy dose of excitement into the field that will eagerly await advancements on the newly discovered MET–PVR pathway.