Signaling through tyrosine kinase receptors (TKRs) is thought to be modulated by receptor-mediated endocytosis and degradation of the receptor in the lysosome. Factors that regulate endosomal sorting of TKRs are largely unknown, however Hrs (Hepatocyte growth factor-regulated tyrosine kinase substrate) has been identified as one such factor. Electron microscopy studies of hrs mutant larvae reveal an impairment in endosome membrane invagination and formation of multivesicular bodies (MVBs). hrs mutant animals fail to degrade active Epidermal growth factor receptor (Egfr) and Torso, leading to enhanced signaling and altered embryonic patterning. These data suggest that Hrs and MVB formation function to downregulate TKR signaling (Lloyd, 2002).

Membrane trafficking events are tightly regulated to ensure proper spatial and temporal delivery of membrane bound cargo. Fusion of intracellular vesicles with their target membrane requires the formation of a highly stable core complex. Regulation of the formation of this complex may modulate vesicle fusion. One proposed regulator of core complex assembly is Hrs; mammalian Hrs binds to the plasma membrane t-SNARE SNAP-25 and inhibits core complex formation in vitro (Bean, 1997; Kwong, 2000). Addition of Hrs to a neuroendocrine cell assay inhibits neurotransmitter release, suggesting that Hrs may regulate Ca²⁺-triggered exocytosis. Notably, Hrs is predominantly localized to early endosomes (Komada, 1997), and Hrs mutant mice have enlarged endosomes (Komada, 1999). Furthermore, Hrs interacts with Eps15, a protein implicated in receptor-mediated endocytosis (Bean, 2000). Thus, Hrs has been proposed to play roles in both exo- and endocytosis (Lloyd, 2002).

Hrs is homologous to yeast Vps27p (vacuolar protein sorting), which regulates protein trafficking from a prevacuolar compartment to the vacuole (Piper, 1995). Vps27p belongs to the Class E subset of VPS proteins, which are implicated in sorting proteins into the vacuole lumen (Odorizzi, 1998). Hrs and Vps27p contain a FYVE domain which binds specifically to phosphatidyl-inositol-3-phosphate (PI3P), and this domain has been demonstrated to localize many proteins to the early endosome. Several FYVE domain-containing proteins have been implicated in endosomal trafficking, including early endosome autoantigen 1 (EEA1), which is essential for early endosome fusion, and Fab1p, which is required for sorting into MVBs. Thus, the FYVE domain may allow proteins to mediate membrane trafficking from or to the endosome through its interaction with PI3P (Lloyd, 2002 and references therein).

In addition to a role in vesicle trafficking, Hrs has been proposed to play different roles in several signal transduction pathways. Hrs binds to Stam, a protein implicated in cytokine signaling (Asao, 1997), and Hrs and Stam both contain VHS (Vps27p, Hrs, Stam) domains present in several proteins implicated in membrane trafficking or signal transduction. Overexpression of Hrs inhibits IL-2-mediated cell growth, suggesting that Hrs may function with STAM to negatively regulate cytokine signaling (Asao, 1997). In contrast to this inhibitory role, Hrs has recently been proposed to play positive roles in both TGF-β and EGFR signaling. Hrs binds to SMAD-2, and hrs mutant mouse embryos exhibit a reduced response to activin and TGF-β (Miura, 2000). Furthermore, overexpression of Hrs in HeLa cells inhibits ligand-induced degradation of EGFR (Chin, 2001), suggesting that Hrs may normally promote EGFR signaling by inhibiting endosome to lysosome trafficking of the receptor (Lloyd, 2002 and references therein).

Thus, although numerous data suggest Hrs may play a role in vesicle trafficking and signal transduction, the precise function of Hrs in these processes is unclear. To further investigate the function of Hrs, the effects of the loss of Hrs was analyzed in Drosophila. The data suggest that Hrs regulates inward budding of endosome membrane and MVB formation. More importantly, hrs mutant animals are unable to degrade active EGFR and Torso TKRs leading to enhanced TKR signaling (Lloyd, 2002).

A mutant allele of hrs that contains only the amino terminal third of the protein behaves genetically as a null mutation, because all phenotypes observed (e.g., enlarged endosomes and increased Egfr tyrosine phosphorylation) were equivalent in hrs/hrs and hrs/Df animals (Lloyd, 2002).

Hrs has been proposed to play a role in at least three vesicle trafficking events: exocytosis, endocytosis, and endosome to lysosome trafficking. Each of these trafficking steps was examined in hrs mutant larvae, and a role for Hrs was found only for endosomal trafficking. Fluid-phase tracer studies and TEM of garland cells reveal a dramatic enlargement of early endosomes, a phenotype that is also seen in mice lacking Hrs (Komada, 1999). Furthermore, TEM analysis of garland cells indicates that the enlarged endosomes result from an inability to invaginate endosomal membrane. A defect in endosomal invagination is consistent with the reduction in MVBs at hrs mutant synapses and with a proposed role for yeast VPS27 (Odorizzi, 1998) in sorting proteins inside the vacuole lumen (Lloyd, 2002).

Although the data demonstrate that Hrs regulates endosomal maturation enroute to lysosomes, several pieces of evidence suggest that Hrs is not essential for endosome to lysosome trafficking. (1) Absence of Hrs does not lead to a generalized increase in expression levels of surface membrane proteins, as would be expected if degradation was blocked. (2) Internalized avidin partially colocalizes with the low-pH indicator dye lysotracker in mutant garland cells at similar time points as in wild-type cells, suggesting that internalized proteins may be delivered to lysosomes. Thus, the data suggest that Hrs specifically functions in endosome invagination, separating membrane and membrane-associated proteins that are destined for lysosomes from those destined for recycling (Lloyd, 2002).

Extracellular signals are communicated to cells with remarkable temporal and spatial resolution. The rapid kinetics of signal amplification and termination are critical to the precision of signal transduction. One mechanism thought to mediate signal downregulation is the internalization and degradation of cell surface receptors. Although internalization of receptors may inhibit ligand binding, many receptors are still active, or in some cases, more active, after internalization. Once inside the early endosome, TKRs may either be recycled back to the surface of the cell or sorted into the multivesicular body (MVB) for degradation in the lysosome (Lloyd, 2002).

It has long been proposed that lysosomal delivery of cell surface receptors is a negative feedback mechanism for downregulation of receptor signaling. However, there is little in vivo evidence for this model, and it remains possible that deactivation of the receptor or downstream components may compensate for a failure to downregulate active receptor. The data suggest that trafficking of TKRs into the MVB plays an important role in signal attenuation. Interestingly, several of the morphological phenotypes observed in hrs mKO embryos are also seen in mutations affecting the torso pathway. For example, posterior cellularization defects are also observed in fs(1)polehole and l(1)polehole/D-raf embryos, and twisted gastrulation phenotypes are also observed in torso embryos (Lloyd, 2002).

Recently, overexpression of Hrs in HeLa cells has been shown to inhibit ligand-mediated degradation of EGFR, suggesting that Hrs may function to prolong EGFR signaling (Chin, 2001). In contrast, the data suggest the opposite function for Hrs, namely that it functions to attenuate TKR signaling by promoting degradation of the tyrosine-phosphorylated, or active, receptor. Interestingly, although active Egfr is upregulated in hrs mutants, total levels of the receptor are decreased, suggesting that Hrs is specifically required for degradation of active receptors. This reduction in total receptor is likely due to a well-characterized negative feedback mechanism whereby Egfr hyperactivation inhibits receptor transcription (Lloyd, 2002).

In summary, the following model is proposed for Hrs function. (1) Endocytosis of activated tyrosine kinase receptors (2) leads to the phosphorylation of Hrs on the early endosome membrane (Urbe, 2000). Phosphorylation may enhance the activity of Hrs, which then (3) leads to localized invagination of endosomal membrane. Ubiquitinated receptors may be sorted into the invagination directly via an interaction with the UIM of Hrs or indirectly through an interaction with Hrs binding proteins SNX1, Clathrin, or Eps15 (Bean, 2000; Chin, 2001; Raiborg, 2001b). Finally, (4) the membrane is pinched off to form a MVB, and (5) the internalized vesicles are trafficked to the lysosome for degradation. This process of MVB formation leads to a reversal of membrane topology such that the cytoplasmic portion of TKRs is now inside the MVB and unable to signal to downstream components. In this model, receptor-mediated activation of Hrs and MVB formation serves a critical role in attenuating tyrosine kinase receptor signaling (Lloyd, 2002).

GENE STRUCTURE

cDNA clone length - 2697 bp

Bases in 5' UTR - 123

Exons - 6

Bases in 3' UTR - 291

PROTEIN STRUCTURE

Amino Acids - 760

Structural Domains

The 2 A X-ray structure of the 219-residue N-terminal VHS and FYVE tandem domain unit of Drosophila Hrs have been determined. The unit assumes a pyramidal structure in which the much larger VHS domain (residues 1-153) forms a rectangular base and the FYVE domain occupies the apical end. The VHS domain is comprised of an unusual 'superhelix' of eight alpha helices, and the FYVE domain is mainly built of loops, two double-stranded antiparallel sheets, and a helix stabilized by two tetrahedrally coordinated zinc atoms. The two-domain structure forms an exact 2-fold-related homodimer through antiparallel association of mainly FYVE domains. Dimerization creates two identical pockets designed for binding ligands with multiple negative charges such as citrate or phosphatidylinositol 3-phosphate (Mao, 2000).

A single Hrs homolog has been identified in the Drosophila genome, and sequence analysis of a 2.7 kb hrs cDNA predicts an open reading frame of 760 amino acids with several well-conserved domains (Mao, 2000). The hrs gene was mapped to cytological band 23A and is removed by deficiency Df(2L)N19. Alleles of several complementation groups mapping to Df(2L)N19 were previously isolated in an EMS mutagenesis screen for mutations in synaptotagmin. An 11.5 kb genomic DNA fragment containing the hrs gene or the hrs cDNA driven by the hsp70 promoter (hs-hrs) fully rescues the early pupal lethality of l(2)23Ad^D28/Df and l(2)23Ad^D28/l(2)23Ad^D28 animals but not other mutations in this region. These data demonstrate that the l(2)23Ad^D28 chromosome (hereafter referred to as hrs) contains a mutation in the hrs gene. Sequencing of DNA from mutant animals revealed a nonsense mutation at amino acid Q2 (Lloyd, 2002).

The FYVE domain mediates the recruitment of proteins involved in membrane trafficking and cell signaling to phosphatidylinositol 3-phosphate [PtdIns(3)P]-containing membranes. To elucidate the mechanism by which the FYVE domain interacts with PtdIns(3)P-containing membranes, the membrane binding of the FYVE domains of yeast Vps27p and Drosophila hepatocyte growth factor-regulated tyrosine kinase substrate and their mutants were measured by surface plasmon resonance and monolayer penetration analyses. These measurements as well as electrostatic potential calculation show that PtdIns(3)P specifically induces the membrane penetration of the FYVE domains and increases their membrane residence time by decreasing the positive charge surrounding the hydrophobic tip of the domain and causing local conformational changes. Mutations of hydrophobic residues located close to the PtdIns(3)P-binding pocket or an Arg residue directly involved in PtdIns(3)P binding abrogate the penetration of the FYVE domains into the monolayer, the packing density of which is comparable with that of biological membranes and large unilamellar vesicles. Based on these results, a mechanism of the membrane binding of the FYVE domain is proposed in which the domain first binds to the PtdIns(3)P-containing membrane by specific PtdIns(3)P binding and nonspecific electrostatic interactions; this is then followed by the PtdIns(3)P-induced partial membrane penetration of the domain (Stahelin, 2002).

date revised: 2 September 2005

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.