EGF receptor

EVOLUTIONARY HOMOLOGS

Table of contents

EGFR in C. elegans

In C. elegans, genes involved in targeting LET-23 RTK to the basolateral membrane domain can be identified in genetic screens for vulvaless mutants. Vulval signaling requires basolateral expression of LET-23 RTK, since cells that lack LET-23 RTK in the basolateral membrane domain presumably cannot respond to LIN-3 EGF in the basal extracellular space. Mutations in lin-2, lin-7, and lin-10 were initially isolated because they decrease vulval signaling and cause a vulvaless phenotype. Subsequently, these mutations were shown to result in apical mislocalization of LET-23 RTK. LIN-7, LIN-2, and LIN-10 each contain protein domains that may mediate interactions with other proteins. LIN-7 contains a single PDZ domain, and PDZ domains are known to mediate protein-protein interactions with C-terminal tails of transmembrane proteins as well as with other PDZ domains. LIN-2 contains a CaM kinase domain, a calmodulin-binding domain, a PDZ domain, an SH3 domain, and a guanylate kinase domain. LIN-2 is highly similar to mammalian Lin2/CASK (52% identical overall). Lin2/CASK is expressed in epithelia and neurons and has been shown to bind to neurexin, syndecan, and protein 4.1. LIN-2 is related to a family of proteins called membrane-associated guanylate kinases (MAGUKs) that includes discs-large (DlgA) and PSD-95. In mammals, DlgA binds the cytoplasmic tail of the Shaker-type K+ channel; in Drosophila, dlg mutations prevent synaptic localization of the Shaker channel. In C. elegans, the LET-23 receptor tyrosine kinase is localized to the basolateral membranes of polarized vulval epithelial cells. lin-2, lin-7, and lin-10 are required for basolateral localization of LET-23, since LET-23 is mislocalized to the apical membrane in lin-2, lin-7, and lin-10 mutants. Yeast two-hybrid, in vitro binding, and in vivo coimmunoprecipitation experiments show that LIN-2, LIN-7, and LIN-10 form a protein complex. Furthermore, compensatory mutations in lin-7 and let-23 exhibit allele-specific suppression of apical mislocalization and signaling-defective phenotypes. These results present a mechanism for basolateral localization of LET-23 receptor tyrosine kinase by direct binding to the LIN-2/LIN-7/LIN-10 complex. Each of the binding interactions within this complex is conserved, suggesting that this complex may also mediate basolateral localization in mammals (Kaech, 1998).

Development of the C. elegans egg-laying systems requires the formation of a connection between the uterine lumen and the developing vulva lumen, thus allowing a passage of eggs and sperm. Development of the connection requires that cells in both tissues become specialized to participate in the connection, and that the specialized cells are brought in register. A single cell, the anchor cell, acts to induce and organize specialization of the epidermal and uterine epithelia, and registrates these tissues. The anchor cell induces the vulva from ventral epithelial cells via the LIN-3 growth factor, an Epidermal growth factor homolog. LIN-3 acts through LET-23, the C. elegans homolog of EGF-R. It then induces surrounding uterine intermediate precursors via the receptor LIN-12, a member of the Notch family of receptors. LET-23 acts via the Ras pathway, which targets LIN-1, an ETS-domain class transcription factor homologous to Drosophila Yan and Pointed, and LIN-31, an HNF3/forkhead transcription factor, both of which act to prevent vulval differentiation (Newman, 1996 and references).

The let-23 gene encodes a C. elegans homolog of the epidermal growth factor receptor (EGF-R) necessary for vulval development. A mutation of let-23 activates the receptor and downstream signal transduction, leading to excess vulval differentiation. This mutation alters a conserved cysteine residue in the extracellular domain. Mutation of a different cysteine in the same subdomain causes a strong loss-of-function phenotype, suggesting that cysteines in this region are each important for function and not equivalent to one another. Vulval precursor cells can generate either of two subsets of vulval cells (distinct fates) in response to sa62 activity. The fates produced depend on the copy number of the mutation, suggesting that quantitative differences in receptor activity influence the decision between these two fates (Katz, 1996).

During induction of the Caenorhabditis elegans hermaphrodite vulva, a signal from the anchor cell activates the LET-23 epidermal growth factor receptor (EGFR)/LET-60 Ras/MPK-1 MAP kinase signaling pathway in the vulval precursor cells. Two mechanisms have been characterized that limit the extent of vulval induction. (1) gap-1 may directly inhibit the LET-60 Ras signaling pathway. The gap-1 gene was identified in a genetic screen for inhibitors of vulval induction. gap-1 is predicted to encode a protein similar to GTPase-activating proteins that likely functions to inhibit the signaling activity of LET-60 Ras. A loss-of-function mutation in gap-1 suppresses the vulvaless phenotype of mutations in the let-60 ras signaling pathway, but a gap-1 single mutant does not exhibit excess vulval induction. (2) It was found that let-23 EGFR prevents vulval induction in a cell-nonautonomous manner, in addition to its cell-autonomous role in activating the let-60 ras/mpk-1 signaling pathway. Using genetic mosaic analysis, it has been shown that let-23 activity in the vulval precursor cell closest to the anchor cell (P6.p) prevents induction of vulval precursor cells further away from the anchor cell (P3.p, P4.p, and P8.p). This result suggests that LET-23 in proximal vulval precursor cells might bind and sequester the inductive signal LIN-3 EGF, thereby preventing diffusion of the inductive signal to distal vulval precursor cells (Hajnal, 1997).

Signaling through an epidermal growth factor receptor in the nematode C. elegans stimulates a Ca2+ release pathway that is independent of Ras. Activity of LET-23, the C. elegans homolog of the epidermal growth factor receptor, is required in multiple tissues. RAS activation is necessary and sufficient for certain LET-23 functions. Two sets of experiments suggest that LIN-3 (a C. elegans homolog of the epidermal growth factor) and LET-23 function in the hermaphrodite gonad are RAS independent. (1) Mutations in components of the RAS pathway capable of rescuing defects in vulval development and viability caused by reduction-of-function let-23 mutations are unable to rescue the sterility caused by such mutations. (2) Mutational analysis of the let-23 gene suggests that distinct domains of the receptor mediate LET-23 function in the vulva, versus the hermaphrodite gonad. In particular, while certain putative binding sites for Src Homology-2 domain-containing proteins are required for LET-23 function in viability and vulval induction, a distinct pair of sites is necessary and sufficient for function in the hermaphrodite gonad (Clandinin, 1998).

Mutations in two loci, lfe-1/itr-1 and lfe-2 are tissue-specific suppressors of reduced LIN-3/LET-23-mediated signaling. Mutations in lfe-1/itr-1 are likely to be gain-of-function while a suppressing mutation in lfe-2 is likely loss-of-function. lfe-1/itr-1 and lfe-2 appear to function downstream of let-23 for hermaphrodite fertility because genetic epistasis tests using both a reduction-of-function allele and a null allele of let-23 demonstrate that mutations in lfe-1/itr-1 or lfe-2 can bypass LET-23 function in the gonad. In addition to their suppression phenotype, the activities of lfe-1/itr-1 and lfe-2 appear to be involved in normal hermaphrodite fertility because lfe-1/itr-1(gf); lfe-2(lf) double-mutant animals display ovulation defects similar to those observed in animals bearing reduction of function mutations in either let-23 or lin-3. It is believed that the fertility function of these two genes lies in the adult somatic gonad, based on an analysis of LFE-2. In particular, LFE-2 is expressed in the adult spermatheca, and misexpression of LFE-2 in adult animals is sufficient to induce defects in spermathecal function (Clandinin, 1998).

Molecular characterization of LFE-1/ITR-1 and LFE-2 argues strongly that LET-23 regulates intracellular calcium levels in the spermatheca. LFE-1/ITR-1 encodes a C. elegans homolog of the mammalian IP3R (see Drosophila IP3R), an established effector of intracellular calcium release. LFE-2 encodes a nematode homolog of IP3 kinase, whose in vivo function in mammalian cells is unknown but which is very likely to play a regulatory role in calcium release based on its substrate specificity. Thus, an inositol trisphosphate receptor can act as a RAS-independent, tissue-specific positive effector of LET-23. An inositol trisphosphate kinase negatively regulates this transduction pathway. Signals transduced by LET-23 control ovulation through changes in spermathecal dilation, possibly dependent upon calcium release regulated by the second messengers IP3 and IP4. It is likely that LET-23 functions by activation of phospholipase Cgamma, which promotes release of intracellular calcium through production of IP3. These results demonstrate that one mechanism by which receptor tyrosine kinases can evoke tissue-specific responses is through activation of distinct signal transduction cascades in different tissues (Clandinin, 1998).

In C. elegans, the epithelial Pn.p cells adopt either a vulval precursor cell fate or fuse with the surrounding hypodermis (the F fate). Two pathways that control vulval precursor cell fate converge on the Hox gene lin-39: the Ras pathway, functioning downstream of the LET-23 Epidermal growth factor receptor and the Wnt pathway. LIN-39 is an Antennapedia class homeodomain, most similar to those of the Drosophila homeotic genes Deformed and Sex combs reduced. The Wnt signal is transduced through a pathway involving the beta-catenin homolog BAR-1 and controls whether P3.p through P8.p adopt the vulval precursor cell fate. In bar-1 mutants, P3.p through P8.p can adopt F fates instead of vulval precursor cell fates. The Wnt/bar-1 signaling pathway acts by regulating the expression of the Hox gene lin-39, since bar-1 is required for LIN-39 expression and forced lin-39 expression rescues the bar-1 mutant phenotype. LIN-39 activity is also regulated by the anchor cell signal/let-23 receptor tyrosine kinase/let-60 Ras signaling pathway. These genetic and molecular experiments show that the vulval precursor cells can integrate the input from the BAR-1 and LET-60 Ras signaling pathways by coordinately regulating activity of the common target, LIN-39 Hox (Eisenmann, 1998).

In Caenorhabditis elegans, vulval induction is mediated by the let-23 receptor tyrosine kinase (RTK)/ Ras signaling pathway. The precise localization of let-23 RTK at epithelial junctions is essential for the vulval induction, and requires three genes, including lin-2, -7, and -10. The mammalian homolog of lin-2 has been identified as CASK, a protein interacting with neurexin, a neuronal adhesion molecule. CASK has recently been reported to interact with syndecans and an actin-binding protein, band 4.1, at epithelial and synaptic junctions, and to play central roles in the formation of cell-cell junctions. The product of C. elegans lin-7 directly interacts with let-23 RTK and localizes let-23 RTK at epithelial junctions. Three rat homologs of lin-7 are ubiquitously expressed in various tissues. These homologs accumulate at the junctional complex region in cultured Madin-Darby canine kidney cells, and are also localized at the synaptic junctions in neurons. The mammalian homologs of lin-7 may be implicated in the formation of cell-cell junctions (Irie, 1999).

In Caenorhabditis elegans, the EGF receptor (encoded by let-23) is localized to the basolateral membrane domain of the epithelial vulval precursor cells, where it acts through a conserved Ras/MAP kinase signaling pathway to induce vulval differentiation. lin-10 acts in LET-23 receptor tyrosine kinase basolateral localization, because lin-10 mutations result in mislocalization of LET-23 to the apical membrane domain and cause a signaling defective (vulvaless) phenotype. The previous molecular identification of lin-10 (Kim, 1990) is incorrect, and a new gene corresponding to the lin-10 genetic locus has now been identified. lin-10 encodes a protein with regions of similarity to mammalian X11/mint proteins, containing a phosphotyrosine-binding and two PDZ domains. A nonsense lin-10 allele that truncates both PDZ domains only partially reduces lin-10 gene activity, suggesting that these protein interaction domains are not essential for LIN-10 function in vulval induction. Immunocytochemical experiments show that LIN-10 is expressed in vulval epithelial cells and in neurons. LIN-10 is present at low levels in the cytoplasm and at the plasma membrane and at high levels at or near the Golgi. LIN-10 may function in secretion of LET-23 to the basolateral membrane domain, or it may be involved in tethering LET-23 at the basolateral plasma membrane once it is secreted (Whitfield, 1999).

LIN-10 is expressed at high levels in neurons, including ring neurons, ventral cord neurons, dorsal cord neurons, lateral neurons, and neurons in the tail. This result supports previous results showing that LIN-10 functions to localize the glutamate receptor GLR-1 to the postsynaptic elements in neurons. LIN-10 appears in neuronal processes and cell bodies. In neural cell bodies, a small amount of LIN-10 appears diffusely throughout the cytoplasm, whereas the majority of LIN-10 is concentrated in discrete perinuclear structures, similar to perinuclear structures observed in vulval epithelial cells. To determine whether these perinuclear structures correspond to Golgi, Sialyltransferase-green fluorescent protein (ST-GFP) as was used as a marker for the trans-cisterna of the Golgi. ST-GFP was expressed in transgenic worms using a heat shock promoter and the subcellular localizations of LIN-10 and ST-GFP were examined using anti-LIN-10 and anti-GFP antibodies. In single neurons expressing both endogenous LIN-10 and transgenic ST-GFP, the subcellular pattern of LIN-10 staining is similar to that of ST-GFP staining. Deconvolution of images obtained in double-staining experiments reveals that LIN-10 staining is closely associated with ST-GFP staining, but LIN-10 staining is consistently offset (by 0.2-0.5 µm) from ST-GFP staining. These results indicate that LIN-10 is localized in the trans-cisterna of the Golgi or is localized in a compartment closely associated with the trans-cisterna, such as the trans-Golgi network (Whitfield, 1999).

In addition to lin-10, two other genes (lin-2 and lin-7) function in basolateral localization of LET-23. Furthermore, recent evidence indicates that LIN-10 is part of a protein complex with LIN-2 and LIN-7, and that LIN-7 directly binds to the cytoplasmic C terminus of LET-23. These results suggest that the mechanism of LET-23 basolateral localization involves direct protein interactions between trans-acting factors (LIN-2, LIN-7, and LIN-10) and a cytoplasmic, cis-acting element in the LET-23 C terminus. LIN-2, LIN-7 and LIN-10 are evolutionarily conserved. C. elegans LIN-2 is highly similar to mammalian CASK and contains a Ca2+/calmodulin-dependent kinase II domain, a conserved LIN-7 binding region, a PDZ domain, an Src homlogy region 3 domain, and a guanylate kinase domain. C. elegans LIN-7 is highly similar to three mammalian homologs (termed mLin-7A, mLin-7B, and mLin-7C) and contains a PDZ domain and a conserved LIN-2 binding region. As discussed above, C. elegans LIN-10 has regions of similarity with mammalian X11/mint proteins. These mammalian homologs have been found to form a ternary complex similar to the C. elegans LIN-2/LIN-7/LIN-10 proteins. Specifically, mammalian LIN-7 binds to mammalian LIN-2/CASK, and mammalian LIN-2/CASK binds to mammalian LIN-10/X11/mint proteins (Whitfield, 1999 and references).

Negative regulation of receptor tyrosine kinase (RTK)/RAS signaling pathways is important for normal development and the prevention of disease in humans. A genetic screen has been used in C. elegans to identify genes that antagonize the activity of activated LET-23, a member of the EGFR family of RTKs. Two loss-of-function mutations in dpy-22, previously cloned as sop-1, have been identified that promote the ability of activated LET-23 to induce ectopic vulval fates. DPY-22 is a glutamine-rich protein that is most similar to human TRAP230, a component of a transcriptional mediator complex. DPY-22 has been shown to regulate WNT responses through inhibition of the ß-catenin-like protein BAR-1. Evidence is provided that DPY-22 also inhibits RAS-dependent vulval fate specification independently of BAR-1, and probably regulates the activities of multiple transcription factors during development. Furthermore, it has been demonstrated that although inhibition of BAR-1-dependent gene expression has been shown to require the C-terminal glutamine-rich region, this region is dispensable for inhibition of RAS-dependent cell differentiation. Thus, the glutamine-rich region contributes to specificity of this class of mediator protein (Moghal, 2003).

ErbB-2/HER2 drives epithelial malignancies by forming heterodimers with growth factor receptors. The primordial invertebrate receptor is sorted to the basolateral epithelial surface by binding of the PDZ domain of Lin-7 (Drosophila homolog: Veli) to the receptor's tail. All four human ErbBs are basolaterally expressed, even when the tail motif is absent. Mutagenesis of hLin-7 unveiled a second domain, KID, that binds to the kinase region of ErbBs. The PDZ interaction mediates stabilization of ErbB-2 at the basolateral surface. On the other hand, binding of KID is involved in initial delivery to the basolateral surface, and in its absence, unprocessed ErbB-2 molecules are diverted to the apical surface. Hence, distinct domains of Lin-7 regulate receptor delivery to and maintenance at the basolateral surface of epithelia (Shelly, 2003).

Inactivation of the C. elegans lin-7 gene, or preventing binding of LIN-7 to LET-23 by deleting the tail motif of the receptor, mislocalizes LET-23 and results in a vulva-less phenotype. While these lines of evidence identify LIN-7 as a component crucial for basolateral receptor localization in worms, the situation in mammals is complicated by the existence of several other PDZ-containing proteins that bind to the tail of ErbB-2. Therefore, to approach the issue of ErbB-2 targeting, the degenerate PDZ target motif was deleted and the tail-less receptors were found to not only retain basolateral expression, but a significant fraction of them localized to intracellular vesicles. These results indicate that the vesicular population reflects rapid endocytosis of the truncated receptors from the basolateral surface, ineffective recycling, and efficient degradation in lysosomes. Hence, the PDZ target motif of ErbB-2 may selectively stabilize the receptor at the basolateral surface, rather than actively target the receptor to this surface. Presumably, the PDZ-mediated retention process acts in concert with cis-acting signals for basolateral sorting that often resemble tyrosine- and dileucine-based motifs that specify receptor endocytosis via clathrin-coated pits. Likewise, a specific clathrin adaptor complex mediates basolateral targeting, and cells deficient for the complex mislocalize a large proportion of ErbB-2 to the apical surface (Shelly, 2003).

Whether or not hLin-7 is the PDZ-bearing adaptor that inhibits ErbB-2 internalization in this cellular system remains unknown. However, it is worth noting that Lin-7 regulates endocytosis and recycling of a chimeric p75 neurotrophin receptor fused to the tail of LET-2. By analogy to the proposed inhibitory role of hLin-7, several other PDZ-bearing proteins inhibit internalization of their targets. Taken together with lessons learned from other receptors, these results imply that PDZ modules recruited to the tail of ErbB-2 mobilize retention mechanisms, which inhibit entrapment of the receptor in the clathrin coat and enhance recycling of internalized receptor molecules (Shelly, 2003).

Because ErbB proteins bind with hLin-7 irrespective of the presence of a PDZ target motif, the existence of a second, PDZ-independent binding site in hLin-7 was inferred. As expected, the identified site binds a region shared by the four ErbBs, namely the kinase domain. Deletion analyses have localized the kinase binding site to a short motif (denoted KID) of hLin-7. This motif is well conserved in evolution, from insects to mammals (the domain is present in Drosophila Lin-7). Because KID mutants are unstable, a relatively large deletion mutant lacking the whole N-terminal part of hLin-7 was tested. Unlike wt-hLin-7, the ΔN mutant localizes to the ER and arrests maturation of receptors containing the PDZ target motif. Most revealing is the ability of the monovalent mutant to alter the apical:basolateral distribution of ErbB-2, similar to the effect of ER retention on the distribution of E-cadherin (Shelly, 2003).

The exit of nascent proteins from the ER depends on cis-acting sequences and interactions with sorting proteins. For example, the ER-associated PDZ protein syntenin/TACIP18 controls membrane targeting of proTGFα, and PSD-95 similarly regulates the NR1 subunit of NMDA receptors. The latter masks an ER retention signal in the cytoplasmic domain of NR1. Presumably, the N-terminal part of hLin-7 fulfils a similar role: by folding over the PDZ domain, it may inhibit an intrinsic ER retention signal, whereas the adjacent KID and L27 domains respectively recognize cargoes (e.g., ErbB-2), as well as effectors (e.g., Lin-2) actively regulating basolateral targeting (Shelly, 2003).

In the absence of a basolateral signal, proteins are sent to the apical surface by default. Alternatively, signals involved in apical sorting of transmembrane proteins rely on glycosyl-phosphatidylinositol anchor modifications, asparagine (N)-linked carbohydrates, or O-glycosylated serine/threonine residues. Only a relatively small fraction of each ErbB (5%-10%) resides at the apical surface of MDCK cells under steady-state conditions. While this fraction of fully processed molecules may represent basolateral receptors rerouted to the apical face, a different mechanism seems to direct Endo-H-sensitive precursors of ErbB-2 to the apical surface of cells coexpressing ΔN-hLin-7. According to one model, the ER and the plasma membrane form a continuum that allows, for example, phagocytosis of extremely large particles. Thus, when arrested in the ER, nascent ErbB-2 molecules can directly reach the plasma membrane. If correct, this model predicts uncoupling of the basolateral surface from the ER (Shelly, 2003).

Finally, understanding polar distribution of ErbB proteins is relevant to many other receptor tyrosine kinases, and it may shed light on human diseases. Progression of polycystic kidney disease depends on aberrant distribution of ErbB-1, and persistent ErbB signaling predicts poor prognosis of several types of malignancies. Moreover, mAbs to ErbB-2 and low-molecular weight kinase inhibitors specific to ErbB-1 are already used in the treatment of breast and lung cancer patients, respectively, and both agents modulate receptor trafficking and stability. Future studies will address the possibility that the breakdown of cellular polarity, which frequently accompanies epithelial transformation, can modulate the PDZ-containing tripartite complex in a way that augments tumorigenicity (Shelly, 2003).

Genetic analysis has shown that dos/soc-1/Gab1 functions positively in receptor tyrosine kinase (RTK) stimulated Ras/Map kinase signaling, through the recruitment of csw/ptp-2/Shp2. Using sensitised assays in C. elegans for let-23/Egfr and daf-2/InsR (Insulin receptor-like) signaling, it has been shown that soc-1/Gab1 inhibits phospholipase C-gamma (PLCgamma) and phosphatidylinositol 3'-kinase (PI3K) mediated signaling. Furthermore, as well as stimulating Ras/Map kinase signaling, soc-1/Gab1 stimulates a poorly defined signaling pathway that represses class 2 daf-2 phenotypes. In addition, it is shown that SOC-1 binds the C-terminal SH3 domain of SEM-5. This binding is likely to be functional because the sem-5(n2195)G201R mutation, which disrupts SOC-1 binding, behaves in a qualitatively similar manner to a soc-1 null allele in all assays for let-23/Egfr and daf-2/InsR signaling examined. Further genetic analysis suggests that ptp-2/Shp2 mediates the negative function of soc-1/Gab1 in PI3K mediated signaling, as well as the positive function in Ras/Map kinase signaling. Other effectors of soc-1/Gab1 are likely to inhibit PLCgamma mediated signaling and stimulate the poorly defined signaling pathway that represses class 2 daf-2 phenotypes. Thus, the recruitment of soc-1/Gab1, and its effectors, into the RTK signaling complex modifies the cellular response by enhancing Ras/Map kinase signaling while inhibiting PI3K and PLCgamma mediated signaling (Hopper, 2006).

The epidermal growth factor receptor (EGFR)/ErbB receptor tyrosine kinases regulate several aspects of development, including the development of the mammalian nervous system. ErbB signaling also has physiological effects on neuronal function, with influences on synaptic plasticity and daily cycles of activity. However, little is known about the effectors of EGFR activation in neurons. This study shows that EGF signaling has a nondevelopmental effect on behavior in Caenorhabditis elegans. Ectopic expression of the EGF-like ligand LIN-3 at any stage induces a reversible cessation of feeding and locomotion. These effects are mediated by neuronal EGFR (also called LET-23) and phospholipase C-gamma (PLC-gamma), diacylglycerol-binding proteins, and regulators of synaptic vesicle release. Activation of EGFR within a single neuron, ALA, is sufficient to induce a quiescent state. This pathway modulates the cessation of pharyngeal pumping and locomotion that normally occurs during the lethargus period that precedes larval molting. These results reveal an evolutionarily conserved role for EGF signaling in the regulation of behavioral quiescence (Van Buskirk, 2007).

Table of contents