These ligands differ from one another in their regulated secretion, tissue distribution, and binding properties. Studies in which specific ligands have been ablated, either surgically or genetically, indicate that often one ligand can compensate for another Tsutsumi et al. When only one or two of the three ligands were ablated, there were only subtle phenotypes. These findings suggest there are complementary roles for these ligands and underscore the difficulty in understanding receptor physiology in the context of a whole animal.
Inactive EGFR exist as monomers on the plasma membrane. The active kinase domain of one EGFR monomer transphosphorylates tyrosine residues on carboxyl terminus of its receptor pair. Once activated, the phosphotyrosines serve as docking site for downstream effectors, which include enzymes, adaptor proteins, and other regulatory molecules.
Signaling from effectors integrates to modulate cell physiology, some of which are indicated. The EGFR is also critical to the development of tissues of epithelial, mesenchymal, and neuronal origin. EGFR knockout mice have been bred from different genetic backgrounds, showing either embryonic or postnatal lethality with multiple organ defects Miettinen et al. These findings point to the essential role of the EGF receptor in the development and tissue homeostasis of organisms in which it is expressed.
Thus, the complete loss of EGFR function is deleterious to the organism. Ligand binding initiates receptor activation by inducing a conformational change and allowing for dimerization of two EGF receptor monomers Ferguson et al. Dimerization leads to transphosphorylation of tyrosine residues on the cytoplasmic tail of one receptor by the intracellular kinase domain of the corresponding dimer Lammers et al.
Tyrosine phosphorylation is the essential activation step in EGFR signal transduction as these residues serve as docking sites for downstream signaling molecules containing Src homology 2 SH2 or phosphotyrosine binding PTB domains.
The activities and biochemical changes induced by these signaling pathways integrate to mediate the specific modulation in cell biology such as cell growth, proliferation, differentiation, migration, and regulation of apoptosis Jorissen et al. Several years later, a direct link between increased signaling from the EGFR and malignant growth was found in Epstein-Barr virus infected cells, a condition already known to lead to epithelial malignancies Miller et al.
Clinical studies provided supporting evidence that many human tumors and cell lines derived from human tumors overexpress EGFRs. Overexpression is used as an indicator of poor prognosis in breast, ovarian, and head and neck cancers Fischer-Colbrie et al.
In the clinic, increased EGFR expression has also been implicated with resistance to hormonal therapies in advanced stage breast cancers Newby et al. Although, it remains controversial as to whether EGFR overexpression is the cause of the cancer or a secondary consequence, the strong association between the EGFR and cancer has made it a natural candidate as an anti-cancer chemotherapeutic.
Enhanced EGFR signaling can arise from a variety of mechanisms including receptor overexpression, mutations leading to constitutive activation, increased ligand production, or defective inactivation Zandi et al.
Since then, the EGFR has been a major molecular target in the treatment of cancer. From the work in tissue culture, two strategies have emerged for inhibiting uncontrolled cell growth arising from EGFR overexpression or hyperactivation—monoclonal antibodies MAbs and tyrosine kinase inhibitors TKIs. These approaches share the same goal of inhibition of receptor activity but differ in their molecular mechanism.
MAbs target the extracellular portion of the receptor whereas the TKIs inhibit the intracellular portion. Multiple drugs from each class have been approved by the FDA for the treatment of certain types of cancers. These antibodies are used therapeutically for the treatment of metastatic colorectal cancer and cancers of the head and neck. Both Cetuximab and Vectibix inhibit binding of ligands to the EGFR causing a decrease in basal and ligand mediated receptor activation Harari et al.
The decreased receptor activity inhibits cell growth, induces apoptosis, and decrease the production of other cellular factors associated with cancer progression and metastasis, such as matrix metalloproteinases, and vascular endothelial growth factor Astsaturov et al.
Currently, the MAbs are used in combination with other agents or alone when the cancer is refractory to standard therapy. Like the MAbs the mechanism of action for the TKIs is to block the activation of downstream signaling pathways. While these are effective approaches in tissue culture models, the methodology for delivering RNAi, oligonucleotides, and ribozymes to patients requires further development.
However, both classes of drugs are currently enrolling patients in Phase 2 and Phase 3 trials for the treatment of other EGFR positive cancers, such as cervical, skin, myelogenous leukemia, prostate and glioblastomas ClinicalTrials. Dermatological rash, light sensitivity, and acne and gastrointestinal diarrhea, loss of appetite, and nausea side effects have been reported for both therapeutic approaches Rocha-Lima et al. Alternative approaches to targeting the EGFR need to take into consideration the empirically determined strengths and weaknesses of the current therapies.
Rather than inhibit the activation of the EGFR, an alternative approach would be to accelerate the rate of receptor inactivation. Strategies to attenuate the activated EGFR in cancers by accelerating the endocytic process have been largely overlooked. Components of the endocytic pathway could be stimulated to accelerate the normal rate of signal attenuation.
At this time, there are no agents under consideration, but that likely reflects the lack of molecular details regarding the EGFR signal termination and which molecules would be the best candidates. Despite the incomplete understanding of this process, there are some obvious advantages to this strategy.
By decreasing the duration of the active EGFR, the signaling necessary for cellular homeostasis would still be permitted while uncontrolled cell growth and replication would be inhibited. Third, by targeting receptor inactivation, there is no discrimination between uncontrolled cell growth that arises due to receptor overexpression versus receptor hyperactivation.
This may allow such compounds have a broader applicability. Below we discuss four potential mechanisms for signal termination of the activated EGFR from within the endocytic pathway: 1 dissociation of the ligand:receptor complex, 2 phosphatase-mediated receptor dephosphorylation 3 sequestration of the activated EGFR from effector molecules, and 4 lysosomal degradation of the receptor Fig.
These molecular mechanisms have been shown to be effective ways of attenuating EGFR signaling in tissue culture models, but it remains unclear which ones are physiologically important. Nevertheless, at this point, they all remain as viable pharmacological targets. This discussion will include the pros and cons of each mechanism.
Ligand stimulation accelerates the rate of EGFR internalization via clathrin-coated pits. Following invagination and pinching off, the resulting clathrin-coated vesicle sheds its clathrin and delivers its cargo to the early endosome. In the early endosome, the cargo is sorted for delivery to its appropriate cellular location.
In most cases, the EGFR is delivered to the lysosome for degradation. Indicated with dashed arrows are other possible routes of endocytic trafficking. In addition, unliganded EGFR will traffic from the early endosome to the plasma membrane via a recycling endosome upon ligand dissociation.
Ligand binding mediates two related functions. First, as mentioned above, is the initiation of downstream signaling pathways. Second, the ligand: receptor complex is internalized. In addition, the internalization of the receptor physically moves the receptor through various endocytic compartments, and thereby changes the downstream effectors with which it has contact. Ligand-mediated receptor endocytosis has historically been overlooked as a molecular mechanism to attenuate the signaling of the EGFR.
However, ligand binding accelerates this process and induces a more dramatic redistribution of the receptor from the plasma membrane and directs the activated EGFR to the lysosome for degradation Fig.
In cells that express multiple ErbB family members, receptors that heterodimerize with the EGFR prevent endocytosis of the entire ligand:receptor complex Baulida et al. At the plasma membrane, ligand-bound EGFRs move laterally along the cell surface to a plasma membrane domain whose intracellular face is enriched with clathrin.
The membrane domain invaginates to give rise to a clathrin-coated pit that eventually pinches off to form a clathrin-coated vesicle containing the ligand:receptor complex. Once inside the cell, the clathrin is shed from the vesicle and is now referred to as a primary endocytic vesicle. Through endosomal maturation, the cargo arrives in a second endosomal compartment often referred to interchangeably as the multivesicular body MVB or late endosome. During this maturation, membrane structures form in the lumen, called intralumenal vesicles.
In these internal vesicles, the EGFR is oriented such that the carboxyl terminal phosphotyrosines no longer have access to effector proteins. Finally, cargo is transferred to the lysosome by fusion of the endosome with lysosome, and degraded in the acidic, protease rich environment Sorkin and von Zastrow, Ligand-mediated endocytosis has always been recognized as a means of regulating EGFR signaling, but the mechanisms of regulation are still being discovered.
Early studies by Wells et al. This idea was challenged by Vieira et al. Dynamin regulates the internalization of clathrin-coated pits, and the expression of dominant negative dynamin allowed activation of the EGFR and retention at the plasma membrane. In these experiments, it was determined that full activation of MAPK and PI3-K could not be achieved if the receptor were retained on the cell surface.
Conversely, the activity of some effectors i. Thus, receptor internalization both positively and negatively regulates signaling Vieira et al. The compartmentalization of the EGFR as it moves through the endocytic pathway provides additional mechanisms to regulate receptor:effector interactions.
It remains to be seen whether the signaling from a given endocytic compartment can be attributed to a specific cell physiology. If this does prove to be the case, inhibition of EGFR signaling from distinct cellular locations may be a new way to modulate receptor response.
Within 5—10 minutes of ligand stimulation, the ligand:receptor complex enters the cell and traffics to the early endosome. The early endosome, as well as all subsequent endosomes, is characterized by its increasingly acidic environment. The early endosome is recognized as a point of sorting in the cell where cargoes are directed to a variety of cellular locations such as the late endosome, endoplasmic reticulum, or to the plasma membrane.
For the EGFR, it has been shown that all three routes are viable options and dependent on the cell type. However, ligand-stimulated EGFR degradation is a saturable process, and trafficking to the lysosome may be the primary destination of the ligand:receptor complex in cells with the physiologic levels of receptor and trafficking proteins French et al.
Similarly, recycling of the stimulated EGFR back to the plasma membrane may be the consequence of receptor overexpression Masui et al. Targeting to the ER and onto the nucleus is a relatively new model, but has been shown in multiple cell lines and affects the transcription of cyclin D, and important regulator of cell cycle regulation Liao and Carpenter, The molecular mechanisms of some aspects of early endosomal sorting have been well-established.
Binding of ligand to the EGFR is pH sensitive, with optimal binding occurring at physiological pH and dissociation occurring at lower pHs. The lower pH of the early endosome can cause dissociation of the receptor from the ligand. Once free of ligand, the receptor becomes rapidly desphosphorylated, thereby inhibiting interactions with downstream effectors, such as c-Cbl Lenferink et al. In the absence of c-Cbl association and receptor ubiquitylation, the receptor is not properly targeted to the lysosome for destruction and instead recycles to the plasma membrane.
To date, it has not been shown that unique or significant signaling originates from the late endosomes, although this remains a formal possibility. Overall, ligand:receptor dissociation has limited potential as a mechanism for inhibiting EGFR signaling, unless additional measures could be taken to ensure the unbound receptor were targeted for degradation. A second mechanism by which EGFRs are inactivated is the catalyzed dephosphorylation of the receptor by phosphatases.
The discovery of protein-tyrosine phosphatases PTP came several years after that of tyrosine kinases, but they were immediately recognized as important regulatory components of signaling Tonks et al.
Disruption of the reciprocal interactions between kinases and phosphatases can result in dramatic changes in cell physiology Tonks and Neel, All PTPs share a central catalytic domain, while the differences in their amino and carboxy terminus confer unique cellular locations and binding partners Barford et al.
PTPs dephosphorylate substrates through the formation of a covalent bond between a phosphatase cysteine residue and the substrate phosphate followed by hydrolysis Cirri et al. There are approximately members of the PTP superfamily.
To date, more is known about tyrosine phosphatase regulation of EGFR signaling than regulation by dual specificity phosphatases. The tyrosine-only phosphatases can be further divided into the transmembrane receptor-like PTPs and the intracellular non-receptor PTPs Andersen et al. Both receptor and non-receptor tyrosine phosphatases have been shown to directly regulate EGFR activity Suarez Pestana et al. This regulation can occur at multiple levels: by choice of protein substrate receptor , recognition sequence, and subcellular localization.
Through mutation of either critical residues of the active site cysteine or the catalytic domain asparigine , these mutants overcome the transient nature of the enzyme-substrate complex to retain high affinity binding of phosphatases to their substrates Flint et al.
These mutatants were key in the identification of the EGFR as a substrate for numerous phosphatases. Subsequently it was shown that overexpression of transfected TCPTP, but not the substrate-trapping mutant, dephophorylated the receptor in an EGF-dependent manner, indicating the presence of a regulatory feedback mechanism Tiganis et al. Together, these studies illustrate how the targeted dephosphorylation of the EGFR can decrease the activity of effectors that lead to cell growth.
The specificity of a phosphatase can also be intramolecular. The existence of site-specific phosphatases that differentially dephosphorylate particular phospho-tyrosines on the carboxy terminus of the EGFR is well established.
The loss of these phosphotyrosines allows for regulation of individual EGFR signaling pathways. The third mechanism by which phosphatase activity is regulated is by restriction of its cellular distribution, thereby controlling where in the cell EGFR signaling is terminated.
The authors propose this to be the site of dephosphorylation, and suggest this occurs prior to the EGFR being targeted to the lysosome. Intriguingly, they propose that endocytosis of the receptor may potentiate signaling by removing the receptor from plasma membrane localized PTPs. It is important to note that increases in phosphatase activity are not always associated with decreased EGFR signaling.
Increased phosphatase activity can also positively regulate EGFR signaling. When considering strategies to attenuate EGFR signaling, there are a number of options including enhanced phosphatase recruitment, expression, and activation of receptor specific inactivating phosphatases and inhibition of phosphatases, such as SHP-2, that positively regulate signaling. However, prior to phosphatase becoming a therapeutic target, several important questions regarding molecular mechanism must be answered.
For instance, is the phosphatase specific for the EGFR or will other receptors also be affected? In such cases, the binding of a signaling molecule to the membrane receptor activates the receptor's inherent enzymatic activity. Of the various receptors that exhibit this capability, receptor tyrosine kinases RTKs make up the largest class. These cell surface receptors bind and respond to growth factors and other locally released proteins that are present at low concentrations.
RTKs play important roles in the regulation of cell growth, differentiation, and survival. When signaling molecules bind to RTKs, they cause neighboring RTKs to associate with each other, forming cross-linked dimers. Cross-linking activates the tyrosine kinase activity in these RTKs through phosphorylation — specifically, each RTK in the dimer phosphorylates multiple tyrosines on the other RTK. This process is called cross-phosphorylation. Once cross-phosphorylated, the cytoplasmic tails of RTKs serve as docking platforms for various intracellular proteins involved in signal transduction.
These proteins have a particular domain — called SH2 — that binds to phosphorylated tyrosines in the cytoplasmic RTK receptor tails. More than one SH2-containing protein can bind at the same time to an activated RTK, allowing simultaneous activation of multiple intracellular signaling pathways. Ultimately, RTK activation brings about changes in gene transcription. Signaling becomes complex as signals travel from the membrane to the nucleus, due to crosstalk between intermediates in various signaling pathways in the cell Figure 1.
Figure 1: RTK activation involves the joining together and phosphorylation of proteins. On the left, an unactivated RTK receptor pink encounters a ligand red. Upon binding, the receptor forms a complex of proteins that phosphorylate each other. In turn, this phosphorylation affects other proteins in the cell that change gene transcription not shown.
Figure Detail. One of the most common intracellular signaling pathways triggered by RTKs is known as the mitogen-activated protein MAP kinase cascade , because it involves three serine-threonine kinases. The pathway starts with the activation of Ras , a small G protein anchored to the plasma membrane. In its inactive state, Ras is bound to GDP. Each of the three kinases in this cascade then activates the next by phosphorylating it. Because all three kinases in this pathway phosphorylate multiple substrates, the initial signal is amplified at each step.
Then, the final enzyme in the pathway phosphorylates transcription regulators, leading to a change in gene transcription Figure 2. Many growth factors, including nerve growth factor and platelet-derived growth factor, use this pathway. For example, insulin-like growth factor receptors activate the enzyme phosphoinositide 3-kinase, which phosphorylates inositol phospholipids in the cell membrane, leading in turn to a protein kinase cascade distinct from the MAP kinase cascade that relays the signal to the nucleus.
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