Graham Carpenter

The EGF receptor: a nexus for trafficking and signaling.

Ligand binding to the EGF receptor initiates both the activation of mitogenic signal transduction pathways plus trafficking events that relocalize the receptor on the cell surface and within intracellular compartments. The trafficking compartments include caveolae, clathrin‐coated pits, and various endosome populations prior to receptor degradation in lysosomes. Evidence is presented that distinct signaling pathways are initiated from these different compartments. These include the Ras/MAP kinase cascade and the PLC‐dependent hydrolysis of PI‐4,5 P2. Multiple tyrosine kinase substrates that facilitate EGF receptor trafficking between these various compartments, as well as the participation of phosphoinositides and Ras‐like G proteins in the trafficking pathway are also described. BioEssays 22:697–707, 2000.

The Juxtamembrane Region of the EGF Receptor Functions as an Activation Domain

In several growth factor receptors, the intracellular juxtamembrane (JM) region participates in autoinhibitory interactions that must be disrupted for tyrosine kinase activation. Using alanine scanning mutagenesis and crystallographic approaches, we define a domain within the JM region of the epidermal growth factor receptor (EGFR) that instead plays an activating--rather than autoinhibitory--role. Mutations in the C-terminal 19 residues of the EGFR JM region abolish EGFR activation. In a crystal structure of an asymmetric dimer of the tyrosine kinase domain, the JM region of an acceptor monomer makes extensive contacts with the C lobe of a donor monomer, thus stabilizing the dimer. We describe how an uncharacterized lung cancer mutation in this JM activation domain (V665M) constitutively activates EGFR by augmenting its capacity to act as an acceptor in the asymmetric dimer. This JM mutant promotes cellular transformation by EGFR in vitro and is tumorigenic in a xenograft assay.

ErbB-4: mechanism of action and biology

The most recently described member of the ErbB receptor tyrosine kinase family is ErbB-4. In general, the structure of this receptor and its mechanism of action is similar to that described for ErbB-1. However, significantly less is known about ErbB-4 and there are several novel aspects to its structure, mechanism of action, and biology. This includes the spectrum of ligands that activate ErbB-4, the presence of functionally distinct isoforms, a proteolytic processing pathway to the nucleus, and the capacity to induce a spectrum of cellular responses such as mitogenesis, differentiation, growth inhibition, and survival.

Selective Cleavage of the Heregulin Receptor ErbB-4 by Protein Kinase C Activation

The 180-kDa transmembrane tyrosine kinase ErbB-4 is a receptor for the growth factor heregulin. 125I-Heregulin binding to NIH 3T3 cells overexpressing the ErbB-4 receptor is rapidly decreased by 12-O-tetradecanoylphorbol-13-acetate (TPA) pretreatment. Immunologic analysis demonstrates that TPA treatment of cells induces the proteolytic cleavage of ErbB-4, producing an 80-kDa cytoplasmic domain fragment, which contains a low level of phosphotyrosine, and a 120-kDa ectodomain fragment, which is released into the extracellular medium. Cleavage of ErbB-4 was also enhanced by other protein kinase C activators, i.e. platelet-derived growth factor, ionomycin, and synthetic diacylglycerol, while protein kinase C inhibition or down-regulation suppressed the TPA stimulation of ErbB-4 degradation. TPA did not induce the degradation of related receptors (ErbB-1, ErbB-2, and ErbB-3) in the EGF receptor family. The phorbol ester-induced cleavage of ErbB-4 occurs within or close to the ectodomain, as the 80-kDa cytoplasmic domain fragment is recognized by antibody to the ErbB-4 carboxyl terminus and is membrane-associated. Coprecipitation experiments show that, while the 80-kDa ErbB-4 fragment is associated with the SH2-containing molecules PLC-γ1 and Shc, TPA did not induce the phosphorylation of these substrates in intact cells. In addition, kinase assays in vitro indicate that the 80-kDa fragment is not an active tyrosine kinase. These results show that protein kinase C negatively regulates heregulin signaling through the ErbB-4 receptor by the activation of a selective proteolytic mechanism.

Epidermal growth factor, its receptor, and related proteins

An interesting aspect of the developments forthcoming from the study of cell growth control by growth factors is the structural and functional homologies that have been found to exist between growth factors or their receptors and other molecules. The epidermal growth factor (EGF) system has been particularly fruitful in this regard. The information in this article is meant to summarize the relationships that have been recently described between EGF and other EGF-like molecules and between the EGF receptor and related macromolecules.

Platelet-derived growth factor induces rapid and sustained tyrosine phosphorylation of phospholipase C-gamma in quiescent BALB/c 3T3 cells.

Platelet-derived growth factor (PDGF) stimulates the proliferation of quiescent fibroblasts through a series of events initiated by activation of tyrosine kinase activity of the PDGF receptor at the cell surface. Physiologically significant substrates for this or other growth factor receptor or oncogene tyrosine kinases have been difficult to identify. Phospholipase C (PLC), a key enzyme of the phosphoinositide pathway, is believed to be an important site for hormonal regulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate, which produces the intracellular second-messenger molecules inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. Treatment of BALB/c 3T3 cells with PDGF led to a rapid (within 1 min) and significant (greater than 50-fold) increase in PLC activity, as detected in eluates of proteins from a phosphotyrosine immunoaffinity matrix. This PDGF-stimulated increase in phosphotyrosine-immunopurified PLC activity occurred for up to 12 h after addition of growth factor to quiescent cells. Interestingly, the PDGF stimulation occurred at 3 as well as 37 degrees C and in the absence or presence of extracellular Ca2+. Immunoprecipitation of cellular proteins with monoclonal antibodies specific for three distinct cytosolic PLC isozymes demonstrated the presence of a 145-kilodalton isozyme, PLC-gamma (formerly PLC-II), in BALB/c 3T3 cells. Furthermore, these immunoprecipitation studies showed that PLC-gamma is rapidly phosphorylated on tyrosine residues after PDGF stimulation. The results suggest that mitogenic signaling by PDGF is coincident with tyrosine phosphorylation of PLC-gamma.

Nuclear localization and possible functions of receptor tyrosine kinases.

Recent data have renewed interest in the possible nuclear localization of receptor tyrosine kinases, as well as their ligands. In one case, that of ErbB-4, the receptor is processed by two membrane-localized proteases to produce a soluble cytoplasmic domain fragment that includes the tyrosine kinase domain. This fragment, generated by a metalloprotease-dependent ectodomain cleavage followed by gamma-secretase cleavage within the transmembrane domain, is also found in the nucleus. Three other receptor tyrosine kinases have been detected in the nucleus in the absence of proteolytic processing. In some instances, nuclear localization of receptor tyrosine kinases is growth-factor-dependent and tentative evidence suggests a role in transcription.

The Role of Individual SH2 Domains in Mediating Association of Phospholipase C-γ1 with the Activated EGF Receptor

The two SH2 (Src homology domain 2) domains present in phospholipase C-γ1 (PLC-γ1) were assayed for their capacities to recognize the five autophosphorylation sites in the epidermal growth factor receptor. Plasmon resonance and immunological techniques were employed to measure interactions between SH2 fusion proteins and phosphotyrosine-containing peptides. The N-SH2 domain recognized peptides in the order of pY1173 > pY992 > pY1068 > pY1148 ≫ pY1086, while the C-SH2 domain recognized peptides in the order of pY992 > pY1068 > pY1148 ≫ pY1086 and pY1173. The major autophosphorylation site, pY1173, was recognized only by the N-SH2 domain. Contributions of the N-SH2 and C-SH2 domains to the association of the intact PLC-γ1 molecule with the activated epidermal growth factor (EGF) receptor were assessedin vivo. Loss of function mutants of each SH2 domain were produced in a full-length epitope-tagged PLC-γ1. After expression of the mutants, cells were treated with EGF and association of exogenous PLC-γ1 with EGF receptors was measured. In this context the N-SH2 is the primary contributor to PLC-γ1 association with the EGF receptor. The combined results suggest an association mechanism involving the N-SH2 domain and the pY1173 autophosphorylation site as a primary event and the C-SH2 domain and the pY992 autophosphorylation site as a secondary event.

Receptor tyrosine kinase substrates: src homology domains and signal transduction.

Among the intracellular milieu of proteins are molecules with defined biochemical functions that serve as substrates for ligand‐activated tyrosine kinase receptors. It seems likely that some of these substrate molecules are elements of a critical signaling pathway used by growth factors to control cell proliferation and subverted by oncogenes to deregulate this process. Although the process of cell growth and division is relatively slow compared with other hormonally regulated responses, homeostasis in a human being requires approximately 20 × 106 cell divisions per second for the renewal of various cell populations. This review summarizes the present understanding of tyrosine kinase substrates that seem likely to have key roles in the signal transduction pathway that regulates cell proliferation. This includes structural features of these molecules, the influence of tyrosine phosphorylation on their functions, the biological roles of these proteins, and the capacity of these substrates to associate with activated receptor tyrosine kinases.— Carpenter, G. Receptor tyrosine kinase substrates: src homology domains and signal transduction. FASEB J. 6: 3283‐3289; 1992.

Epidermal growth factor receptor juxtamembrane region regulates allosteric tyrosine kinase activation.

Structural studies of the extracellular and tyrosine kinase domains of the epidermal growth factor receptor (ErbB-1) provide considerable insight into facets of the receptor activation mechanism, but the contributions of other regions of ErbB-1 have not been ascertained. This study demonstrates that the intracellular juxtamembrane (JM) region plays a vital role in the kinase activation mechanism. In the experiments described herein, the entire ErbB-1 intracellular domain (ICD) has been expressed in mammalian cells to explore the significance of the JM region in kinase activity. Deletion of the JM region (ΔJM) results in a severe loss of ICD tyrosine phosphorylation, indicating that this region is required for maximal activity of the tyrosine kinase domain. Coexpression of ΔJM and dimerization-deficient kinase domain ICD mutants revealed that the JM region is indispensable for allosteric kinase activation and productive monomer interactions within a dimer. Studies with the intact receptor confirmed the role of the JM region in kinase activation. Within the JM region, Thr-654 is a known protein kinase C (PKC) phosphorylation site that modulates kinase activity in the context of the intact ErbB-1 receptor; yet, the mechanism is not known. Whereas a T654A mutation promotes increased ICD tyrosine phosphorylation, the phosphomimetic T654D mutant generates a 50% reduction in ICD tyrosine phosphorylation. Similar to the ΔJM mutants, the T654D mutant ICD failed to interact with a wild-type monomer. This study reveals an integral role for the intracellular JM region of ErbB-1 in allosteric kinase activation.