A stopped-flow fluorescence anisotropy method for measuring hormone binding and dissociation kinetics with cell-surface receptors in living cells

We have developed a system for extending stopped-flow analysis to the kinetics of ligand capture and release by cell surface receptors in living cells. While most mammalian cell lines cannot survive the shear forces associated with turbulent, stopped-flow mixing, we determined that 32D cells, murine hematopoietic precursor cells, can survive rapid mixing, even at the high flow rates necessary to achieve dwell times as short as 10 msec. In addition, 32D cells do not express any member of the ErbB family of receptors, providing a null background for studying this receptor family. We have established a series of stable, monoclonal 32D-derived cell lines that express the epidermal growth factor (EGF) receptor, ErbB2, or a combination of both at different ratios. Using these cell lines and a homogeneous fluorescent derivative of H22Y-mEGF modified with fluorescein at the amino terminus (F-EGF), we have measured association and dissociation of F-EGF with its receptor. Association was measured by following the time-dependent changes in fluorescence anisotropy after rapidly mixing cells at various cell densities with F-EGF at 1-15nM. Dissociation was measured both by chase experiments in which unlabeled EGF was mixed with cells pre-equilibrated with F-EGF or by dilution of cells equilibrated with F-EGF. Comparison of these dissociation experiments demonstrated that little or no ligand-induced dissociation occurs in the chase dissociation experiments. For each cell line, data from a series of association experiments and dilution dissociation experiments were subjected to global analysis using a two independent receptor-class model. Our analysis is consistent with the presence of two distinct receptor populations, even in cells bearing only the EGF receptor. Increasing the relative expression of ErbB2 leads to an increase in the fraction of high affinity class receptors observed, without altering the total number of EGF binding sites.     

A STOPPED-FLOW FLUORESCENCE ANISOTROPY METHOD FOR MEASURING HORMONE BINDING AND DISSOCIATION KINETICS WITH CELL-SURFACE RECEPTORS IN LIVING CELLS

ABSTRACT

We have developed a system for extending stopped-flow analysis to the kinetics of ligand capture and release by cell surface receptors in living cells. While most mammalian cell lines cannot survive the shear forces associated with turbulent, stopped-flow mixing, we determined that 32D cells, murine hematopoietic precursor cells, can survive rapid mixing, even at the high flow rates necessary to achieve dwell times as short as 10?msec. In addition, 32D cells do not express any member of the ErbB family of receptors, providing a null background for studying this receptor family. We have established a series of stable, monoclonal 32D-derived cell lines that express the epidermal growth factor (EGF) receptor, ErbB2, or a combination of both at different ratios. Using these cell lines and a homogeneous fluorescent derivative of H22Y-mEGF modified with fluorescein at the amino terminus (F-EGF), we have measured association and dissociation of F-EGF with its receptor. Association was measured by following the time-dependent changes in fluorescence anisotropy after rapidly mixing cells at various cell densities with F-EGF at 1–15?nM. Dissociation was measured both by chase experiments in which unlabeled EGF was mixed with cells pre-equilibrated with F-EGF or by dilution of cells equilibrated with F-EGF. Comparison of these dissociation experiments demonstrated that little or no ligand-induced dissociation occurs in the chase dissociation experiments. For each cell line, data from a series of association experiments and dilution dissociation experiments were subjected to global analysis using a two independent receptor-class model. Our analysis is consistent with the presence of two distinct receptor populations, even in cells bearing only the EGF receptor. Increasing the relative expression of ErbB2 leads to an increase in the fraction of high affinity class receptors observed, without altering the total number of EGF binding sites.     

Real-time kinetics of ligand/cell surface receptor interactions in living cells: binding of epidermal growth factor to the epidermal growth factor receptor

We describe a system for extending stopped-flow analysis to the kinetics of ligand capture and release by cell surface receptors in living cells. While most mammalian cell lines cannot survive the shear forces associated with turbulent stopped-flow mixing, we determined that a murine hematopoietic precursor cell line, 32D, is capable of surviving rapid mixing using flow rates as great as 4.0 mL/s, allowing rapid processes to be quantitated with dead times as short as 10 ms. 32D cells do not express any endogenous epidermal growth factor (EGF) receptor or other ErbB family members and were used to establish monoclonal cell lines stably expressing the EGF receptor. Association of fluorescein-labeled H22Y-murine EGF (F-EGF) to receptor-expressing 32D cells was observed by measuring time-dependent changes in fluorescence anisotropy following rapid mixing. Dissociation of F-EGF from EGF-receptor-expressing 32D cells was measured both by chase experiments using unlabeled mEGF and by experiments in which equilibrium was perturbed by dilution. Comparison of these dissociation experiments showed that little, if any, ligand-induced dissociation occurs in the chase dissociation experiments. Data from a series of association and dissociation experiments, performed at various concentrations of F-EGF in the nanomolar range and at multiple cell densities, were simultaneously analyzed using global analysis techniques and fit to a two independent receptor-class model. Our analysis is consistent with the presence of two distinct receptor populations having association rate constants of k(on1) = 8.6 x 10(6) M(-1) s(-1) and k(on2) = 2.4 x 10(6) M(-1) s(-1) and dissociation rate constants of k(off1) = 0.17 x 10(-2) s(-1) and k(off2) = 0.21 x 10(-2) s(-1). The magnitudes of these parameters suggest that under physiological conditions, in which cells are transiently exposed to nanomolar concentrations of ligand, ligand capture and release may function as the first line of regulation of the EGF receptor-induced signal transduction cascade.     

Quantitative characterization of the large-scale association of ErbB1 and ErbB2 by flow cytometric homo-FRET measurements

The association of receptor tyrosine kinases is a key step in the initiation of growth factor-mediated signaling. Although the ligand-induced dimerization of inactive, monomeric receptors was the central dogma of receptor tyrosine kinase activation for decades, the existence of larger oligomers is now accepted. Both homoassociations and heteroassociations are of extreme importance in the epidermal growth factor (EGF) receptor family, leading to diverse and robust signaling. We present a statistically reliable, flow-cytometric homo-fluorescence resonance energy transfer method for the quantitative characterization of large-scale receptor clusters. We assumed that a fraction of a certain protein species is monomeric, whereas the rest are present in homoclusters of N-mers. We measured fluorescence anisotropy as a function of the saturation of fluorescent antibody binding, and fitted the model to the anisotropy data yielding the fraction of monomers and the cluster size. We found that ErbB2 formed larger homoclusters than ErbB1. Stimulation with EGF and heregulin led to a decrease in ErbB2 homocluster size, whereas ErbB1 homoclusters became larger after EGF stimulation. The activation level of ErbB2 was inversely proportional to its homocluster size. We conclude that homoclusters of ErbB1 and ErbB2 behave in a fundamentally different way. Whereas huge ErbB2 clusters serve as a reservoir of inactive coreceptors and dissociate upon stimulation, small ErbB1 homoclusters form higher-order oligomers after ligand binding.     

Epidermal growth factor mutant with wild-type affinity for both ErbB1 and ErbB3

The family of epidermal growth factor (EGF)-like ligands binds to ErbB receptors in a highly selective manner. Previous studies indicated that both linear regions of the ligand play a major role in determining receptor selectivity, and phage display studies showed that each region could be optimized independently for enhanced affinity. In this study, we broadened the ErbB binding specificity of EGF by introducing the optimal sequence requirements for ErbB3 binding in both the N- and C-terminal linear regions. One such EGF mutant, designated WVR/EGF/IADIQ, gained high affinity for ErbB3 and showed concomitant ErbB3 activation through ErbB2.ErbB3 heterodimers similar to the natural ErbB3 ligand NRG1beta, while the capacity to bind and activate ErbB1 was fully maintained. Despite its high affinity for ErbB1 and ErbB3, this mutant was unable to activate ErbB1.ErbB3 heterodimers, as shown by the cell survival and receptor phosphorylation analysis. We concluded that despite the fact that no naturally occurring ligand exists with this dual-specificity, high-affinity binding to both ErbB1 and ErbB3 is not mutually exclusive. This mutant can be useful in a direct structural comparison of the ligand-binding characteristics of ErbB1 and ErbB3.     

Enhanced drug resistance in cells coexpressing ErbB2 with EGF receptor or ErbB3

Overexpression of ErbB2 has been found in approximately 25-30% of human breast cancers and has been shown to render the cancer cells more resistant to chemotherapy. However, it is not clear whether ErbB2 overexpression renders the cells more resistant to specific anti-cancer drugs or renders the cells more resistant to a broad range of anti-cancer drugs. It is not clear how the function of ErbB2 in drug resistance is related to expression and activation of the other ErbB receptors. In this communication, we showed that several breast cancer cell lines including BT20, BT474, MCF-7, MDA-MB-453, and SKBR-3 cells had a similar pattern of resistance to a broad range of anti-cancer drugs including 5-Fluorouracil, Cytoxan, Doxorubincin, Taxol, and Vinorelbin, suggesting a mechanism of multidrug resistance. High expression of P-glycoprotein and the ErbB receptors contribute to drug resistance of these breast cancer cells; however, overexpression of ErbB2 alone is not a major factor in determining drug resistance. To further determine the role of the ErbB receptors in drug resistance, we selected various NIH 3T3 cell lines that specifically expressed EGF receptor (EGFR), ErbB2, ErbB3, EGFR/ErbB2, EGFR/ErbB3, or ErbB2/ErbB3. A cytotoxicity assay showed that expression of ErbB2 alone did not significantly enhance drug resistance, whereas coexpression of either EGFR or ErbB3 with ErbB2 significantly enhanced drug resistance. Moreover, ErbB2 was highly phosphorylated in NIH 3T3 cells that coexpress ErbB2 with either EGFR or ErbB3, but not in NIH 3T3 cells that express ErbB2 alone. Together, our results suggest that coexpression of EGFR or ErbB3 with ErbB2 induces high phosphorylation of ErbB2 and renders the cells more resistant to various anti-cancer drugs.     

ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling.

We have analyzed ErbB receptor interplay induced by the epidermal growth factor (EGF)-related peptides in cell lines naturally expressing the four ErbB receptors. Down-regulation of cell surface ErbB-1 or ErbB-2 by intracellular expression of specific antibodies has allowed us to delineate the role of these receptors during signaling elicited by: EGF and heparin binding EGF (HB-EGF), ligands of ErbB-1; betacellulin (BTC), a ligand of ErbB-1 and ErbB-4; and neu differentiation factor (NDF), a ligand of ErbB-3 and ErbB-4. Ligand-induced ErbB receptor heterodimerization follows a strict hierarchy and ErbB-2 is the preferred heterodimerization partner of all ErbB proteins. NDF-activated ErbB-3 or ErbB-4 heterodimerize with ErbB-1 only when no ErbB-2 is available. If all ErbB receptors are present, NDF receptors preferentially dimerize with ErbB-2. Furthermore, EGF- and BTC-induced activation of ErbB-3 is impaired in the absence of ErbB-2, suggesting that ErbB-2 has a role in the lateral transmission of signals between other ErbB receptors. Finally, ErbB-1 activated by all EGF-related peptides (EGF, HB-EGF, BTC and NDF) couples to SHC, whereas only ErbB-1 activated by its own ligands associates with and phosphorylates Cbl. These results provide the first biochemical evidence that a given ErbB receptor has distinct signaling properties depending on its dimerization.     

Rotational mobility of high-affinity epidermal growth factor receptors on the surface of living A431 cells.

The rotational diffusion of epidermal growth factor (EGF) bound to its specific receptor on the surface of human carcinoma A431 cells was studied by means of time-resolved phosphorescence anisotropy measurements. The rotational mobility was measured on the total population of EGF receptors by using a saturating concentration of EGF conjugated with a phosphorescent label, erythrosin, or on the subpopulation of high-affinity EGF receptors by using a low concentration of labeled EGF. At 4 degrees C, the rotational correlation times for both the high-affinity and total (mostly low affinity) receptor populations were in the range of 60-100 microns. Elevation of the temperature to 37 degrees C resulted in a lengthening of the rotational correlation time of the total receptor population to 200-300 microns, confirming a previous study of receptor microaggregation. The high-affinity EGF receptors were completely immobilized at 37 degrees C (rotational correlation time greater than 500 microns). The data are consistent with a model involving association of the cytoskeleton with the high-affinity receptors at 37 degrees C, but not at 4 degrees C.     

Quantitation of the Effect of ErbB2 on Epidermal Growth Factor Receptor Binding and Dimerization

The epidermal growth factor (EGF) receptor is a member of the ErbB family of receptors that also includes ErbB2, ErbB3, and ErbB4. These receptors form homo- and heterodimers in response to ligand with ErbB2 being the preferred dimerization partner. Here we use (125)I-EGF binding to quantitate the interaction of the EGF receptor with ErbB2. We show that the EGFR/ErbB2 heterodimer binds EGF with a 7-fold higher affinity than the EGFR homodimer. Because it cannot bind a second ligand, the EGFR/ErbB2 heterodimer is not subject to ligand-induced dissociation caused by the negatively cooperative binding of EGF to the second site on the EGFR homodimer. This increases the stability of the heterodimer relative to the homodimer and is associated with enhanced and prolonged EGF receptor autophosphorylation. These effects are independent of the kinase activity of ErbB2 but require back-to-back dimerization of the EGF receptor with ErbB2. Back-to-back dimerization is also required for phosphorylation of ErbB2. These findings provide a molecular explanation for the apparent preference of the EGF receptor for dimerizing with ErbB2 and suggest that the phosphorylation of ErbB2 occurs largely in the context of the EGFR/ErbB2 heterodimer, rather than through lateral phosphorylation of isolated ErbB2 subunits.     

Regulation of the EGFR/ErbB signalling by clathrin in response to various ligands in hepatocellular carcinoma cell lines

Membrane receptor intracellular trafficking and signalling are frequently altered in cancers. Our aim was to investigate whether clathrin‐dependent trafficking modulates signalling of the ErbB receptor family in response to amphiregulin (AR), EGF, heparin‐binding EGF‐like growth factor (HB‐EGF) and heregulin‐1β (HRG). Experiments were performed using three hepatocellular carcinoma (HCC) cell lines, Hep3B, HepG2 and PLC/PRF/5, expressing various levels of EGFR, ErbB2 and ErbB3. Inhibition of clathrin‐mediated endocytosis (CME), by down‐regulating clathrin heavy chain expression, resulted in a cell‐ and ligand‐specific pattern of phosphorylation of the ErbB receptors and their downstream effectors. Clathrin down‐regulation significantly decreased the ratio between phosphorylated EGFR (pEGFR) and total EGFR in all cell lines when stimulated with AR, EGF, HB‐EGF or HRG, except in HRG‐stimulated Hep3B cells in which pEGFR was not detectable. The ratio between phosphorylated ErbB2 and total ErbB2 was significantly decreased in clathrin down‐regulated Hep3B cells stimulated with any of the ligands, and in HRG‐stimulated PLC/PRF/5 cells. The ratio between phosphorylated ErbB3 and total ErbB3 significantly decreased in clathrin down‐regulated cell lines upon stimulation with EGF or HB‐EGF. STAT3 phosphorylation levels significantly increased in all cell lines irrespective of stimulation, while that of AKT remained unchanged, except in AR‐stimulated Hep3B and HepG2 cells in which pAKT was significantly decreased. Finally, ERK phosphorylation was insensitive to clathrin inhibition. Altogether, our observations indicate that clathrin regulation of ErbB signalling in HCC is a complex process that likely depends on the expression of ErbB family members and on the autocrine/paracrine secretion of their ligands in the tumour environment.     

Modeling the effects of HER/ErbB1-3 coexpression on receptor dimerization and biological response

The human epidermal growth factor receptor (HER/ErbB) system comprises the epidermal growth factor receptor (EGFR/HER1) and three other homologs, namely HERs 2-4. This receptor system plays a critical role in cell proliferation and differentiation and receptor overexpression has been associated with poor prognosis in cancers of the epithelium. Here, we examine the effect of coexpressing varying levels of HERs 1-3 on the receptor dimerization patterns using a detailed kinetic model for HER/ErbB dimerization and trafficking. Our results indicate that coexpression of EGFR with HER2 or HER3 biases signaling to the cell surface and retards signal downregulation. In addition, simultaneous coexpression of HERs 1-3 leads to an abundance of HER2-HER3 heterodimers, which are known to be potent inducers of cell growth and transformation. Our new approach to use parameter dependence analysis in experimental design reveals that measurements of HER3 phosphorylation and HER2 internalization ratio may prove to be especially useful for the estimation of critical model parameters. Further, we examine the effect of receptor dimerization patterns on biological response using a simple phenomenological model. Results indicate that coexpression of EGFR with HER2 and HER3 at low to moderate levels may enable cells to match the response of a high HER2 expresser.     

Ligand depletion negatively controls the mitogenic activity of epidermal growth factor

EGF activates the ErbB1 receptor, but there appears only a limited correlation between its receptor binding affinity and mitogenic activity. This is indicated by our present observation that in cells with high ErbB1 expression, including SUM102 breast tumor cells, low affinity EGF/Notch chimeras have similarly high mitogenic activity as EGF, in spite of the fact that EGF is superior in inducing receptor tyrosine phosphorylation and p42/p44 MAP-kinase activity. However, as a result of receptor-mediated internalisation high-affinity ligands such as EGF are depleted much more rapidly from the extracellular medium than low-affinity EGF/Notch chimeras. As a consequence, the mitogenic activity of EGF on ErbB1 overexpressing cells is limited by substantial degradation of internalised ligand in the period before cells enter S-phase, a phenomenon that is not observed for low affinity mutant ligands. The mitogenic activity of EGF on ErbB1 overexpressing cells does therefore not only depend on the applied concentration but also on the total amount of ligand added, and is strongly underestimated when tested in a limited assay volume. No such dependence on the incubation volume was observed for EGF activity on cells with low ErbB1 expression levels and on cells for which EGF is growth inhibitory.     

Transmembrane peptides as inhibitors of ErbB receptor signaling

Receptor tyrosine kinases have a single transmembrane (TM) segment that is usually assumed to play a passive role in ligand-induced dimerization and activation of the receptor. However, mutations within some of these receptors, and recent studies with the epidermal growth factor (EGF) and ErbB2 receptors have indicated that interactions between TM domains do contribute to stabilization of ligand-independent and/or ligand-induced receptor dimerization and activation. One consequence of the importance of these interactions is that short hydrophobic peptides corresponding to these domains should act as specific inhibitors. To test this hypothesis, we constructed expression vectors encoding short fusion peptides encompassing native or mutated TM domains of the EGF, ErbB2, and insulin receptors. In human cell lines overexpressing the wild-type EGF receptor or ErbB2, we observed that the peptides are expressed at the cell surface and that they inhibit specifically the autophosphorylation and signaling pathway of their cognate receptor. Identical results were obtained with peptides chemically synthesized. Mechanism of action involves inhibition of dimerization of the receptors as shown by the lack of effects of mutant nondimerizing sequences, completed by density centrifugation and covalent cross-linking experiments. Our findings stress the role of TM domain interactions in ErbB receptor function, and possibly for other single-spanning membrane proteins.     

The leucine-rich repeat protein LRIG1 is a negative regulator of ErbB family receptor tyrosine kinases

The molecular mechanisms by which mammalian receptor tyrosine kinases are negatively regulated remain largely unexplored. Previous genetic and biochemical studies indicate that Kekkon-1, a transmembrane protein containing leucine-rich repeats and an immunoglobulin-like domain in its extracellular region, acts as a feedback negative regulator of epidermal growth factor (EGF) receptor signaling in Drosophila melanogaster development. Here we tested whether the related human LRIG1 (also called Lig-1) protein can act as a negative regulator of EGF receptor and its relatives, ErbB2, ErbB3, and ErbB4. We observed that in co-transfected 293T cells, LRIG1 forms a complex with each of the ErbB receptors independent of growth factor binding. We further observed that co-expression of LRIG1 with EGF receptor suppresses cellular receptor levels, shortens receptor half-life, and enhances ligand-stimulated receptor ubiquitination. Finally, we observed that co-expression of LRIG1 suppresses EGF-stimulated transformation of NIH3T3 fibroblasts and that the inducible expression of LRIG1 in PC3 prostate tumor cells suppresses EGF- and neuregulin-1-stimulated cell cycle progression. Our observations indicate that LRIG1 is a negative regulator of the ErbB family of receptor tyrosine kinases and suggest that LRIG1-mediated receptor ubiquitination and degradation may contribute to the suppression of ErbB receptor function.     

Differential association of phosphatidylinositol-5-phosphate 4-kinase with the EGF/ErbB family of receptors.

Phosphatidylinositol-5-phosphate 4-kinase (PIP4K) is required for the production of phosphoinositol-4,5-hisphosphate (PIP2), which has been closely associated with growth factor signalling. Here we have tested the possibility that phosphoinositide kinases may be take part in signal transduction through interactions with the epidermal growth factor (EGF) receptor and the ErbB family of tyrosine kinase receptors. Interactions of the Type IIbeta isoform of PIP4K were observed with the EGF receptor family members in a number of diverse cell lines, including A431, PC12 and MCF7 cells but not with the N6F TrkA receptor. Co-immunoprecipitation experiments indicate that PIP4K interacts with not only the EGF receptor, but also selectively with members of the ErbB tyrosine kinase family. These results demonstrate another enzyme substrate for EGF receptors that facilitates the production of phosphoinositides at the cell membrane.     

Heparin-binding EGF-like growth factor interacts with mouse blastocysts independently of ErbB1: a possible role for heparan sulfate proteoglycans and ErbB4 in blastocyst implantation

Blastocyst implantation requires molecular and cellular interactions between the uterine luminal epithelium and blastocyst trophectoderm. We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) is induced in the mouse luminal epithelium solely at the site of blastocyst apposition at 16:00 hours on day 4 of pregnancy prior to the attachment reaction (22:00-23:00 hours), and that HB-EGF promotes blastocyst growth, zona-hatching and trophoblast outgrowth. To delineate which EGF receptors participate in blastocyst activation, the toxicity of chimeric toxins composed of HB-EGF or TGF-(&agr;) coupled to Pseudomonas exotoxin (PE) were used as measures of receptor expression. TGF-(&agr;) or HB-EGF binds to EGF-receptor (ErbB1), while HB-EGF, in addition, binds to ErbB4. The results indicate that ErbB1 is inefficient in mediating TGF-(&agr;)-PE or HB-EGF-PE toxicity as follows: (i) TGF-(&agr;)-PE was relatively inferior in killing blastocysts, 100-fold less than HB-EGF-PE, (ii) analysis of blastocysts isolated from cross-bred egfr+/- mice demonstrated that HB-EGF-PE, but not TGF-(&agr;)-PE, killed egfr-/- blastocysts, and (iii) blastocysts that survived TGF-(&agr;)-PE were nevertheless killed by HB-EGF-PE. HB-EGF-PE toxicity was partially mediated by cell surface heparan sulfate proteoglycans (HSPG), since a peptide corresponding to the heparin-binding domain of HB-EGF as well as heparitinase treatment protected the blastocysts from the toxic effects of HB-EGF-PE by about 40%. ErbB4 is a candidate for being an HB-EGF-responsive receptor since RT-PCR analysis demonstrated that day 4 mouse blastocysts express two different erbB4 isoforms and immunostaining with anti-ErbB4 antibodies confirmed that ErbB4 protein is expressed at the apical surface of the trophectoderm cells. It is concluded that (i) HB-EGF interacts with the blastocyst cell surface via high-affinity receptors other than ErbB1, (ii) the HB-EGF interaction with high-affinity blastocysts receptors is regulated by heparan sulfate, and (iii) ErbB4 is a candidate for being a high-affinity receptor for HB-EGF on the surface of implantation-competent blastocysts.     

ERBB2 is a target for USP8-mediated deubiquitination

Overexpression and poor downregulation of ErbB receptor tyrosine kinases are associated with enhanced signaling and tumorigenesis. Attenuation of EGF-receptor (EGFR) signaling is mediated by endocytosis and ubiquitination by the E3-ligase Cbl. En route to lysosomes, but before incorporation of the EGFR into internal vesicles of MVBs, the EGFR undergoes Usp8-mediated deubiquitination. ErbB2 displays enhanced recycling back to the cell surface, and therefore we hypothesized that Usp8 is not part of the ErbB2 trafficking pathway. Here, we demonstrate, in the context of a chimeric EGFR-ErbB2 receptor, that (i) EGF induces pY1091 Cbl binding site-dependent K63-polyubiquitination of EGFR-ErbB2, (ii) Cbl is tyrosine phosphorylated upon stimulation of EGFR-ErbB2 wt and Y1091F mutant receptor, (iii) EGF-induced activation of EGFR-ErbB2 induces Usp8 tyrosine phosphorylation, and (iv) ubiquitination of the EGFR-ErbB2 wt and Y1091F mutant is enhanced upon coexpression of catalytically inactive Usp8-C748A in the presence and absence of EGF. We further show that Usp8 tyrosine phosphorylation upon stimulation of EGFR-ErbB2 is (a) independent of Y1091, (b) dependent on Src- and EGFR-ErbB2-kinase activity, (c) enhanced upon coexpression of Usp8-C748A, and (d) partly dependent on the Microtubule Interacting and Transport (MIT) domain of Usp8. Our findings demonstrate that Usp8 is part of the ErbB2 endosomal trafficking pathway.     

Functional effects of glycosylation at Asn-579 of the epidermal growth factor receptor

We have investigated functional effects of glycosylation at N(579) of the epidermal growth factor receptor (EGFR). Our previous study showed that the population of cell-surface expressed EGFRs in A431 cells, a human epidermoid carcinoma cell line, is composed of two subpopulations that differ by glycosylation at N(579) [Zhen et al. (2003) Biochemistry 42, 5478-5492]. To characterize the subpopulation of receptors not glycosylated at N(579), we established a 32D cell line expressing a point mutant of the EGFR (N579Q), which cannot be glycosylated at this position. Analysis of epitope accessibility suggests that the lack of glycosylation at N(579) weakens auto-inhibitory tether interactions, and cross-linking experiments suggest a somewhat elevated level of preformed N579Q-EGFR dimers in the absence of ligand relative to wild-type EGFR (WT-EGFR). However, ligand drives the majority of N579Q-EGFR dimerization, suggesting that untethering, while necessary, is not sufficient to drive dimerization. Ligand-binding experiments reveal a much greater fraction of N579Q-EGFRs in a high-affinity state compared to the fraction of WT-EGFRs in a high-affinity state. However, differences in the kinetic association and dissociation rates indicate that the high-affinity states of the WT and the N579Q receptors are distinct. EGF-stimulated phosphorylation in cells expressing N579Q-EGFRs results in notable differences in the pattern of tyrosine phosphorylated proteins compared with that obtained in cells expressing WT-EGFRs. Moreover, although WT-EGFRs confer cell survival in 32D cells in the absence of interleukin-3 and EGF, we found that receptors lacking glycosylation at N(579) do not. This is the first study of which we are aware to show that selective glycosylation of a specific N-glycosylation site can produce two functionally distinct receptors.