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Abstract Background Over the last decade, kinases have emerged as attractive therapeutic targets for a number of different diseases, and numerous high throughput screening efforts in the pharmaceutical community are directed towards discovery of compounds that regulate kinase function. The emerging utility of systems biology approaches has necessitated the development of multiplex tools suitable for proteomic-scale experiments to replace lower throughput technologies such as mass spectroscopy for the study of protein phosphorylation. Recently, a new approach for identifying substrates of protein kinases has applied the miniaturized format of functional protein arrays to characterize phosphorylation for thousands of candidate protein substrates in a single experiment. This method involves the addition of protein kinases in solution to arrays of immobilized proteins to identify substrates using highly sensitive radioactive detection and hit identification algorithms. Results To date, the factors required for optimal performance of protein array-based kinase substrate identification have not been described. In the current study, we have carried out a detailed characterization of the protein array-based method for kinase substrate identification, including an examination of the effects of time, buffer compositions, and protein concentration on the results. The protein array approach was compared to standard solution-based assays for assessing substrate phosphorylation, and a correlation of greater than 80% was observed. The results presented here demonstrate how novel substrates for protein kinases can be quickly identified from arrays containing thousands of human proteins to provide new clues to protein kinase function. In addition, a pooling-deconvolution strategy was developed and applied that enhances characterization of specific kinase-substrate relationships and decreases reagent consumption. Conclusion Functional protein microarrays are an important new tool that enables multiplex analysis of protein phosphorylation, and thus can be utilized to identify novel kinase substrates. Integrating this technology with a systems biology approach to cell signalling will help uncover new layers in our understanding of this essential class of enzymes.

Abstract Background Gab1 is a docking protein that recruits phosphatidylinositol-3 kinase (PI-3 kinase) and other effector proteins in response to the activation of many receptor tyrosine kinases (RTKs). As the autophosphorylation sites on EGF-receptor (EGFR) do not include canonical PI-3 kinase binding sites, it is thought that EGF stimulation of PI-3 kinase and its downstream effector Akt is mediated by an indirect mechanism. Results We used fibroblasts isolated from Gab1-/- mouse embryos to explore the mechanism of EGF stimulation of the PI-3 kinase/Akt anti-apoptotic cell signaling pathway. We demonstrate that Gab1 is essential for EGF stimulation of PI-3 kinase and Akt in these cells and that these responses are mediated by complex formation between p85, the regulatory subunit of PI-3 kinase, and three canonical tyrosine phosphorylation sites on Gab1. Furthermore, complex formation between Gab1 and the protein tyrosine phosphatase Shp2 negatively regulates Gab1 mediated PI-3 kinase and Akt activation following EGF-receptor stimulation. We also demonstrate that tyrosine phosphorylation of ErbB3 may lead to recruitment and activation of PI-3 kinase and Akt in Gab1-/- MEFs. Conclusions The primary mechanism of EGF-induced stimulation of the PI-3 kinase/Akt anti-apoptotic pathway occurs via the docking protein Gab1. However, in cells expressing ErbB3, EGF and neuroregulin can stimulate PI-3 kinase and Akt activation in a Gab1-dependent or Gab1-independent manner.

The low incidence of ovarian cancer (OC) dictates that any screening strategy needs to be both highly sensitive and highly specific. This study explored the utility of detecting multiple colocalized proteins or glycosylation epitopes on single tumor-associated extracellular vesicles from blood. The novel Mercy Halo Ovarian Cancer Test (OC Test) uses immunoaffinity capture of tumor-associated extracellular vesicles, followed by proximity-ligation real-time quantitative PCR to detect combinations of up to three biomarkers to maximize specificity, and measures multiple combinations to maximize sensitivity. A high-grade serous carcinoma (HGSC) case-control training set of EDTA plasma samples from 397 women was used to lock down the test design, the data interpretation algorithm, and the cutoff between cancer and noncancer. Performance was verified and compared with cancer antigen 125 in an independent blinded case-control set of serum samples from 390 women (132 controls, 66 HGSC, 83 non-HGSC OC, and 109 benign). In the verification study, the OC Test showed a specificity of 97.0% (128/132; 95% CI, 92.4%-99.6%), a HGSC sensitivity of 97.0% (64/66; 95% CI, 87.8%-99.2%), and an area under the curve of 0.97 (95% CI, 0.93-0.99) and detected 73.5% (61/83; 95% CI, 62.7%-82.6%) of the non-HGSC OC cases. This test exhibited fewer false positives in subjects with benign ovarian tumors, nonovarian cancers, and inflammatory conditions when compared with cancer antigen 125. The combined sensitivity and specificity of this new test suggests that it may have potential in OC screening., Competing Interests: Disclosure Statement L.T.B., A.D.C., D.P.S., S.B., I.O.Z., D.M.B., M.S.K., L.T.C., B.J.M., T.B.H., T.G., and D.M. are current employees of Mercy BioAnalytics Inc. E.S.W.-D. is a retired Mercy BioAnalytics employee and is currently a paid consultant of Mercy BioAnalytics Inc. She was a full-time employee when this work was performed. K.M.B., P.A.D., J.G., D.G., B.F.H., and C.R.S. are former employees of Mercy BioAnalytics Inc., who were active employees at the time this work was performed. S.J.S. and K.C. are paid consultants for Mercy BioAnalytics Inc. A.J., J.N.M., and D.H. are employees of University of British Columbia and provided the patient samples used for the training study. A.G.-M., S.A., and U.M. are employees of University College London and provided the patient samples used for the verification study. They also report research collaborations with Cambridge University, QIMR Berghofer Medical Research Institute, Intelligent Lab on Fiber, RNA Guardian, Micronoma, Imperial College London, University of Innsbruck, and Dana Farber USA in the area of early detection of cancer. U.M. had stock ownership (2011 to 2021) awarded by University College London in Abcodia, which held the license for the Risk of Ovarian Cancer Algorithm. She has received grant funding from the Medical Research Council, Cancer Research UK, the National Institute for Health Research UK, the Eve Appeal, and the Australian National Health and Medical Research Council. She is also a member of Tina's Wish Scientific Advisory Board (United States) and the Research Advisory Panel, Yorkshire Cancer Research (United Kingdom). L.T.B. and D.P.S. are inventors on US patent number 11,085,089 B2, Systems, Compositions and Methods for Target Entity Detection (issued August 10, 2021). L.T.B., D.P.S., E.S.W.-D., D.G., K.M.B., and A.D.C. are inventors on US patent application 63/417309, Composition and Methods for Detection of Ovarian Cancer (filed October 18, 2022). U.M. holds patent number EP10178345.4 for Breast Cancer Diagnostics., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. All rights reserved.)

Traditional cellular and live-virus methods for detection of SARS-CoV-2 neutralizing antibodies (nAbs) are labor- and time-intensive, and thus not suited for routine use in the clinical lab to predict vaccine efficacy and natural immune protection. Here, we report the development and validation of a rapid, high throughput method for measuring SARS-CoV-2 nAbs against native-like trimeric spike proteins. This assay uses a blockade of human angiotensin converting enzyme 2 (hACE-2) binding (BoAb) approach in an automated digital immunoassay on the Quanterix HD-X platform. BoAb assays using Wuhan-WT (vaccine strain), delta (B.1.167.2), omicron BA1 and BA2 variant viral strains showed strong correlation with cell-based pseudovirus neutralization activity (PNA) and live-virus neutralization activity. Importantly, we were able to detect similar patterns of delta and omicron variant resistance to neutralization in samples with paired vaccine strain and delta variant BoAb measurements. Finally, we screened clinical samples from patients with or without evidence of SARS-CoV-2 exposure by a single-dilution screening version of our assays, finding significant nAb activity only in exposed individuals. Importantly, this completely automated assay can be performed in 4 h to measure neutralizing antibody titers for 16 samples over 8 serial dilutions or, 128 samples at a single dilution with replicates. In principle, these assays offer a rapid, robust, and scalable alternative to time-, skill-, and cost-intensive standard methods for measuring SARS-CoV-2 nAb levels., Competing Interests: N.C., H.V., S.P., K.B.M., W.H.Os., H.W., A.S.N., and J.D.R. are co-inventors of BoAb assay technology. Emory University filed a patent on this technology., (© 2023 The Authors.)

The Seraph ® 100 Microbind ® Affinity Blood Filter (Seraph ® 100) is a hemoperfusion device designed to adsorb bacteria, viruses, and toxins when added to extracorporeal circuits. The FDA granted emergency use authorization in adults, but this device had never been utilized in children. A 17-year-old patient with asthma presented with respiratory distress due to COVID-19. His course was complicated by respiratory failure, rhabdomyolysis, and stage 3 AKI requiring initiation of continuous kidney replacement therapy (CKRT) on ICU day 3. The Seraph ® 100 filter was added on ICU day 4. He was treated with 3 filters from ICU day 4 to 8. On ICU day 8, he was extubated and CKRT discontinued. He required no further kidney replacement therapy but did not have laboratory work post-discharge. In conclusion, this adolescent patient with COVID-19 and AKI requiring CKRT tolerated treatment with the Seraph ® 100 Microbind ® Affinity Blood Filter without significant adverse events., Competing Interests: Stuart Goldstein serves on the Scientific Advisory Board for ExThera Medical Corporation who developed the Seraph® 100 Microbind® Affinity Blood Filter. Dawn Mattoon and Dandan Shan work for Quanterix Corporation who provided laboratory testing for COVID-19 N-protein and anti-spike IgG levels for authorship in this case report. Quanterix provided testing of patient serum for COVID 19 N-protein levels and COVID anti-spike IgG levels., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

The presence of neutralizing antibodies against SARS-CoV-2 correlates with protection against infection and severe COVID-19 disease courses. Understanding the dynamics of antibody development against the SARS-CoV-2 virus is important for recommendations on vaccination strategies and on control of the COVID-19 pandemic. This study investigates the dynamics and extent of α-Spike-Ab development by different vaccines manufactured by Johnson & Johnson, AstraZeneca, Pfizer-BioNTech and Moderna. On day 1 after vaccination, we observed a temporal low-grade inflammatory response. α-Spike-Ab titers were reduced after six months of vaccination with mRNA vaccines and increased 14 days after booster vaccinations to a maximum that exceeded titers from mild and critical COVID-19 and Long-COVID patients. Within the group of critical COVID-19 patients, we observed a trend for lower α-Spike-Ab titers in the group of patients who survived COVID-19. This trend accompanied higher numbers of pro-B cells, fewer mature B cells and a higher frequency of T follicular helper cells. Finally, we present data demonstrating that past infection with mild COVID-19 does not lead to long-term increased Ab titers and that even the group of previously infected SARS-CoV-2 patients benefit from a vaccination six months after the infection.

Background and Objectives: Although patients hospitalized with COVID-19 frequently present with encephalopathy, those with mild initial COVID-19 disease who never required hospitalization also often develop neurologic symptoms as part of postacute sequelae of severe acute respiratory coronavirus type 2 (SARS-CoV-2) infection (neuro-PASC). The pathogenic mechanisms of COVID-19 encephalopathy and neuro-PASC are unknown. We sought to establish biochemical evidence of CNS injury in those patients and their association with neuropsychiatric manifestations and SARS-CoV-2 antigenemia., Methods: We recruited hospitalized, posthospitalized, and nonhospitalized patients with confirmed diagnosis of COVID-19 with neurologic symptoms in addition to healthy control (HC) subjects. Plasma neurofilament light chain (pNfL), plasma glial fibrillary acidic protein (pGFAP), and plasma SARS-CoV-2 Nucleocapsid antigen (pN Ag) were measured by HD-X Simoa analyzer (Quanterix) and compared with neuropsychiatric symptoms, patient-reported quality-of-life measures, and standardized cognitive assessments. Neuroglial scores (pGFAP/pNfL) were calculated to estimate the relative contribution of astroglial and neuronal involvement., Results: We enrolled a total of 64 study participants, including 9 hospitalized patients with COVID-19 encephalopathy (CE), 9 posthospitalization neuro-PASC (PNP) patients, 38 nonhospitalized neuro-PASC (NNP) patients, and 8 HC subjects. Patients with CE were older, had higher pNfL and pGFAP concentrations, and more frequent pN Ag detection than all neuro-PASC groups. PNP and NNP patients exhibited similar PASC symptoms, decreased quality-of-life measures, and cognitive dysfunction, and 1 of the 38 (2.6%) NNP patients had pN Ag detectable 3 weeks postsymptoms onset. Patients with neuro-PASC presenting with anxiety/depression had higher neuroglial scores, which were correlated with increased anxiety on quality-of-life measures., Discussion: pNfL, pGFAP, and pN Ag measurements indicate neuronal dysfunction and systemic involvement in hospitalized COVID-19 patients with encephalopathy. Detection of SARS-CoV-2 N Ag in blood 3 weeks after symptoms onset in a nonhospitalized patient suggests that prolonged antigenic stimulation, or possibly latent infection, may occur. Anxiety was associated with evidence of astroglial activation in patients with neuro-PASC. These data shed new light on SARS-Cov-2 neuropathogenesis and demonstrate the value of plasma biomarkers across the COVID-19 disease spectrum., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Traditional cellular and live-virus methods for detection of SARS-CoV-2 neutralizing antibodies (nAbs) are labor- and time-intensive, and thus not suited for routine use in the clinical lab to predict vaccine efficacy and natural immune protection. Here, we report the development and validation of a rapid, high throughput method for measuring SARS-CoV-2 nAbs against native-like trimeric spike proteins. This assay uses a blockade of hACE-2 binding (BoAb) approach in an automated digital immunoassay on the Quanterix HD-X platform. BoAb assays using vaccine and delta variant viral strains showed strong correlation with cell-based pseudovirus and live-virus neutralization activity. Importantly, we were able to detect similar patterns of delta variant resistance to neutralization in samples with paired vaccine and delta variant BoAb measurements. Finally, we screened clinical samples from patients with or without evidence of SARS-CoV-2 exposure by a single-dilution screening version of our assays, finding significant nAb activity only in exposed individuals. In principle, these assays offer a rapid, robust, and scalable alternative to time-, skill-, and cost-intensive standard methods for measuring SARS-CoV-2 nAb levels., Competing Interests: Declaration of Interests N.C., H.V., S.P., K.B.M., W.H.Os., H.W., A.S.N., and J.D.R. are co-inventors of BoAb assay technology. Emory University filed a patent on this technology.

The COVID-19 pandemic continues to have an unprecedented impact on societies and economies worldwide. There remains an ongoing need for high-performance SARS-CoV-2 tests which may be broadly deployed for infection monitoring. Here we report a highly sensitive single molecule array (Simoa) immunoassay in development for detection of SARS-CoV-2 nucleocapsid protein (N-protein) in venous and capillary blood and saliva. In all matrices in the studies conducted to date we observe >98% negative percent agreement and >90% positive percent agreement with molecular testing for days 1-7 in symptomatic, asymptomatic, and pre-symptomatic PCR+ individuals. N-protein load decreases as anti-SARS-CoV-2 spike-IgG increases, and N-protein levels correlate with RT-PCR Ct-values in saliva, and between matched saliva and capillary blood samples. This Simoa SARS-CoV-2 N-protein assay effectively detects SARS-CoV-2 infection via measurement of antigen levels in blood or saliva, using non-invasive, swab-independent collection methods, offering potential for at home and point of care sample collection.

Objective: The purpose of this project was to use an in vivo method to discover riboswitches that are activated by new ligands. We employed phage-assisted continuous evolution (PACE) to evolve new riboswitches in vivo. We started with one translational riboswitch and one transcriptional riboswitch, both of which were activated by theophylline. We used xanthine as the new target ligand during positive selection followed by negative selection using theophylline. The goal was to generate very large M13 phage populations that contained unknown mutations, some of which would result in new aptamer specificity. We discovered side products of three new theophylline translational riboswitches with different levels of protein production., Results: We used next generation sequencing to identify M13 phage that carried riboswitch mutations. We cloned and characterized the most abundant riboswitch mutants and discovered three variants that produce different levels of translational output while retaining their theophylline specificity. Although we were unable to demonstrate evolution of new riboswitch ligand specificity using PACE, we recommend careful design of recombinant M13 phage to avoid evolution of "cheaters" that short circuit the intended selection pressure.

The development of autoantibodies is observed in autoimmune disorders and numerous cancers. Consequently, autoantibodies form the basis of potential diagnostic and prognostic assays, as well as approaches for monitoring disease progression and treatment response. The effective use of autoantigen biomarkers for these applications, however, is contingent upon the identification of not one but multiple biomarkers. This is a consequence of the observation that the development of autoantibodies to any given protein is typically seen only in a fraction of patients. We have previously demonstrated the utility of functional protein microarrays containing thousands of different human proteins (ProtoArrays) for discovering novel autoimmune biomarkers in serum and plasma. Here, we describe a protocol for detecting autoantibodies in urine.

Protein microarrays are similar to DNA microarrays; both enabling the parallel interrogation of thousands of probes immobilized on a surface. Consequently, they have benefited from technologies previously developed for DNA microarrays. However, assumptions for the analysis of DNA microarrays do not always translate to protein arrays, especially in the case of normalization. Hence, we have developed an experimental and computational framework to assess normalization procedures for protein microarrays. Specifically, we profiled two sera with markedly different autoantibody compositions. To analyze intra- and interarray variability, we compared a set of control proteins across subarrays and the corresponding spots across multiple arrays, respectively. To estimate the degree to which the normalization could help reveal true biological separability, we tested the difference in the signal between the sera relative to the variability within replicates. Next, by mixing the sera in different proportions (titrations), we correlated the reactivity of proteins with serum concentration. Finally, we analyzed the effect of normalization procedures on the list of reactive proteins. We compared global and quantile normalization, techniques that have traditionally been employed for DNA microarrays, with a novel normalization approach based on a robust linear model (RLM) making explicit use of control proteins. We show that RLM normalization is able to reduce both intra- and interarray technical variability while maintaining biological differences. Moreover, in titration experiments, RLM normalization enhances the correlation of protein signals with serum concentration. Conversely, while quantile and global normalization can reduce interarray technical variability, neither is as effective as RLM normalization in maintaining biological differences. Most importantly, both introduce artifacts that distort the signals and affect the correct identification of reactive proteins, impairing their use for biomarker discovery. Hence, we show RLM normalization is better suited to protein arrays than approaches used for DNA microarrays.

Small molecules possess the ability to interact with proteins and perturb their specific functions, a property that has been exploited for numerous research applications and to produce therapeutic agents in disease treatment. However, commonly utilized mass spectrometry-based approaches for identifying the target proteins for a small molecule have a number of limitations, particularly in terms of throughput and time and resource consumption. In addition, current technologies lack a mechanism to broadly assess the selectivity profile of the small molecule, which may be important for understanding off-target effects of the compound. Protein microarray technology has emerged as a powerful tool in the systems biology arsenal. Here, we describe how protein microarray technology can be applied to the study of small molecule protein interactions, with sensitivity sufficient to detect interactions with low muM affinity. These assays are highly reproducible, sensitive, and scalable, and provide an enabling technology for small molecule selectivity profiling in the context of drug development.

Protein microarrays represent an important new tool in proteomic systems biology. This review focuses on the contributions of protein microarrays to the discovery of novel disease biomarkers through antibody-based assays. Of particular interest is the use of protein microarrays for immune response profiling, through which a disease-specific antibody repertoire may be defined. The antigens and antibodies revealed by these studies are useful for clinical assay development, with enormous potential to aid in diagnosis, prognosis, disease staging and treatment selection. The discovery and characterization of novel biomarkers specifically tailored to disease type and stage are expected to enable personalized medicine by facilitating preventative medicine, predictive diagnostics and individualized curative therapies.

Antibody cross-reactivity can compromise interpretation of experiments and derail therapeutic antibody development. Standard techniques such as immunohistochemistry or Western analysis provide important but often inadequate approaches to assess antibody specificity. Protein microarrays are providing a new approach to rapidly characterize antibody cross-reactivity against 1,000s of proteins simultaneously. This review will focus on reported examples of antibody cross-reactivity, methods used to characterize them, and the recent development and use of protein microarrays for assessing antibody specificity.

Quantitative epidermal growth factor (EGF)-binding experiments have shown that the EGF-receptor (EGFR) is displayed on the surface of intact cells in two forms, a minority of high-affinity and a majority of low-affinity EGFRs. On the basis of the three-dimensional structure of the extracellular ligand binding domain of the EGFR, it was proposed that the intramolecularly tethered and autoinhibited configuration corresponds to the low-affinity receptor, whereas the extended configuration accounts for the high-affinity EGFRs on intact cells. Here we test this model by analyzing the properties of EGFRs mutated in the specific regions responsible for receptor autoinhibition and dimerization, respectively. Our results show that mutagenic disruption of the autoinhibitory tether in EGFR results in a decrease in the dissociation rate of EGF without a detectable change in EGFR activation and signaling through EGFR even in response to stimulation with low concentrations of EGF. Mutagenic disruption of the dimerization arm, on the other hand, increased the rate of EGF dissociation and impaired EGFR activation and signaling via the EGFR. This study demonstrates that the extended configuration of EGFR does not account for the apparent high-affinity EGF-binding to EGFR on intact cells. Furthermore, the autoinhibition conferred by the tethered configuration of the extracellular ligand-binding domain provides only a limited control of EGFR function.

On the basis of the 3D structures of the extracellular ligand-binding domains of the epidermal growth factor (EGF) receptor (EGFR) and ErbB3, a mechanism has been proposed for how the extracellular region of the EGFR is maintained in an autoinhibited configuration and for how EGF binding induces EGFR dimerization and activation. We have attempted to derive a mathematical model for EGF binding to the EGFR and for ligand-induced receptor dimerization and activation that uses this structural information and can explain the characteristic concave-up curvilinear Scatchard plots seen when EGF binding to intact EGFR is studied in living cells. We show that these curvilinear plots cannot be accounted for by simply ascribing different affinities to the autoinhibited and extended (dimeric) configurations of the receptor seen in structural studies. Concave-up plots can only be obtained by including in the mathematical model an additional binding event in which occupied EGFR dimers bind to an "external site." The external site may represent receptor interactions with coated-pit regions in the cell membrane or with other cellular components involved in receptor endocytosis and turnover. We conclude in this study and in the accompanying article that the active extended EGFR configuration binds EGF 5- to 20-fold more strongly than the autoinhibited monomeric receptor configuration. However, these extended receptors do not correspond directly with the "high-affinity" EGF-binding sites seen in EGF-binding studies on intact cells.

The papillomavirus E5 proteins are short, hydrophobic transforming proteins. The transmembrane E5 protein encoded by bovine papillomavirus transforms cells by activating the platelet-derived growth factor beta receptor tyrosine kinase in a ligand-independent fashion. The bovine papillomavirus E5 protein forms a stable complex with the receptor, thereby inducing receptor dimerization and activation, trans-phosphorylation, and recruitment of cellular signaling proteins to the receptor. The E5 proteins of the human papillomaviruses also appear to affect the activity of growth factor receptors and their signaling pathways. The interaction of papillomavirus E5 proteins with a subunit of the vacuolar ATPase may also contribute to transformation. Further analysis of these unique mechanisms of viral transformation will yield new insight into the regulation of growth factor receptor activity and cellular signal transduction pathways.

We have developed a genetic method to determine the active orientation of dimeric transmembrane protein helices. The bovine papillomavirus E5 protein, a 44-amino acid homodimeric protein that appears to traverse membranes as a left-handed coiled-coil, transforms fibroblasts by binding and activating the platelet-derived growth factor (PDGF) beta receptor. A heterologous dimerization domain was used to force E5 monomers to adopt all seven possible symmetric coiled-coil registries relative to one another within the dimer. Focus formation assays demonstrated that dimerization of the E5 protein is required for transformation and identified a single preferred orientation of the monomers. The essential glutamine residue at position 17 resided in the dimer interface in this active orientation. The active chimera formed complexes with the PDGF beta receptor and induced receptor tyrosine phosphorylation. We also identified E5-like structures that underwent non-productive interactions with the receptor.

The 44-amino acid E5 protein of bovine papillomavirus is a homo-dimeric, transmembrane protein that transforms cells by activating the platelet-derived growth factor ss receptor in a ligand-independent fashion. The E5 protein induces receptor activation by forming a stable complex with the receptor, thereby inducing receptor dimerization, trans-phosphorylation of tyrosine residues in the cytoplasmic domain of the receptor, and recruitment of cellular SH2 domain-containing proteins into a signal transduction complex. Direct interactions between specific transmembrane and juxtamembrane amino acids in the E5 protein and the PDGF ss receptor appear to drive complex formation and dimerization of the receptor. Further analysis of this unique mechanism of viral transformation promises to yield new insight into the regulation of growth factor receptor activity and cellular signal transduction pathways.