Decoding Complexity in Synthetic Oligonucleotides: Unraveling Coeluting Isobaric Impurity Ions by High Resolution Mass Spectrometry.

Analyzing coeluting impurities with similar masses in synthetic oligonucleotides by liquid chromatography-mass spectrometry (LC-MS) poses challenges due to inadequate separation in either dimension. Herein, we present a direct method employing fully resolved isotopic envelopes, enabled by high resolution mass spectrometry (HRMS), to identify and quantify isobaric impurity ions resulting from the deletion or addition of a uracil (U) or cytosine (C) nucleotide from or to the full-length sequence. These impurities may each encompass multiple sequence variants arising from various deletion or addition sites. The method utilizes a full or targeted MS analysis to measure accurate isotopic distributions that are chemical formula dependent but nucleotide sequence independent. This characteristic enables the quantification of isobaric impurity ions involving sequence variants, a capability typically unavailable in sequence-dependent MS/MS methods. Notably, this approach does not rely on standard curves to determine isobaric impurity compositions in test samples; instead, it utilizes the individual isotopic distributions measured for each impurity standard. Moreover, in cases where specific impurity standards are unavailable, the measured isotopic distributions can be adequately replaced with the theoretical distributions (calculated based on chemical formulas of standards) adjusted using experiment-specific correction factors. In summary, this streamlined approach overcomes the limitations of LC-MS analysis for coeluting isobaric impurity ions, offering a promising solution for the in-depth profiling of complex impurity mixtures in synthetic oligonucleotide therapeutics.

US FDA public meeting: identification of concepts and terminology for multicomponent biomarkers.

The US FDA convened a virtual public workshop with the goals of obtaining feedback on the terminology needed for effective communication of multicomponent biomarkers and discussing the diverse use of biomarkers observed across the FDA and identifying common issues. The workshop included keynote and background presentations addressing the stated goals, followed by a series of case studies highlighting FDA-wide and external experience regarding the use of multicomponent biomarkers, which provided context for panel discussions focused on common themes, challenges and preferred terminology. The final panel discussion integrated the main concepts from the keynote, background presentations and case studies, laying a preliminary foundation to build consensus around the use and terminology of multicomponent biomarkers.

Co-Culture of Human Primary Hepatocytes and Nonparenchymal Liver Cells in the Emulate® Liver-Chip for the Study of Drug-Induced Liver Injury.

Drug-induced liver injury (DILI) is a significant public health issue, but standard animal tests and clinical trials sometimes fail to predict DILI due to species differences and the relatively low number of human subjects involved in preapproval studies of a new drug, respectively. In vitro models have long been used to aid DILI prediction, with primary human hepatocytes (PHHs) being generally considered the gold standard. However, despite many efforts and decades of work, traditional culture methods have been unsuccessful in either fully preserving essential liver functions after isolation of PHHs or in emulating interactions between PHHs and hepatic nonparenchymal cells (NPCs), both of which are essential for the development of DILI under in vivo conditions. Recently, various liver-on-a-chip (Liver-Chip) systems have been developed to co-culture hepatocytes and NPCs in a three-dimensional environment on microfluidic channels, enabling better maintenance of primary liver cells and thus improved DILI prediction. The Emulate® Liver-Chip is a commercially available system that can recapitulate some in vivo DILI responses associated with certain compounds whose liver safety profile cannot be accurately evaluated using conventional approaches involving PHHs or animal models due to a lack of innate immune responses or species-dependent toxicity, respectively. Here, we describe detailed procedures for the use of Emulate® Liver-Chips for co-culturing PHHs and NPCs for the purpose of DILI evaluation. First, we describe the procedures for preparing the Liver-Chip. We then outline the steps needed for sequential seeding of PHHs and NPCs in the prepared Liver-Chips. Lastly, we provide a protocol for utilizing cells maintained in perfusion culture in the Liver-Chips to evaluate DILI, using acetaminophen as an example. In all, use of this system and the procedures described here allow better preservation of the functions of human primary liver cells, resulting in an improved in vitro model for DILI assessment. © 2022 Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: Liver-Chip preparation Basic Protocol 2: Seeding primary human hepatocytes and nonparenchymal cells on Liver-Chips Basic Protocol 3: Perfusion culture for the study of acetaminophen-induced liver injury.

Tandem mass spectrometric sequence characterization of synthetic thymidine-rich oligonucleotides.

Tandem mass spectrometry (MS/MS) can provide direct and accurate sequence characterization of synthetic oligonucleotide drugs, including modified oligonucleotides. Multiple factors can affect oligonucleotide MS/MS sequencing, including the intrinsic properties of oligonucleotides (i.e., nucleotide composition and structural modifications) and instrument parameters associated with the ion activation for fragmentation. In this study, MS/MS sequencing of a thymidine (T)-rich and phosphorothioate (PS)-modified DNA oligonucleotide was investigated using two fragmentation techniques: trap-type collision-induced dissociation ("CID") and beam-type CID also termed as higher-energy collisional dissociation ("HCD"), preceded by a hydrophilic interaction liquid chromatography (HILIC) separation. A low to moderate charge state (-4), which predominated under the optimized HILIC-MS conditions, was selected as the precursor ion for MS/MS analysis. Comparison of the two distinctive ion activation mechanisms on the same precursor demonstrated that HCD was superior to CID in promoting higher sequence coverage and analytical sensitivity in sequence elucidation of T-rich DNA oligonucleotides. Specifically, HCD provided more sequence-defining fragments with higher fragment intensities than CID. Furthermore, the direct comparison between unmodified and PS-modified DNA oligonucleotides demonstrated a loss of MS/MS fragmentation efficiency by PS modification in both CID and HCD approaches, and a resultant reduction in sequence coverage. The deficiency in PS DNA sequence coverage observed with single collision energy HCD, however, was partially recovered by applying HCD with multiple collision energies. Collectively, this work demonstrated that HCD is advantageous to MS/MS sequencing of T-rich PS-modified DNA oligonucleotides.

Development and Regulatory Challenges for Peptide Therapeutics.

There has been an increased interest in and activity for the use of peptide therapeutics to treat a variety of human diseases. The number of peptide drugs entering clinical development and the market has increased significantly over the past decade despite inherent challenges of peptide therapeutic discovery, development, and patient-friendly delivery. Disparities in interpretation and application of existing regulatory guidances to innovative synthetic and conjugated peptide assets have resulted in challenges for both regulators and sponsors. The Symposium on Development and Regulatory Challenges for Peptide Therapeutics at the 40th Annual Meeting of the American College of Toxicology held in November of 2019 focused on the following specific topics: (1) peptide therapeutic progress and future directions, and approaches to discover, optimize, assess, and deliver combination peptide therapeutics for treatment of diseases; (2) toxicological considerations to advance peptide drug-device combination products for efficient development and optimal patient benefit and adherence; (3) industry and regulatory perspectives on the regulation of synthetic and conjugated peptide products, including exploration of regulatory classifications, interpretations, and application of the existing guidances International Council for Harmonisation (ICH) M3(R2) and ICH S6(R1) in determining nonclinical study recommendations; and (4) presentation of the 2016 Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee working group assessment of genotoxicity testing requirements. Perspectives were shared from industry and regulatory scientists working in the peptide therapeutics field followed by an open forum panel discussion to discuss questions drafted for the peptide therapeutics scientific community, which will be discussed in more detail.

Elucidating Interactions Between SARS-CoV-2 Trimeric Spike Protein and ACE2 Using Homology Modeling and Molecular Dynamics Simulations.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). As of October 21, 2020, more than 41.4 million confirmed cases and 1.1 million deaths have been reported. Thus, it is immensely important to develop drugs and vaccines to combat COVID-19. The spike protein present on the outer surface of the virion plays a major role in viral infection by binding to receptor proteins present on the outer membrane of host cells, triggering membrane fusion and internalization, which enables release of viral ssRNA into the host cell. Understanding the interactions between the SARS-CoV-2 trimeric spike protein and its host cell receptor protein, angiotensin converting enzyme 2 (ACE2), is important for developing drugs and vaccines to prevent and treat COVID-19. Several crystal structures of partial and mutant SARS-CoV-2 spike proteins have been reported; however, an atomistic structure of the wild-type SARS-CoV-2 trimeric spike protein complexed with ACE2 is not yet available. Therefore, in our study, homology modeling was used to build the trimeric form of the spike protein complexed with human ACE2, followed by all-atom molecular dynamics simulations to elucidate interactions at the interface between the spike protein and ACE2. Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) and in silico alanine scanning were employed to characterize the interacting residues at the interface. Twenty interacting residues in the spike protein were identified that are likely to be responsible for tightly binding to ACE2, of which five residues (Val445, Thr478, Gly485, Phe490, and Ser494) were not reported in the crystal structure of the truncated spike protein receptor binding domain (RBD) complexed with ACE2. These data indicate that the interactions between ACE2 and the tertiary structure of the full-length spike protein trimer are different from those between ACE2 and the truncated monomer of the spike protein RBD. These findings could facilitate the development of drugs and vaccines to prevent SARS-CoV-2 infection and combat COVID-19.

U.s. Food and Drug Administration approval: carfilzomib for the treatment of multiple myeloma.

The U.S. Food and Drug Administration (FDA) review leading to accelerated approval of carfilzomib is described. A single-arm trial enrolled 266 patients with multiple myeloma refractory to the most recent therapy who had received prior treatment with bortezomib and an immunomodulatory agent (IMID). Patients received carfilzomib by intravenous infusion over 2 to 10 minutes at a dose of 20 mg/m2 on days 1, 2, 8, 9, 15, and 16 of the 28 days of cycle 1, and at a dose of 27 mg/m2 on the same schedule in cycle 2 and subsequent cycles. The primary efficacy endpoint was overall response rate (ORR) as determined by an independent review committee using International Myeloma Working Group Uniform Response Criteria. The safety of carfilzomib was evaluated in 526 patients with multiple myeloma treated with various dosing regimens. The ORR was 23%. The median duration of response was 7.8 months. The most common adverse reactions associated with carfilzomib infusion were fatigue, anemia, nausea, thrombocytopenia, dyspnea, diarrhea, and fever. The most common serious adverse events were pneumonia, acute renal failure, fever, and congestive heart failure. Infusion reactions to carfilzomib could be reduced by pretreatment with dexamethasone and intravenous fluids. On July 20, 2012, the FDA granted accelerated approval of carfilzomib for the treatment of patients with multiple myeloma who have received at least two prior therapies including bortezomib and an IMID and who have shown disease progression while on therapy or within 60 days of completion of the last therapy.

Control of proliferation in astrocytoma cells by the receptor tyrosine kinase/PI3K/AKT signaling axis and the use of PI-103 and TCN as potential anti-astrocytoma therapies.

A growing body of work suggests that astrocytomas and glioblastoma multiforme will require carefully tailored, molecularly targeted therapy for successful treatment. Recent efforts to comprehensively identify mutations and gene expression changes in glioblastoma have shown that mutation of NF1 is a common alteration in human glioblastoma. We have developed and characterized a panel of 14 tumor lines from grades II through IV astrocytomas developed from our Nf1-/+;Trp53-/+cis mouse model and have used this panel to characterize signal transduction pathways and inhibitors that are candidate therapeutic targets for astrocytoma and glioblastoma. We show that these tumors express platelet-derived growth factor receptor-α, epidermal growth factor receptor, and their respective ligands to varying degrees. We find that both the MEK and PI3K signaling pathways downstream of epidermal growth factor receptor and platelet-derived growth factor receptor-α are necessary for full proliferation of astrocytoma cells; however, inhibition of the PI3K pathway is more effective than inhibition of MEK at blocking cell growth. We have examined inhibitors of the PI3K/Akt/mTOR signaling pathway and find that PI-103 and TCN show particular promise for inhibiting growth in Nf1 and Trp53 mutant astrocytoma cells.

Genetically engineered mouse models in cancer research.

Mouse models of human cancer have played a vital role in understanding tumorigenesis and answering experimental questions that other systems cannot address. Advances continue to be made that allow better understanding of the mechanisms of tumor development, and therefore the identification of better therapeutic and diagnostic strategies. We review major advances that have been made in modeling cancer in the mouse and specific areas of research that have been explored with mouse models. For example, although there are differences between mice and humans, new models are able to more accurately model sporadic human cancers by specifically controlling timing and location of mutations, even within single cells. As hypotheses are developed in human and cell culture systems, engineered mice provide the most tractable and accurate test of their validity in vivo. For example, largely through the use of these models, the microenvironment has been established to play a critical role in tumorigenesis, since tumor development and the interaction with surrounding stroma can be studied as both evolve. These mouse models have specifically fueled our understanding of cancer initiation, immune system roles, tumor angiogenesis, invasion, and metastasis, and the relevance of molecular diversity observed among human cancers. Currently, these models are being designed to facilitate in vivo imaging to track both primary and metastatic tumor development from much earlier stages than previously possible. Finally, the approaches developed in this field to achieve basic understanding are emerging as effective tools to guide much needed development of treatment strategies, diagnostic strategies, and patient stratification strategies in clinical research.

Bioluminescent approaches for measuring tumor growth in a mouse model of neurofibromatosis.

Neurofibomatosis (NF1) patients are susceptible to multiple tumors of the nervous system including neurofibromas, optic glioma, malignant peripheral nerve sheath tumors (MPNSTs), and astrocytoma. The Nf1+/-;Trp53+/- (NPcis) mouse model of NF1 spontaneously develops astrocytoma and MPNSTs that are very similar to human NF1 tumors. To use this model for testing potential therapeutics, we have developed systems that take advantage of bioluminescent reporters of tumor growth. We have generated E2F1 promoter-driving luciferase (ELUX) reporter mice to detect proliferating tumors in NPcis mice in vivo using bioluminescence. The power of this system is that it enables looking at tumor evolution and detecting spontaneous tumors at early stages of development as they evolve within their natural haploinsufficient microenvironment. This system can be used to identify tumors at different stages of tumorigenesis and to examine where spontaneous NF1 tumors initiate. The ability to rapidly screen multiple animals at a time increases the potential for use of this model in preclinical trials. This model will be valuable for the characterization of spontaneous NF1 tumors and will be important for studying the treatment and prevention of NF1 tumors in vivo.

Novel dual-reporter preclinical screen for antiastrocytoma agents identifies cytostatic and cytotoxic compounds.

Astrocytoma/glioblastoma is the most common malignant form of brain cancer and is often unresponsive to current pharmacological therapies and surgical interventions. Despite several potential therapeutic agents against astrocytoma and glioblastoma, there are currently no effective therapies for astrocytoma, creating a great need for the identification of effective antitumor agents. The authors have developed a novel dual-reporter system in Trp53/Nf1-null astrocytoma cells to simultaneously and rapidly assay cell viability and cell cycle progression as evidenced by activity of the human E2F1 promoter in vitro. The dual-reporter high-throughput assay was used to screen experimental therapeutics for activity in Trp53/Nf1-null astrocytoma. Several compounds were identified demonstrating selectivity for astrocytoma over primary astrocytes. The dual-reporter system described here may be a valuable tool for identifying potential antitumor treatments that specifically target astrocytoma.

Galanin protects against behavioral and neurochemical correlates of opiate reward.

The mechanisms underlying responses to drugs of abuse have been widely investigated; however, less is known about pathways normally protective against the development of drug reinforcement. These pathways are also important since they may regulate individual differences in vulnerability to addiction. The neuropeptide galanin and its binding sites are expressed in brain areas important for drug reward. Previous studies have shown that centrally infused galanin attenuates morphine place preference and peripheral injection of galnon, a galanin agonist, decreases opiate withdrawal signs. The current studies in galanin knockout (GKO) mice examined the hypothesis that galanin is an endogenous negative regulator of opiate reward and identified downstream signaling pathways regulated by galanin. We show that GKO mice demonstrate increased locomotor activation following morphine administration, which is inhibited by acute administration of galnon. GKO mice also show enhanced morphine place preference, supporting the idea that galanin normally antagonizes opiate reward. In addition, morphine-induced ERK1/2 phosphorylation was increased in the VTA of both wild-type and GKO mice, but only the GKO mice showed increases in ERK1/2 and CREB phosphorylation in the amygdala or nucleus accumbens. Furthermore, a single systemic injection of galnon in GKO mice was sufficient to reverse some of the biochemical changes brought about by morphine administration. These data suggest that galanin normally attenuates behavioral and neurochemical effects of opiates; thus, galanin agonists may represent a new class of therapeutic targets for opiate addiction.

Nf1 expression is dependent on strain background: implications for tumor suppressor haploinsufficiency studies.

Neurofibromatosis type 1 (NF1) is the most common cancer predisposition syndrome affecting the nervous system, with elevated risk for both astrocytoma and peripheral nerve sheath tumors. NF1 is caused by a germline mutation in the NF1 gene, with tumors showing loss of the wild type copy of NF1. In addition, NF1 heterozygosity in surrounding stroma is important for tumor formation, suggesting an additional role of haploinsufficiency for NF1. Studies in mouse models and NF1 families have implicated modifier genes unlinked to NF1 in the severity of the disease and in susceptibility to astrocytoma and peripheral nerve sheath tumors. To determine if differences in Nf1 expression may contribute to the strain-specific effects on tumor predisposition, we examined the levels of Nf1 gene expression in mouse strains with differences in tumor susceptibility using quantitative polymerase chain reaction. The data presented in this paper demonstrate that strain background has as much effect on Nf1 expression levels as mutation of one Nf1 allele, indicating that studies of haploinsufficiency must be carefully interpreted with respect to strain background. Because expression levels do not correlate entirely with the susceptibility or resistance to tumors observed in the strain, these data suggest that either variation in Nf1 levels is not responsible for the differences in astrocytoma and peripheral nerve sheath tumor susceptibility in Nf1-/+;Trp53-/+cis mice, or that certain mouse strains have evolved compensatory mechanisms for differences in Nf1 expression.

Galanin and galanin-like peptide modulate neurite outgrowth via protein kinase C-mediated activation of extracellular signal-related kinase.

The neuropeptide galanin is widely distributed in the central nervous system and plays a role in a number of processes in the adult brain. Galanin also has neurotrophic effects in the developing nervous system and after nerve injury. The current study investigated the mechanism by which galanin promotes neurite outgrowth in the neuronal cell line PC12 and in neurospheres derived from adult hippocampal progenitor cells. We demonstrated that galanin can induce extracellular signal-related kinase (ERK) phosphorylation transiently in a concentration-dependent manner in neurons. Galanin-like peptide, which is thought to signal primarily through the GalR2 receptor subtype, induced ERK phosphorylation with similar kinetics to galanin. In functional studies, the ability of galanin and galanin-like peptide to induce neurite outgrowth was dependent on activation of both protein kinase C and ERK. This study identified a novel physiological role for galanin-induced ERK phosphorylation and identified ERK and protein kinase C as important signaling components in the galanin-mediated modulation of neurite outgrowth.

Galanin attenuates cyclic AMP regulatory element-binding protein (CREB) phosphorylation induced by chronic morphine and naloxone challenge in Cath.a cells and primary striatal cultures.

Repeated morphine administration leads to molecular alterations of the neural circuitry in the locus coeruleus and nucleus accumbens. These changes include increased activity of several components of the cAMP signaling pathway that are thought to be associated with psychological and somatic signs of opiate withdrawal. The neuropeptide galanin has been shown to attenuate cAMP signaling in multiple cell types. The current study demonstrates that acute galanin treatment blocks the consequences of increased cAMP signaling following chronic opiate administration and withdrawal in Cath.a cells and primary cultures of striatal neurons as measured by phosphorylation of the transcription factor cAMP regulatory element-binding protein (CREB). In addition, galanin-mediated attenuation of CREB phosphorylation is independent of galanin-induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in Cath.a cells. These data suggest that galanin receptors may serve as an additional potential therapeutic target for the treatment of opiate withdrawal.

Galanin can attenuate opiate reinforcement and withdrawal.

Galanin and its receptors are expressed in brain areas associated with opiate reinforcement and withdrawal. An emerging body of data suggests that galanin can attenuate the neurochemical, physiological and behavioral signs of opiate reinforcement and withdrawal. Experiments in transgenic mice overexpressing galanin and knockout mice lacking the peptide support a role for endogenous galanin in modulating the actions of opiates on brain regions associated with reinforcement and withdrawal. These studies suggest that galanin receptor agonists could be useful therapeutic agents to combat opiate addiction. Further, genetic variation in the genes encoding galanin and its receptors could be associated with altered susceptibility to opiate dependence.

GalR1, but not GalR2 or GalR3, levels are regulated by galanin signaling in the locus coeruleus through a cyclic AMP-dependent mechanism.

The galanin receptors GalR1, GalR2 and GalR3 are widely expressed throughout the mouse brain and are enriched in catecholaminergic nuclei. Here, we show that GalR1 protein levels are regulated by neuronal activity and changes in cAMP levels. GalR1, but not GalR2 or GalR3, is specifically up-regulated in the LC-like Cath.a cell line in a cAMP-dependent manner. GalR1 protein and mRNA levels are also up-regulated in the LC of galanin knockout mice, whereas GalR2 and GalR3 are not. Lack of galanin-maintained cAMP tone in the galanin knockout mouse appears to result in a loss of negative feedback resulting in increased levels of CREB phosphorylation and increased GalR1 expression. These findings suggest that changes in levels of GalR1 may play an important role in modulating signaling events and neuroplasticity underlying physiological functions of the LC.

Characterization of GalR1, GalR2, and GalR3 immunoreactivity in catecholaminergic nuclei of the mouse brain.

The distribution of immunoreactivity for the three identified neuropeptide galanin receptors, GalR1, GalR2, and GalR3, was determined in areas of the mouse brain involved in drug addiction, including the ventral tegmental area (VTA), substantia nigra (SN), nucleus accumbens (NA), and locus coeruleus (LC). All three galanin receptors are found in the VTA, SN, NA, and LC; however, GalR1 protein is most highly represented in the VTA, NA, and SN, suggesting that GalR1 may play a predominant role in galanin-mediated regulation of dopamine neurotransmission. GalR1 and GalR3 protein levels are high in the LC, suggesting that these isoforms may be important for galanin-mediated regulation of noradrenergic transmission during opiate withdrawal. Although the distribution of GalR1, GalR2, and GalR3 largely recapitulates the pattern of galanin binding throughout the brain, some discrepancies exist, suggesting that another galanin receptor(s) may be present in some brain areas. Overall, GalR1, GalR2, and GalR3 are distributed widely throughout the brain, correlate with widespread galanin binding, and colocalize with tyrosine hydroxylase in catecholaminergic brain areas.

The neuropeptide galanin modulates behavioral and neurochemical signs of opiate withdrawal.

Much research has focused on pathways leading to opiate addiction. Pathways opposing addiction are more difficult to study but may be critical in developing interventions to combat drug dependence and withdrawal. Galanin decreases firing of locus coeruleus neurons, an effect hypothesized to decrease signs of opiate withdrawal. The current study addresses whether galanin affects morphine withdrawal signs by using a galanin agonist, galnon, that crosses the blood-brain barrier, and mice genetically engineered to under- or overexpress galanin peptide. Galnon significantly decreased morphine withdrawal signs in C57BL/6 mice. Further, knockout mice lacking galanin showed exacerbated morphine withdrawal signs, suggesting that endogenous galanin normally counteracts opiate withdrawal. Transgenic mice overexpressing galanin in noradrenergic neurons also showed decreased morphine withdrawal signs, suggesting a possible neuroanatomical locus for these effects of galanin. Both c-fos immunoreactivity, a marker of neuronal activity, and phosphorylation of tyrosine hydroxylase at Ser-40, a marker of cAMP levels, are decreased in the locus coeruleus by galnon treatment after morphine withdrawal, suggesting a possible molecular mechanism for the behavioral effects of galanin. These studies suggest that galanin normally acts to counteract opiate withdrawal and that small molecule galanin agonists could be effective in diminishing the physical signs of withdrawal.

The docking protein FRS2alpha controls a MAP kinase-mediated negative feedback mechanism for signaling by FGF receptors.

The docking protein FRS2alpha functions as a major mediator of signaling by FGF and NGF receptors. Here we demonstrate that, in addition to tyrosine phosphorylation, FRS2alpha is phosphorylated by MAP kinase on multiple threonine residues in response to FGF stimulation or by insulin, EGF, and PDGF, extracellular stimuli that do not induce tyrosine phosphorylation of FRS2alpha. Prevention of FRS2alpha threonine phosphorylation results in constitutive tyrosine phosphorylation of FRS2alpha in unstimulated cells and enhanced tyrosine phosphorylation of FRS2alpha, MAPK stimulation, cell migration, and proliferation in FGF-stimulated cells. Expression of an FRS2alpha mutant deficient in MAPK phosphorylation sites induces anchorage-independent cell growth and colony formation in soft agar. These experiments reveal a novel MAPK-mediated, negative feedback mechanism for control of signaling pathways that are dependent on FRS2 and a mechanism for heterologous control of signaling via FGF receptors.