James V. Staros

Enhancement by N-hydroxysulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions☆

Water-soluble carbodiimides are frequently employed in coupling or conjugation reactions, e.g., to link a peptide immunogen to a carrier protein. However, their utility is limited by low coupling yields obtained under some conditions. We have found that addition of N-hydroxysulfosuccinimide to such reactions can greatly enhance the yields obtained.

Evolutionary Analysis of the ErbB Receptor and Ligand Families

Abstract. We have compared all available deduced protein sequences of the ErbB family of receptors and their ligands. Analysis of the aligned sequences of the receptors indicates that there are some differences in the receptors that are specific to invertebrates. In addition, comparison of the vertebrate ErbB receptors suggest that a gene duplication event generated two ancestral receptors, the ErbB3/ErbB4 precursor and the ErbB1/ErbB2 precursor. Subsequent gene duplications of these precursors generated the four receptors present in mammals. Analysis of the sequences for the known ligands of the ErbB receptors suggests that the vertebrate ligands segregate into the ErbB1 ligands and the ErbB3/ErbB4 ligands, paralleling the evolution of the receptors; however, it is difficult to ascertain any correlation between the invertebrate and the vertebrate ligands. Even though ErbB3 is kinase-impaired, there is significant conservation of the kinase domain within the vertebrate lineage (human, rat, and F. rubripes), suggesting some function for this domain other than kinase activity, such as mediating protein–protein interactions that are involved in receptor dimerization and/or activation of the kinase domain of the heterodimerization partner. To date, no ligand for ErbB2 has been identified, and comparison of the extracellular domains of ErbB2 reveals two regions that are not conserved across the mammalian species. These two regions of divergence align with sequences in ErbB1 that have been shown to be proximal to the amino-terminus and to the carboxyl-terminal region, respectively, of bound EGF. Further, one of these regions contains an insertion, relative to the other members of the mammalian ErbB family, which might affect the ligand binding site and provide a structural basis for this receptors apparent inability to bind ligand independently.

Aryl azide photolabels in biochemistry

Abstract When photolabels are appropriately irradiated they 3roduce highly reactive species that can bind covalently to biomolecules. Recent studies of the modes of reaction of the aryl azides, an important class of photolabel precursors, suggest new experimental approaches to their use.

Insights into the evolution of the ErbB receptor family and their ligands from sequence analysis

BackgroundIn the time since we presented the first molecular evolutionary study of the ErbB family of receptors and the EGF family of ligands, there has been a dramatic increase in genomic sequences available. We have utilized this greatly expanded data set in this study of the ErbB family of receptors and their ligands.ResultsIn our previous analysis we postulated that EGF family ligands could be characterized by the presence of a splice site in the coding region between the fourth and fifth cysteines of the EGF module and the placement of that module near the transmembrane domain. The recent identification of several new ligands for the ErbB receptors supports this characterization of an ErbB ligand; further, applying this characterization to available sequences suggests additional potential ligands for these receptors, the EGF modules from previously identified proteins: interphotoreceptor matrix proteoglycan-2, the alpha and beta subunit of meprin A, and mucins 3, 4, 12, and 17. The newly available sequences have caused some reorganizations of relationships among the ErbB ligand family, but they add support to the previous conclusion that three gene duplication events gave rise to the present family of four ErbB receptors among the tetrapods.ConclusionThis study provides strong support for the hypothesis that the presence of an easily identifiable sequence motif can distinguish EGF family ligands from other EGF-like modules and reveals several potential new EGF family ligands. It also raises interesting questions about the evolution of ErbB2 and ErbB3: Does ErbB2 in teleosts function differently from ErbB2 in tetrapods in terms of ligand binding and intramolecular tethering? When did ErbB3 lose kinase activity, and what is the functional significance of the divergence of its kinase domain among teleosts?

Cross-linking and chymotryptic digestion of the extractoplasmic domain of the anion exchange channel in intact human erythrocytes

SummaryWe have applied our new high yield, membraneimpermeant, protein cross-linking reagents (J.V. Staros, 1982.Biochemistry21:3950–3955) together with chymotryptic digestion of the surface of intact erythrocytes (T.L. Steck, B. Ramos, and E. Strapazon, 1976.Biochemistry15:1154–1161) in an investigation of the topology of the extracytoplasmic domain of the anion exchange channel of intact human erythrocytes. In intact erythrocytes, these cross-linking reagents have been shown to cross-link subunits of the anion exchange channel to dimers in the extracytoplasmic domain of the protein. Chymotryptic treatment of intact erythrocytes has been shown to cleave subunits of the anion exchange channel into two fragments of distinctMr. Sequential treatment of intact erythrocytes with either of two membrane-impermeant cross-linkers, followed by digestion with chymotrypsin, yields chymotryptic fragments of the anion exchange channel cross-linked to one another. The cross-linked products observed appear to arise by cross-linking of unlike chymotryptic fragments, whether the cross-links are intersubunit or intrasubunit. These results are consistent with a model of the anion exchange channel in which the subunits form a head-to-head dimer with a twofold center of symmetry perpendicular to the plane of the membrane.

Identification of Residues of the Epidermal Growth Factor Receptor Proximal to Residue 45 of Bound Epidermal Growth Factor

A triple mutant of murine epidermal growth factor (mEGF), N1Q/H22Y/R45K-mEGF, was constructed by site-directed mutagenesis, expressed, purified, and characterized for use in an affinity cross-linking study to identify aminoacyl residues of the EGF receptor adjacent to a residue in the carboxyl-terminal domain of bound EGF thought to be important in distinguishing between EGF and transforming growth factor-α in their recognition by the receptor. Cyclization of Gln1 to form pyroglutamate (pE) limited the site of cross-linking in the mutant to Lys45, permitting identification of receptor residues that are proximal to this residue of bound EGF. The resulting N1pE/H22Y/R45K-mEGF was shown to be comparable to wild-type mEGF in receptor binding and stimulation of receptor autophosphorylation. 125I-Labeled N1pE/H22Y/R45K-mEGF was reacted with the heterobifunctional cross-linking reagent sulfo-N-succinimidyl-4-(fluorosulfonyl)benzoate, and the resulting modified EGF was incubated with A431 membrane vesicles bearing EGF receptors. Incubation resulted in specific cross-linking of the labeled N1pE/H22Y/R45K-mEGF to EGF receptors. The resulting cross-linked complex was then partially purified, denatured, reduced, and carboxyamidomethylated. Digestion with endoprotease LysC resulted in a unique radiolabeled peptide that could be immunoprecipitated using antibodies to mEGF. This immunoprecipitated fragment was purified by gel electrophoresis and subjected to microsequencing. The resulting sequence was matched to that of a LysC fragment of the receptor, which begins with Thr464 and is near the interface of receptor subdomains III and IV. Loss of signal at cycle 2 suggests that the point of attachment of cross-linked N1pE/H22Y/R45K is Lys465 of the receptor.

High-yield trapping of EGF-induced receptor dimers by chemical cross-linking.

The binding of epidermal growth factor (EGF) to its plasma membrane receptor results in the stimulation of a tyrosyl residue‐specific protein kinase, which has been shown to be part of the receptor. The mechanism by which EGF binding gives rise to the stimulation of kinase activity is not understood in detail; however, a number of recent studies have implicated receptor dimerization or oligomerization in this process. We prepared Triton X‐100 extracts of A431 cells in which the concentration of EGF receptors was on the order of 10−7 M. When samples of the extracts were incubated with or without EGF and then treated with the high‐yield cross‐linking reagent bis(sulfosuccinimidyl)suberate (BS3), covalent receptor dimers could be detected in high yield in samples that had been treated with both EGF and BS3, whereas only monomeric receptor was detected in untreated samples or in samples that had been treated with either EGF or BS3. The yield of receptor dimers trapped by cross‐linking correlated with the stimulation of autophosphorylation by EGF and with the concentration of EGF present. EGF‐induced receptor dimers were also efficiently cross‐linked in highly purified receptor preparations, suggesting that EGF‐induced dimerization is a process intrinsic to the receptor, requiring no additional accessory proteins.— Fanger, B. O.; Stephens, J. E.; Staros, J. V. High‐yield trapping of EGF‐induced receptor dimers by chemical cross‐linking. FASEB J. 3: 71‐75; 1989.

Ligand- and kinase activity-independent cell survival mediated by the epidermal growth factor receptor expressed in 32D cells

To investigate the intrinsic activities of the epidermal growth factor receptor and the role of its kinase domain in these functions within a cellular environment lacking endogenous ErbB protein expression, wild-type EGF receptor (WT-EGFR) and two kinase-impaired mutants, D813A and K721R, were expressed in 32D murine hematopoietic cells, a line which is normally dependent on interleukin 3 (IL3) for growth and survival. Addition of EGF in the absence of IL3 stimulates receptor autophosphorylation and, in the presence of serum, mitosis in cells expressing WT-EGFR, but not in cells expressing D813A or K721R. Unexpectedly, cells expressing WT-EGFR or K721R exhibited IL3-independent survival in the presence of fetal bovine serum; parental 32D cells and cells expressing D813A did not survive, apparently undergoing apoptosis in the absence of IL3, whether or not serum was present. Addition of EGF did not prevent the apoptosis of WT-EGFR or K721R cells in serum-free medium. Activation of Akt was not necessary to mediate the prosurvival activity of EGF receptor expression. These results suggest that the EGF receptor can mediate the prevention of apoptosis independently of both receptor-ligand binding and receptor kinase activity, and this activity is disrupted by the D813A mutation.

[35] Electrophoretic transfer of high-molecular-weight proteins for immunostaining

Publisher Summary The technique of electrophoretic transfer of proteins from SDS-polyacrylamide gels to solid supports for the identification of specific proteins by immunological (or other) methods has been widely applied to many biochemical problems. A number of modifications in the original technique have been introduced that facilitate the transfer of high-molecular-weight proteins. This chapter reviews a set of methods that has been applied successfully to the efficient electrophoretic transfer of high-molecular-weight (M r to ∼500,000) or very-high-molecular-weight (M r to ∼2,000,000) membrane and cytoskeletal proteins. Electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels to nitrocellulose or other matrices is being applied to a growing catalog of proteins. This has opened this technique to many proteins of the membrane, cytoskeleton, and contractile apparatus.

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.