James V. Staros

The Staros laboratory focused on mechanisms by which the binding of polypeptide hormones to their cell surface receptors are transduced into signals in the cell and mechanisms by which those signals are regulated.  The primary biological systems studied were the ErbB receptor family and their ligands, the archetypes of which are epidermal growth factor (EGF) and its receptor. The Staros group applied methods from protein chemistry, spectroscopy, and molecular biology in these investigations, often employing new chemical and spectroscopic reagents developed in Staros’ laboratory.

Protein chemical studies in the Staros laboratory in the early 1980’s showed that the EGF receptor and the EGF-stimulable Tyr-specific protein kinase are two functions of a single molecule, making the EGF receptor the first recognized receptor tyrosine kinase.  Using affinity labeling methods, they identified Lys721 as an important residue in the kinase active site.  Subsequently, using site-directed mutagenesis, they showed that Asp813 functions as the catalytic base of the kinase in phosphoryl transfer.  A surprising outcome of these studies was that the kinase-negative mutant receptors with Asp813 replaced with Ala or Lys 721 replaced with Arg, when expressed in cells without endogenous EGF receptors, are still capable of signaling for DNA replication, but only if ErbB2 is present. When the EGF receptor was expressed in 32D cells, a cell line that normally requires interleukin-3 (IL-3) for survival and proliferation and is devoid of endogenous ErbB receptors, EGF binding to the wild-type receptor could replace the functions of IL-3 binding to the IL-3 receptor. In the absence of EGF, the EGF receptor prevented apoptosis in these cells. Unexpectedly, the kinase-negative mutant in which Lys721 is replaced with Arg also prevented apoptosis; however, the kinase-negative mutant with Asp813 replaced with Ala did not retain this function.

A variety of spectroscopic studies were employed to investigate the dynamic interaction of EGF with its receptor and the state of the occupied EGF-receptor complex in the membrane.   For example, the Staros group employed fluorescence homo-transfer, a specialized form of fluorescence resonance energy transfer (FRET) in which the same fluorophore is used as both donor and acceptor, to show that FRET between EGF molecules bound to receptors in cells arises not from transfer within occupied receptor dimers, but between occupied receptors within higher order oligomers.  They built a specialized stopped-flow fluorimeter for investigating the kinetics of EGF-receptor binding and dissociation in living cells by fluorescence anisotropy.  They expressed the EGF receptor in 32D cells, which do not express any endogenous ErbB receptors and showed that binding and dissociation isotherms can best be fit to two classes of receptors, indicating that the two affinity states of the receptor that are commonly observed are an intrinsic property of the receptor and disproving the then current hypothesis that the two states were due to heterodimerization with other members of the ErbB family.  Studies in 32D cells expressing both the EGF receptor and ErbB2 suggested that the main effect of heterodimerization is to increase the population of high affinity receptors; however, the high affinity state of the EGF receptor in the presence of ErbB2 is different from the high affinity state in its absence.   When the EGF receptor was expressed in the absence of ErbB2, the high affinity state is defined by a fast on-rate; however, in the presence of ErbB2, the high affinity state is defined by a very slow off-rate.

Using mass spectrometry to study the glycosylation state of the receptor, they found that Asn579, one of the eleven canonical asparagine-linked glycosylation sites, is not glycosylated in a fraction of the receptors expressed in A431 cells.   This site is especially interesting because Asn579 lies in a part of the receptor that controls the transition between the inactive (tethered) state of the receptor and the active (untethered) state.   By making a site-directed mutant receptor in which Asn579 is substituted with Gln, resulting in a receptor that cannot be glycosylated at that site, they studied the properties of this subclass of receptors.   Kinetic studies showed that the Asn579→Gln mutant EGF receptor when expressed alone in 32D cells has kinetic characteristics more closely resembling those of the wild-type receptor in the presence of ErbB2 than in its absence, i.e., a higher proportion of high affinity receptors than for the wild-type receptor expressed alone, and a high affinity state that is defined by a slow off-rate rather than a fast on-rate.   These results suggest that glycosylation at Asn579 contributes to stabilizing the inactive (tethered) state of the receptor.

In an independent series of studies they employed computational methods to study the molecular evolution of the ErbB family of receptors and of the EGF family of ligands.  One end result of these studies is the prediction of previously unrecognized ligands for the ErbB family of receptors.

Buhrow, S.A., S. Cohen, and J.V. Staros (1982), "Affinity Labeling of the Protein Kinase Associated with the Epidermal Growth Factor Receptor in Membrane Vesicles from A431 Cells", J. Biol. Chem. 257, 4019-4022.

Staros, J.V. (1982), "N-Hydroxysulfosuccinimide Active Esters:  Bis(N-hydroxy-sulfosuccinimide) Esters of Two Dicarboxylic Acids Are Hydrophilic, Membrane-impermeant, Protein Cross-linkers", Biochemistry 21, 3950-3955.

Russo, M.W., T. J. Lukas, S. Cohen, and J.V. Staros (1985), "Identification of Residues in the Nucleotide Binding Site of the Epidermal Growth Factor Receptor/Kinase", J. Biol. Chem. 260, 5205-5208.

Staros, J.V. (1988), "Membrane-Impermeant Cross-Linking Reagents:  Probes of the Structure and Dynamics of Membrane Proteins", Accounts Chem. Res., 21, 435-441.

Anjaneyulu, P.S.R., A. H. Beth, B. J. Sweetman, L.A. Faulkner, and J.V. Staros (1988), "Bis(sulfo-N-succinimidyl) [15N,2H16]Doxyl-2-spiro-4'-pimelate, A Stable Isotope-Substituted, Membrane-Impermeant Bifunctional Spin Label for Studies of the Dynamics of Membrane Proteins:  Application to the Anion-Exchange Channel in Intact Human Erythrocytes", Biochemistry 27, 6844-6851.

Woltjer, R.L., L. Weclas-Henderson, I.A. Papayannopoulos, and J.V. Staros (1992), "High-Yield Covalent Attachment of Epidermal Growth Factor to Its Receptor by Kinetically Controlled, Step-Wise Affinity Cross-Linking", Biochemistry 31, 7341-7346.

Summerfield, A.E., A.K. Hudnall, T.J. Lukas, C.A. Guyer, and J.V. Staros (1996), "Identification of Residues of the Epidermal Growth Factor Receptor Proximal to Residue 45 of Bound Epidermal Growth Factor", J. Biol. Chem. 271, 19656-19659.

Ewald, J.A., Coker, K.J., Price, J.O., Staros, J.V. and Guyer, C.A. (2001), “Stimulation of Mitogenic Pathways through Kinase-Impaired Mutants of the Epidermal Growth Factor Receptor”, Exp. Cell Res. 268, 262-273.

Wilkinson, J.C., Stein, R.A., Guyer, C.A., Beechem, J.M., and Staros, J.V. (2001), “Real-Time Kinetics of Ligand/Cell Surface Receptor Interactions in Living Cells: Binding of Epidermal Growth Factor to the Epidermal Growth Factor Receptor”, Biochemistry 40, 10230-10242.

Wilkinson, J.C., and Staros, J.V. (2002), “Effect of ErbB2 Coexpression on the Kinetic Interactions of Epidermal Growth Factor with Its Receptor in Intact Cells”, Biochemistry 41, 8-14.

Stein, R.A., Hustedt, E.J., Staros, J.V., and Beth, A.H. (2002), “Rotational Dynamics of the Epidermal Growth Factor Receptor”, Biochemistry 41, 1957-1964.

Wilkinson, J.C., Beechem, J.M., and Staros, J.V. (2002), “A Stopped-Flow Fluorescence Anisotropy Method for Measuring Hormone Binding and Dissociation Kinetics with Cell-Surface Receptors in Living Cells”, J. Recept. Signal Transduction 22, 357-371.

Ewald, J.A., Wilkinson, J.C., Guyer, C.A., and Staros, J.V. (2003), “Ligand- and Kinase-Activity-Independent Cell Survival Mediated by the Epidermal Growth Factor Receptor Expressed in 32D Cells”, Exp. Cell Res. 282, 121-131.

Zhen, Y., Caprioli, R.M., and Staros, J.V. (2003), “Characterization of Glycosylation Sites of the Epidermal Growth Factor Receptor”, Biochemistry 42, 5478-5492.

Whitson, K.B., Beechem, J.M., Beth, A.H., and Staros, J.V. (2004), “Preparation and Characterization of Alexa Fluor 594-labeled Epidermal Growth Factor for Fluorescence Resonance Energy Transfer Studies: Application to the Epidermal Growth Factor Receptor”, Anal. Biochem. 324, 227-236.

Whitson, K.B., Whitson, S.R., Red-Brewer, M.L., McCoy, A.J., Vitali, A.A., Walker, F., Johns, T.G., Beth, A.H., and Staros, J.V. (2005), “Functional Effects of Glycosylation at Asn-579 of the Epidermal Growth Factor Receptor”, Biochemistry 44, 14920-14931.

Stein R.A., and Staros, J.V. (2006), “Insights into the evolution of the ErbB receptor family and their ligands from sequence analysis”, BMC Evolutionary Biology 6, 79.