James V. Staros

Professor, Department of Biochemistry & Molecular Biology

PhD: Molecular Biophysics & Biochemistry, Yale University
Postdoc: Bio-organic Chemistry, Harvard University

Research Interests

My laboratory focused on the 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 which we studied were the ErbB receptor family and their ligands, the archetypes of which are epidermal growth factor (EGF) and its receptor. A wide variety of techniques from protein chemistry, spectroscopy, and molecular biology were brought to bear in these investigations.

Protein chemical studies in my laboratory showed almost thirty years ago 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 member of the superfamily of receptor tyrosine kinases.  Using affinity labeling methods, we identified Lys721 as an important residue in the kinase active site.  Subsequently, using site-directed mutagenesis, we 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 the receptor and the state of the occupied EGF-receptor complex in the membrane.   For example, we have 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.  A major part of our more recent efforts centered on the kinetics of ligand capture and release, using stopped-flow fluorescence anisotropy methods that we developed for investigating the kinetics of EGF-receptor binding and dissociation in living cells.  We expressed the EGF receptor in 32D cells, which do not express any endogenous ErbB receptors, and we 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 are not 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.

In a study of the glycosylation state of the receptor, we found that Asp579, 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 Asp579 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 that Asp is substituted with Gln, resulting in a receptor that cannot be glycosylated at that site, we were able to study the properties of this subclass of receptors.   Kinetic studies showed that the Asp579→Gln mutant EGF receptor when expressed alone in 32D cells has kinetic characteristics more closely resembling those of the receptor in the presence of ErB2 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 Asp579 contributes to stabilizing the inactive (tethered) state of the receptor.

In an independent series of studies we 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.