James V. Staros, Ph.D. Professor Department of Biochemistry & Cell Biology Dean of the College of Arts and Sciences Stony Brook University E3320 Frank Melville Jr. Memorial Library Stony Brook, NY 11794-3391 Office telephone: 631-632-6976 Fax:  631-632-6900 E-mail: james.staros@stonybrook.edu
Research Description

Molecular mechanisms of transmembrane signaling.

We are working to elucidate 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 under study are 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 are brought to bear in this investigation.

Protein chemical studies in our laboratory showed more than twenty 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 is 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 can replace the functions of IL-3 binding to the IL-3 receptor. In the absence of EGF, the EGF receptor prevents apoptosis in these cells. Unexpectedly, the kinase-negative mutant in which Lys721 is replaced with Arg also prevents apoptosis; however, the kinase-negative mutant with Asp813 replaced with Ala does not retain this function.

A variety of spectroscopic studies are being carried out 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 used 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 current effort centers on the kinetics of ligand capture and release, using stopped-flow fluorescence anisotropy methods that we have developed for investigating the kinetics of EGF-receptor binding and dissociation in living cells. We have expressed the EGF receptor in 32D cells, which do not express any endogenous ErbB receptors, and we have shown 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 suggest 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 is expressed in the absence of ErbB2, the high affinity state is defined by a fast on-rate; however, in the presence of ErB2, the high affinity state is defined by a very slow off-rate.

In a recent 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 ant 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 have been able to study the properties of this subclass of receptors.   Kinetic studies have shown 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.

We are currently expanding our kinetic studies to investigate the effects of coexpression of other ErbB family receptors with the EGF receptor and to investigate how differences in kinetics of ligand capture and release correlate with changes in downstream signaling.

We are also using computational methods to study the molecular evolution of the ErbB family of receptors and of the EGF family of ligands.

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