My main research goals are to develop a multi-disciplinary approach to study membrane proteins that are relevant to cancer pathology and immunology, and to integrate structural information into biology and medicine. It has been estimated that about one third of the proteins encoded by a typical genome are membrane proteins. However, structures of only less than 100 integral proteins have been solved, comparing to that of more than 25,000 soluble proteins solved by X-ray crystallography and NMR. Thus, elucidation of the structural basis of transmembrane protein signaling remains an important challenge for the future. We will focus on the following three projects in our lab:
1) To study the regulation and bidirectional signaling of aV integrins across the plasma membrane.
aV family of integrins contains five members: aVb1, aVb3, aVb5, aVb6 and aVb8. They bind a group of overlapping ligands which generally contain the canonical tripeptide sequence, arginine-glycine-aspartic acid (RGD). aV integrins play critical roles in vascular development and permeability, the development of central nervous system, and tumor angiogenesis and metastasis. How aV integrins transmit bidirectional signals across the plasma membrane remains elusive, and specifically, it is unknown whether all integrins share a similar mechanism of TM signaling. We will study the ligand binding specificity, the conformational regulation and the outside-in signaling of aV integrins using cell-base assays as well as purified proteins.
2) To identify novel aVb3 small molecule inhibitors.
Most metastatic melanomas have high levels of aVb3 expression. This integrin receptor appears to be important for migration and invasion of the tumor cells, as well as cell proliferation and tumor-induced angiogenesis. Blocking aVb3 ligand binding and function with inhibitors can prevent tumor progression. Integrin antagonists can be classified into two groups. The first group is direct inhibitors which recognize the ligand binding site on the integrin molecules and act competitively. Indeed, most of the small molecule inhibitors are ligand mimetics and they directly block ligand binding. Their binding normally induces integrin conformational change similar to that by the binding of physiologic ligands, which can be recognized by conformation-dependent ligand induced binding site (LIBS) antibodies. The second group is allosteric inhibitors which exert their effect by stabilizing the low affinity state or by preventing conformational change necessary for ligand binding. We will develop high throughput screens for these two classes of inhibitors by using fluorescent ligands and LIBS antibodies.
3) To elucidate the structural basis of transmembrane protein signaling.
Dysregulation of receptor tyrosine kinase (RTK) activation has long been implicated in a variety of human leukemias. RTKs normally consist of an extracellular domain that binds growth factors, a single-span TM domain and a cytoplasmic domain. The cytoplasmic domain undergoes a conformational change upon ligand binding, leading to intracellular signaling. FMS-like tyrosine kinase-3 (FLT3) is a member of the PDGF-R subfamily of RTKs. In acute myeloid leukemia, FLT3 is frequently mutated and inhibitors to impair the oncogenic signaling are in development. In 20-25% of all cases of acute myeloid leukemia there are internal tandem duplications in the juxtamembrane domains of FLT3, resulting in constitutive activation of the FLT3 cytoplasmic domain tyrosine kinase activity. It was hypothesized that ligand binding induced FLT3 homodimerization, leading to autophosphorylation and activation of two kinase domains of the cytoplasmic domains. However, how signals are transmitted across the plasma membrane and specifically, what role of TM and juxtamembrane domains plays on kinase activation, remains elusive. We will use cysteine scanning, site-directed spin labeling EPR, computational structural modeling, crystallography and other biochemical and biophysical methods to study the ligand-induced and mutation-induced TM signaling of FLT3. It is expected that by uncovering the role of this domain we may more completely understand the regulation of FLT3, and potentially RTKs in general, and therefore design agents that modulate its signaling.
Selected Publications
Luo, B.-H., Carman, C. V., Springer, T. A. Structural basis of integrin regulation and signaling. Annu. Rev. Immunol. 2007, 25, 619-647.
Zhu, J., Carman, C. V., Kim, M., Shimaoka, M., Springer, T. A., Luo, B.-H. Requirement of alpha and beta subunit transmembrane helix separation for integrin outside-in signaling. Blood, 2007, 110, 2475- 2483.
Luo, B.-H., Springer, T. A. Integrin structures and conformational signaling. Curr. Opin. Cell Biol. 2006, 18(5), 579-86.
Luo, B.-H., Carman, C. V., Takagi, J., Springer, T. A. Disrupting integrin transmembrane heterodimerization increases ligand binding affinity, not valency or clustering. PNAS. 2005, 102, 3679-84.
Mehboob, S.*, Luo, B.-H.*, Fu, W., Johnson, M. E., Fung, L. W. Conformational studies of the tetramerization site of human erythroid spectrin by cysteine-scanning spin-labeling EPR methods. Biochemistry, 2005, 44(48), 15898-905. (*co-first authors)
Luo, B.-H., Strokovich, K., Walz, W.. Springer, T.A., Takagi, J. Allosteric beta1 integrin antibodies that stabilize the low affinity state by preventing the swing-out of the hybrid domain. J. Biol. Chem. 2004, 279, 27466-27471.
Luo, B.-H., Springer, T.A., Takagi, J. A specific helical interface between integrin alpha and beta subunit transmembrane domains maintains low affinity for ligand. PloS Biol. 2004, 2, 776-786.
Luo, B.-H., Takagi, J., Springer, T. A. Locking the beta3 integrin I-like domain into high and low affinity conformations with disulfides. J. Biol. Chem. 2004, 279: 10215-10221.
Luo, B.-H., Springer, T.A., Takagi, J. Stabilizing the open conformation of the integrin headpiece with a glycan wedge increases affinity for ligand. PNAS. 2003, 100, 2403-2408.
Luo, B.-H., Springer, T. A., Takagi, J. High affinity ligand binding by integrins does not involve head separation. J. Biol. Chem. 2003. 278, 17185-17189.