University of Pittsburgh School of Medicine - Interdisciplinary Biomedical Graduate Program
The focus of the research in the laboratory is currently split into two major directions which are apparently distinct from each other with respect to the biological systems involved, their relation to the human disease, and experimental models used. However, the main idea underlying both directions is conceptually the same - to understand how endocytosis and post-endocytic trafficking regulates function(s) of the transmembrane proteins, such as receptors and transporters.
The elucidation of the molecular mechanisms of endocytosis of growth factor receptors using a prototypic member of the family, epidermal growth factor (EGF) receptor, and analysis of the role of endocytosis in spatial and temporal regulation of signal transduction by the EGF receptor.
EGF receptor is the best studied receptor tyrosine kinase and a member of ErbB family implicated in regulation of key cellular functions in normal and neoplastic cells. EGF receptor is also important prognostic marker and therapeutics target in many types of cancer. Despite that a number of EGF receptor inhibitors are already in clinical use, they currently benefit only a small pool of patients. Thus, a new focus of our current research program is to analyze the mechanisms of intrinsic and acquired resistance of the EGF receptor expressing tumors to clinically-relevant EGF receptor inhibitors.
Elucidation of the mechanisms of EGF receptor endocytosis involves the use of cutting-edge quantitative mass-spectrometry methods (collaboration with Dr. S. Gygi, Harvard Medical School), RNA interference methods (collaboration with Dharmacon, Inc) in combination with kinetics endocytosis assays developed in our laboratory over the years.
The role of endocytosis in spatial regulation of signaling is studied in conjunction with the development and application of a set of technologies allowing visualization of protein interaction and activities in living cells including various FRET methods. In addition, we have developed a new approach that enables analysis of the localization of fluorescently-tagged proteins expressed at physiological levels in living human cells. This is achieved by stable knock-down of the endogenous protein by RNA interference with concurrent constitutive expression of the same proteins tagged with fluorescent protein at the levels similar to that of an endogenous protein.
In the analysis of the mechanisms of cancer resistance to the EGF receptor drugs, the focus is on head-and-neck cancer, where we are using a panel of squamous cell carcinoma cell lines derived from head-and-neck cancer patients.
To elucidate the role of trafficking processes in the regulation of dopaminergic neurotransmission by the plasma membrane dopamine transporter (DAT).
Dopamine (DA) plays an important role in brain reward, both to natural reinforcers and addictive drugs. Removal of DA from the extracellular space and its transport back into DA neurons is an important mechanism controlling DA neurotransmission. This removal occurs via the DAT. DAT plays important roles in psychomotor stimulant behavioral activation and reward. By understanding how DAT activity is regulated, we will better appreciate its contribution to normal neurotransmission and to brain diseases like drug addiction.
Our current research is aimed at characterization of the mechanisms of endocytosis and intracellular trafficking of DAT. The first set of projects involves structure-function studies of heterologously-expressed human DAT. We have performed extensive analysis of the molecular mechanisms of DAT endocytosis induced by PKC activation and identified key players involved. Our immediate goal is to elucidate the molecular mechanisms of PKC-dependent endocytosis and also elucidate the mechanisms that control constitutive PKC-independent endocytosis.
The main focus in the nest several years will be on the development of in vitro and in vivo experimental models to study trafficking of endogenous DAT in dopaminergic neurons. To this end, we are working in two independent tracks. First, we are developing and characterizing midbrain-striatal organotypic cultures prepared from post-natal rats, and developing the reagents that can be used to manipulate DAT trafficking in these cultures. In the same time, we began generation of the knock-in mice with epitope-tagged DAT that should allow quantitative analysis of DAT trafficking in vitro and in vivo in neurons obtained from these mice. Finally, the data obtained using mechanistic analyses will be further developed in experiments with the intact animals to analyze how changes in DAT trafficking at the synapse correlate with the behavior patterns and response of the drugs of abuse.