Biomedical Sciences ETDs

Publication Date

5-1-2011

Abstract

The erbB1 receptor regulates cellular programs including proliferation, migration, and differentiation and is the prototypical receptor tyrosine kinase (RTK). The erbB family consists of four homologous transmembrane receptors (erbB1/HER1/EGFR, erbB2/HER2, erbB3/HER3, erbB4). Canonically, ligand binding leads to an extracellular conformational change that promotes the formation of a receptor-mediated back-to-back dimer, asymmetric orientation of the catalytic kinase domains, and downstream transphosphorylation of cytoplasmic tyrosine residues. Exceptions to this paradigm are the orphan erbB2 and the kinase defective erbB3. The erbB receptors are implicated in mechanisms of carcinogenesis and are, thus, important therapeutic targets. Antibodies and small molecule inhibitors have been used to target cancer cells expressing erbB1 and erbB2, however, tumors often become resistant to treatment. Recent evidence implicates erbB3 in escape from erbB1- and erbB2-targeted pharmacological agents. Therefore, understanding the function of these receptors and their interactions with each other is important for designing better therapeutics. Here, we investigated erbB dimer formation and lifetime using live cell imaging and an analytical three-state Hidden Markov Model (HMM). First, multi-color quantum dot (QD) based probes that label resting or activated receptors were used to directly observe dimerization and quantify diffusion and correlated motion. Second, pairwise analyses of single particle trajectories in our HMM are used to characterize transition rates between free, confined, and dimerized states. We examined preformed, unliganded erbB1 homodimers and demonstrate that these do not display correlated motion and that observed dimers are short lived. Interestingly, liganded erbB1 dimers have the same off rate regardless of the activation status of the kinase domain. We further describe features of membrane organization, in particular demonstrating differential partitioning of activated receptors that alters mobility and permits repeated interactions within domains. Important mechanistic insight comes from our novel observations of short lived erbB2-erbB3 heterodimers and long lived erbB3 homodimers. Prior biochemical studies suggested that the erbB2-erbB3 heterodimer was the functional signaling unit. Our single particle tracking results are consistent with a new model for an active erbB3 kinase domain that is dependent on interactions with erbB2. Furthermore, our data indicate that erbB3 dimers and, ultimately, oligomers may be the principal signaling complex. This work demonstrates the importance of membrane architecture and reorganization in signal transduction and sheds new light on mechanisms of erbB activation with unprecedented spatial and temporal resolution.

Keywords

ErbB, Biophysics, Single Particle Tracking, Hidden Markov Model

Sponsors

National Science Foundation Career Award and IGERT Fellowship; National Cancer Institute

Document Type

Dissertation

Language

English

Degree Name

Biomedical Sciences

Level of Degree

Doctoral

Department Name

Biomedical Sciences Graduate Program

First Advisor

Wilson, Bridget

First Committee Member (Chair)

Lidke, Diane

Second Committee Member

Lidke, Keith

Third Committee Member

Oliver, Janet

Fourth Committee Member

Evans, Deborah

Fifth Committee Member

Edwards, Jeremy

Sixth Committee Member

Timlin, Jerilyn

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