Shalini T. Low-Nam

ErbB1 dimerization is promoted by domain co-confinement and stabilized by ligand binding

The extent to which ligand occupancy and dimerization contribute to erbB1 signaling is controversial. To examine this, we used two-color quantum-dot tracking for visualization of the homodimerization of human erbB1 and quantification of the dimer off-rate (koff) on living cells. Kinetic parameters were extracted using a three-state hidden Markov model to identify transition rates between free, co-confined and dimerized states. We report that dimers composed of two ligand-bound receptors are long-lived and their koff is independent of kinase activity. By comparison, unliganded dimers have a more than four times faster koff. Transient co-confinement of receptors promotes repeated encounters and enhances dimer formation. Mobility decreases more than six times when ligand-bound receptors dimerize. Blockade of erbB1 kinase activity or disruption of actin networks results in faster diffusion of receptor dimers. These results implicate both signal propagation and the cortical cytoskeleton in reduced mobility of signaling-competent erbB1 dimers.

erbB3 Is an Active Tyrosine Kinase Capable of Homo- and Heterointeractions

ABSTRACT Often considered to be a “dead” kinase, erbB3 is implicated in escape from erbB-targeted cancer therapies. Here, heregulin stimulation is shown to markedly upregulate kinase activity in erbB3 immunoprecipitates. Intact, activated erbB3 phosphorylates tyrosine sites in an exogenous peptide substrate, and this activity is abolished by mutagenesis of lysine 723 in the catalytic domain. Enhanced erbB3 kinase activity is linked to heterointeractions with catalytically active erbB2, since it is largely blocked in cells pretreated with lapatinib or pertuzumab. erbB2 activation of erbB3 is not dependent on equal surface levels of these receptors, since it occurs even in erbB3-transfected CHO cells with disproportionally small amounts of erbB2. We tested a model in which transient erbB3/erbB2 heterointeractions set the stage for erbB3 homodimers to be signaling competent. erbB3 homo- and heterodimerization events were captured in real time on live cells using single-particle tracking of quantum dot probes bound to ligand or hemagglutinin tags on recombinant receptors.

Bone marrow stromal and vascular smooth muscle cells have chemosensory capacity via bitter taste receptor expression.

The ability of cells to detect changes in the microenvironment is important in cell signaling and responsiveness to environmental fluctuations. Our interest is in understanding how human bone marrow stromal-derived cells (MSC) and their relatives, vascular smooth muscle cells (VSMC), interact with their environment through novel receptors. We found, through a proteomics screen, that MSC express the bitter taste receptor, TAS2R46, a protein more typically localized to the taste bud. Expression was also confirmed in VSMCs. A prototypical bitter compound that binds to the bitter taste receptor class, denatonium, increased intracellular calcium release and decreased cAMP levels as well as increased the extracellular release of ATP in human MSC. Denatonium also bound and activated rodent VSMC with a change in morphology upon compound exposure. Finally, rodents given denatonium in vivo had a significant drop in blood pressure indicating a vasodilator response. This is the first description of chemosensory detection by MSC and VSMCs via a taste receptor. These data open a new avenue of research into discovering novel compounds that operate through taste receptors expressed by cells in the marrow and vascular microenvironments.

Dynamic Transition States of ErbB1 Phosphorylation Predicted by Spatial Stochastic Modeling

ErbB1 overexpression is strongly linked to carcinogenesis, motivating better understanding of erbB1 dimerization and activation. Recent single-particle-tracking data have provided improved measures of dimer lifetimes and strong evidence that transient receptor coconfinement promotes repeated interactions between erbB1 monomers. Here, spatial stochastic simulations explore the potential impact of these parameters on erbB1 phosphorylation kinetics. This rule-based mathematical model incorporates structural evidence for conformational flux of the erbB1 extracellular domains, as well as asymmetrical orientation of erbB1 cytoplasmic kinase domains during dimerization. The asymmetric dimer model considers the theoretical consequences of restricted transactivation of erbB1 receptors within a dimer, where the N-lobe of one monomer docks with the C-lobe of the second monomer and triggers its catalytic activity. The dynamic nature of the erbB1 phosphorylation state is shown by monitoring activation states of individual monomers as they diffuse, bind, and rebind after ligand addition. The model reveals the complex interplay between interacting liganded and nonliganded species and the influence of their distribution and abundance within features of the membrane landscape.

Determining FcεRI Diffusional Dynamics via Single Quantum Dot Tracking

Single-particle tracking (SPT) using fluorescent quantum dots (QDs) provides high-resolution spatial-temporal information on receptor dynamics that cannot be obtained through traditional biochemical techniques. In particular, the high brightness and photostability of QDs make them ideal probes for SPT on living cells. We have shown that QD-labeled IgE can be used to characterize the dynamics of the high-affinity IgE Receptor. Here, we describe protocols for (1) coupling QDs to IgE, (2) tracking individual QD-bound receptors, and (3) analyzing one- and two-color tracking data.

Single Quantum Dot Tracking Coupled to a Three-State HMM Provides New Mechanistic Insight Into erbB1 Homodimerization

The erbB family of trans-membrane receptor tyrosine kinases serves as the prototypical model for dimerization-induced signal transduction. The current paradigm for signal initiation by erbB1 homo-dimers is based on ligand-induced conformational changes, which expose the dimerization arms of activated receptors. To track erbB1 dynamics at the molecular level, we used two-color single quantum dot (QD) tracking to determine diffusion and dimerization characteristics of resting, ligand-bound and kinase-inhibited receptors. Long-lived erbB1 homo-dimers were directly visualized between EGF-QD-bound receptors, characterized by a 50 nm separation. However, we also observed dimer interactions whose close approach was punctuated by periods of excursion up to hundreds of nanometers apart, suggesting a third domain-confined receptor state. For each condition, transition rates between free, domain-confined, and dimer states were extracted using a modified three-state Hidden Markov Model (HMM). This analytical model uses separation of pairwise QD trajectories to determine probabilities of state transitions, whose rates are fit over many candidate interactions. Diffusional behavior was determined by state and showed slowing upon dimer formation; EGF-bound monomeric receptors confined to the same domain showed two-fold slower diffusion than free receptors and showed a further three and a half-fold slowing upon dimerization. Resting or kinase inhibited receptors did not show this dramatic mobility change. While the small molecule tyrosine kinase inhibitor, PD153035 that blocks receptor phosphorylation altered receptor diffusion, it did not change the dimer off rate. Resting erbB1 dimers tracked via monovalent heavy-chain only antibody fragments (VhH) did not demonstrate correlated motion and had a >6-fold higher off-rate. Our studies provide new mechanistic insight into erbB1 dimerization, demonstrate a role for membrane domains in promoting protein-protein interactions, and suggest a reduction in receptor mobility as a feature of signaling competent erbB1 dimers.