Activation of G Protein-Coupled Receptors and Heterotrimeric G Proteins.

G protein-coupled receptors (GPCRs) are an important class of cell-surface transmembrane receptors that pass an activation signal to the interior of the cell through heterotrimeric G proteins. In this work, we study the human beta-2-adrenergic receptor (B2AR) and stimulatory G protein (Gs) as examples in order to understand the molecular basis of this signal transfer event. We solved a 3.2 angstrom crystal structure of B2AR and Gs in a nucleotide-free, intermediate signaling complex, revealing the interaction between the proteins at atomic resolution. The structure was consistent with previous biochemical knowledge, but also revealed several previously unknown features of the activation process. We used deuterium/hydrogen exchange and electron microscopy in order to find regions in the complex that change conformation during the activation process. These regions are highly conserved within the GPCR and G protein families, and his work shows the central role that they play in the process of GPCR signal transduction. The binding of drugs to the receptor in the fully activated state, as seen in the B2AR-Gs complex, was also characterized by radioligand and antibody fragment binding. A full kinetic model was developed for drug binding to the activated receptor which demonstrated how the ligand is held very tightly in the receptor binding pocket. This tight ligand binding can be relieved by the addition of GDP, demonstrating a direct allosteric link between the G protein nucleotide binding site and the receptor ligand binding site. Overall, this work demonstrates how the GPCR signal transduction machinery operates in high-resolution structural, kinetic, and pharmacological detail. It advances our understanding of how GPCRs and G proteins pass a signal across the cellular membrane.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/99848/1/btdevree_1.pd

Pharmacological Characterization of Purified Full-Length Dopamine Transporter from <i>Drosophila melanogaster</i>

The dopamine transporter (DAT) is a member of the neurotransmitter:sodium symporter (NSS) family, mediating the sodium-driven reuptake of dopamine from the extracellular space thereby terminating dopaminergic neurotransmission. Our current structural understanding of DAT is derived from the resolutions of DAT from Drosophila melanogaster (dDAT). Despite extensive structural studies of purified dDAT in complex with a variety of antidepressants, psychostimulants and its endogenous substrate, dopamine, the molecular pharmacology of purified, full length dDAT is yet to be elucidated. In this study, we functionally characterized purified, full length dDAT in detergent micelles using radioligand binding with the scintillation proximity assay. We elucidate the consequences of Na+ and Cl− binding on [3H]nisoxetine affinity and use this to evaluate the binding profiles of substrates and inhibitors to the transporter. Additionally, the technique allowed us to directly determine a equilibrium binding affinity (Kd) for [3H]dopamine to dDAT. To compare with a more native system, the affinities of specified monoamines and inhibitors was determined on dDAT, human DAT and human norepinephrine transporter expressed in COS-7 cells. With our gathered data, we established a pharmacological profile for purified, full length dDAT that will be useful for subsequent biophysical studies using dDAT as model protein for the mammalian NSS family of proteins

Pharmacological chaperone for α-crystallin partially restores transparency in cataract models

Cataracts reduce vision in 50% of individuals over 70 years of age and are a common form of blindness worldwide. Cataracts are caused when damage to the major lens crystallin proteins causes their misfolding and aggregation into insoluble amyloids. Using a thermal stability assay, we identified a class of molecules that bind α-crystallins (cryAA and cryAB) and reversed their aggregation in vitro. The most promising compound improved lens transparency in the R49C cryAA and R120G cryAB mouse models of hereditary cataract. It also partially restored protein solubility in the lenses of aged mice in vivo and in human lenses ex vivo. These findings suggest an approach to treating cataracts by stabilizing α-crystallins