Date of Award
Doctor of Philosophy (PhD)
G protein-coupled receptors (GPCRs) comprise a large family of related seventransmembrane- helical membrane proteins that bind to specific extracellular ligands, such as hormones or neuromodulators. The active receptor-ligand complex then engages with a heterotrimeric G protein on the cytoplasmic surface of the plasma membrane to facilitate a change in the concentration of an intracellular second messenger, such as cAMP. A number of non-canonical signaling pathways, such as β-arrestin-mediated signaling, also exist for many, if not all, GPCRs. Receptor signaling is attenuated by phosphorylation and receptor internalization. Recent advances in structural studies of GPCRs have revealed high-resolution structures of both inactive and active receptors in complex with various ligands. Endogenous ligands, drugs and the membrane environment, and even oligomerization can affect receptor signaling efficacy, but the mechanistic details underlying these allosteric effects are poorly characterized. To study allosterism of a ligand-receptor complex in a bilayer requires at least partial enrichment or isolation of the basic signaling unit. Many studies have employed biochemical purification and reconstitution strategies, but GPCRs are inherently unstable when extracted from native membranes so conditions must be carefully selected to preserve receptor integrity. To monitor the functional state of GPCRs during purification and reconstitution, a novel homogeneous time-resolved fluorescence resonance energy transfer (FRET)-based analytical assay was developed. To enable the assay, a novel bioconjugation method was invented to prepare microgram quantities of monoclonal antibodies labeled with long-lived lanthanide fluorophores. As a proof-of-concept, the folding and stability of human C-C chemokine receptor 5 (CCR5), the primary coreceptor for HIV-1 cellular entry, was studied. The assay enabled high-throughput detection of femtomole quantities of CCR5. Thermal denaturation measurements demonstrated that small molecule antagonists substantially stabilize CCR5 and also revealed that the ligands induce distinct receptor conformations, consistent with the hypothesis that GPCRs access numerous conformations during signal transduction rather than operating as a binary active-inactive switch. In addition, high-throughput stability screens led directly to the identification of CCR5 mutants that should be sufficiently stable for crystallization and lead to a high-resolution structure of CCR5, which would significantly advance understanding of the structural basis of HIV entry. The FRETbased assay was also applied to devise and optimize a protocol to incorporate CCR5 into an artificial membrane scaffold called nanoscale apolipoprotein bound bilayers (NABBs). CCR5 was shown to retain proper folding in NABBs and proof-of-concept fluorescence correlation spectroscopy (FCS) experiments were carried out to characterize these structures. Novel FCS standard reagents were developed to facilitate these measurements. The biochemical and analytical approaches reported may be adapted to prepare stable, functional samples of other GPCRs for structural and dynamic studies of receptor allostery. GPCRs are known to form dimers and higher-order oligomers, and despite a growing body of evidence that these complexes are functionally important, the structural basis of receptor-receptor interactions remains unknown. To address this problem, a potential dimerization interface of the prototypical GPCR, rhodopsin, was analyzed using a proteomics approach involving chemical crosslinking and liquid chromatography-mass spectrometry. The strategy was devised so that rhodopsin could be probed in the unique native environment of the rod cell disk membrane. Crosslinking results supported a model of rhodopsin dimerization involving contacts in transmembrane helix 1 and an amphipathic cytoplasmic helix at the carboxyl-terminal tail of the receptor. This novel interface is postulated to be relevant for understanding GPCR oligomerization in general.
Knepp, Adam, "Studies of G Protein-Coupled Receptor Stability and Dimerization Using Novel Fluorescence and Crosslinking Approaches" (2013). Student Theses and Dissertations. 230.