Investigations into Allosteric Mechanisms of G Protein Activation
Thaker, Tarjani Mahesh
The G protein coupled receptor (GPCR) family is comprised of ubiquitous, membrane-bound proteins that are highly conserved in structure, yet varied in their cognate ligand. This allows for regulation of diverse physiological processes ranging from cardiac contractility and neurotransmission to olfaction and vision. Signaling events downstream of activated GPCRs are initiated through a conserved allosteric mechanism of receptor-mediated nucleotide exchange of GDP for GTP in the Gα subunit of their intracellular binding partner, the heterotrimeric G protein (Gαβγ). While much is known about GPCR and Gαβγ function, less is understood about the mechanism of GPCR-Gαβγ complex formation and subsequent receptor-mediated Gα activation. Biophysical characterizations have indirectly informed on the dynamic nature of the interaction. Direct investigations, however, have been thwarted due to the intrinsic instability of such complexes both in vivo and in vitro. In lieu of structural data on the GPCR-Gαβγ complex, models derived from structural studies of the Gα subunit mimicking receptor-activated states constitute our understanding of receptor-mediated Gα activation. Many of these studies focus on the endpoints of activation and information on transient features of GPCR-Gαβγ interactions remains sparse. The first part of this work aims to lower the barrier for structural studies on GPCR-G protein complexes by developing and characterizing model membrane systems that recapitulate the native environment of the signaling receptor where complex stability is optimized. In the second part of this work, these findings are applied to the structural characterization of the model system GPCR-G protein pair, rhodopsin-transducin. In the third part, I focus on the biochemical and structural characterization of an allosteric activation intermediate of Gαi1 to better understand how crosstalk between receptor and nucleotide binding sites is facilitated for promoting GDP release during Gα activation. Taken together, the work presented here offers insight into allosteric processes of GPCR-G protein complex formation, complex stabilization, and subsequent activation of G proteins for rhodopsin, transducin, and Gαi1. These results provide a framework for future investigations into the allosteric regulation of additional GPCR-G protein superfamily members.