Dept. of Biochemistry and Molecular Biology
Oregon Health & Science University
G Protein-Coupled Receptors (GPCRs) are critical mediators of transmembrane signaling. Signaling via G proteins, in response to a cue from a ligand, is central to transduction of extracellular signals to the interior of a cell to trigger a cellular response. Equally important is the attenuation of GPCR signal by cytosolic modulators that limit the tone and duration of the response to any stimulus. This dissertation is aimed at understanding better the interactions, with the receptor, of different proteins involved in attenuating GPCR signals. Some of the key findings are outlined below. We report that both visual arrestin and Î²-arrestin 1 can bind monomeric rhodopsin in nanodiscs, which are discoidal membranes structures, encapsulated by a belt of amphipathic lipid carrying proteins. In fact, arrestin bound monomeric rhodopsin in nanodiscs better than rhodopsin oligomers in nanodiscs or liposomes. Using a fast and simple one-step purification method, we purified rhodopsin kinase (RK) expressed in mammalian cells. We found that the kinase, too, could make a functional interaction with monomeric rhodopsin and phosphorylate the receptor. Next, our studies also suggest that both arrestin and RK bind the same region of rhodopsin as the G protein, transducin. These results point to conserved mode of interaction between rhodopsin and its various modulating proteins. Further, fluorescence studies employing tryptophan induced quenching (TrIQ) showed two distinct sites on arrestin interact with the base of TM6 of rhodopsin. Finally, initial characterization of a novel protein (CRIP1a) involved in the attenuation of the constitutive signaling of CB1 receptor, shows that protein is stable and as high Î²-sheet content, with no disulfide bonds. Crystal screens of CRIP1a have provided some conditions, which might require some more fine-tuning to get crystals of CRIP1s for structure determination.
School of Medicine
Sinha, Abhinav, "GPCR signal attenuation : biochemical and spectroscopic analysis of interaction with affiliate proteins" (2012). Scholar Archive. 769.
Available for download on Saturday, July 01, 2017