Date of Award
Doctor of Philosophy (PhD)
Microbiology, Molecular Biology and Biochemistry
Dr. Jeffrey Benovic
Dr. Walter Koch
Dr. Michael Root
Dr. Philip Wedegaertner
Phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) and the subsequent recruitment of arrestin is a well-established paradigm that initiates the process known as desensitization. However, an emerging theme in GPCR regulation is the possibility of differential regulation dictated by the phosphorylation pattern elicited by the different members of the GRK family. Therefore, we have used small interfering RNA-mediated knock down of the GRKs and arrestins in an attempt to better understand how phosphorylation regulates the activity and signaling of the M3 muscarinic acetylcholine receptor (M3 mAChR) and CXCR4, two receptors endogenously expressed in HEK293 cells.
Using a two-pronged approach of assaying calcium mobilization and ERK activation, we were able to define and monitor changes in both the G protein- dependent and –independent signaling pathways. We found that GRK2, 3, and 6, and arrestin2 and 3 each has a distinct and separable role in regulating the activity of each receptor. Interestingly, knock down of GRK5 did not effect signaling via either receptor. Our studies with the M3 mAChR suggest that signaling is strictly through a G protein-dependent manner and relief of inhibitory constraints (GRKs and arrestins) subsequently enhances receptor function. In contrast, CXCR4 uses both a G protein-dependent and –independent (arrestin- dependent) means of signaling. Notably, arrestin-dependent signaling requires both GRK3 and 6.
Busillo, John M., "Characterizing the Dynamics and Functional Role of Site-Specific Phosphorylation of G Protein-Coupled Receptors" (2008). Theses & Dissertations. 5.