Biased ligands of CXCR3 promote differential signaling mediated by the phosphorylation barcode

Biased ligands of CXCR3 promote differential signaling mediated by the phosphorylation barcode

Cole Honeycutt

I started out this year working on a project in the Rajagopal Lab in which we investigated the phosphorylation barcode as a mechanism underlying biased agonism at the G protein-coupled receptor (GPCR) CXCR3. This project was started by the previous MSTP student working in the Rajagopal Lab and continued by Dylan Eiger, the MSTP student with whom which I worked throughout this past year. I really started my scientific interest and experience with this project, as I learned basic cell culture, transfection, BRET assays, western blotting, molecular cloning, confocal microscopy, qPCR, and more. Following completion of this project, for which we are currently writing a manuscript, I started heading off my own project on the intersection of GPCR signaling with the NF-κB pathway, again under the supervision of Dylan. I cloned a suite of novel BRET constructs, with some difficulty, and then assayed their interactions with β-arrestin 2. We initially planned for the project to be about CXCR3, but soon found that while the B2AR displayed some very interesting interactions between these proteins, CXCR3 did not. My PI, Sudar Rajagopal, then gave me the leeway to continue this project and see where it took us. I worked with Dylan over nearly all of the end of the fall semester, winter break, and beginning of spring semester on this project. There were many difficulties with inconsistent data, reagents going bad, and cells being unhappy, and this inconsistency, coupled with the divergent nature of the project compared with that which the lab typically investigates, led to us stopping work on the NF-κB project in favor of a project on CXCR3 endocytosis as a mechanism promoting biased agonism. I was a little downtrodden after this project cancellation, as I had put months of time and effort into it. However, I presented my findings at Experimental Biology 2021 conference and won the 1st place undergraduate poster award in the Division for Drug Discovery and Development, which made me feel a bit better. After that, I worked on the CXCR3 endocytosis project, which really came together rapidly throughout the spring semester. Overall, I had a really great time working in the Rajagopal Lab. I learned a lot, accomplished a lot, and had my career path change dramatically due to my experience therein. Thank you to the Undergraduate Research Support Office for the DSRF award, the opportunity to present my findings at Visible Thinking, and the continued help along the way.