A Novel Interaction between the A2a Adenosine Receptor and Ran Binding Protein 9 (RBP9) and the Role of RBP9 in GPCR Function

Open Access
Frey, Colleen Marie
Graduate Program:
Doctor of Philosophy
Document Type:
Date of Defense:
September 17, 2010
Committee Members:
  • Robert G Levenson, Dissertation Advisor
  • Robert G Levenson, Committee Chair
  • Victor Alan Canfield, Committee Member
  • Robert J Milner, Committee Member
  • Zachary Simmons, Committee Member
  • A2aR
  • GPCR
  • RBP9
Several lines of evidence describe an involvement of the A2a adenosine receptor (A2aR), a seven transmembrane, G protein-coupled receptor (GPCR) in Parkinson’s disease (PD). Caffeine, an antagonist of this receptor has been shown to have a protective effect on the development of PD. Interacting proteins of GPCRs assist in their signaling and regulation. In this work, the yeast two-hybrid assay was used to screen for novel interacting proteins for the A2aR, in order to discover a means of regulating the receptor’s signaling. Several candidate interacting proteins for the A2aR tail were identified. This work characterizes the interaction between the A2aR and one interactor, Ran Binding Protein 9 (RBP9). RBP9 has been described as a scaffolding protein which interacts with at least three (GPCRs). The functional role of RBP9 is still under investigation, but several regulatory roles have been described for its interaction with GPCRs, including mediating internalization, phosphorylation, and receptor expression. Here, the A2aR- RBP9 interaction is confirmed through GST-pulldown and Co-immunoprecipitation experiments conducted in A2aR- transfected HEK-293 cells and SHSY5Y cells. Furthermore, the two proteins are shown to co-localize in both cell lines. The possibility that RBP9 is a promiscuous interactor of GPCR proteins was also investigated. Co-immunoprecipitation experiments demonstrate an interaction between RBP9 and the D2 dopamine receptor (D2R), mu, kappa, and delta opioid receptors (MOR, KOR, and DOR). Because RBP9 interacts with several prominent GPCRs, it is possible that it holds a common function in regulation of these receptors. Through RBP9 siRNA knockdown experiments, a regulatory role for RBP9 in the overall expression of three GPCRs, namely the A2aR, MOR, and D2R in stably-tranfected HEK-293 cells is demonstrated. Specifically, knockdown of RBP9 results in a down-regulation of the A2aR, and an up-regulation of the D2R and MOR. RBP9-dependent up-regulation of D2R expression is confirmed in SHSY5Y neuroblastoma cells. It appears evident that RBP9 is a promiscuous interactor of GPCRs, and has a regulatory role in their overall expression levels. Further studies may expand our understanding of RBP9’s function, and the mechanism by which it regulates GPCR expression, providing a novel pharmaceutical target in GPCR signal regulation.