Use this URL to cite or link to this record in EThOS:
Title: Arrestin interactions with the μ-opioid receptor
Author: Saxena, Kunal
ISNI:       0000 0004 2723 5168
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2012
Availability of Full Text:
Access from EThOS:
Upon agonist binding the u-opioid receptor (MOPr) is phosphorylated and can recruit arrestin-2 (beta-arrestin-l) and arrestin-3 (p-arrestin-2). Apart from promoting the desensitization and intemalization of the MOPr, the binding of arrestins can also lead to the triggering of alternative sigvalling pathways. It has been shown that kinases including GRK2, PKC and CaMKII can regulate MOPr. Here we used GST -fusion constructs of the intracellular regions of MOPr to investigate whether arrestin-2 and -3 can bind to these sequences in vitro, and secondly to determine whether phosphorylation of these sequences by various kinases can alter their ability to bind arrestins. Arrestin-3 bound to the unphosphorylated intracellular loops (ICL2, ICL3) and the COOH-tail of the MOPr, whereas arrestin-2 displayed preferential binding to the COOH-tail. Phosphorylation of the MOPr COOH-tail by PKC and CaMKII increased arrestin-2 and -3 binding but to a lesser extent than GRK2. The full agonist DAMGO was able to increase the interaction of both GFP-arrestin-2 and -3 to the full length MOPr. Previous published work, suggests that mice lacking arrestin-3, showed reduced opiate-induced constipation and respiratory suppression and the development of tolerance to opiates such as morphine. During my masters degree at Moscow State University (Russia), I designed and synthesized small molecule inhibitors (Kt, K2 and K3; see appendix-I-Ill for the chemical structures) of the arrestin-MOPr interaction using computer-aided drug design. In the GST pull down assay, only K3 affected the arrestin-MOPr interaction. It decreased the association of arrestin-2 b~t increased the association of arrestin-3 to the COOH-tail of the MOPr. K3 did not affect the interaction of arrestin-3 with the GRK2-phosphorylated MOPr COOH-tail fusion protein. Similar results were obtained in the full length receptor, where the association of GFP- arrestin-3 with the HA-tagged-MOPr remained unaffected in the presence of K3 and DAMGO stimulation. On the other hand, in the absence of DAMGO, K3 did increase the interaction of GFP-arrestin-3 with MOPr. In addition, K3 did not bind to the MOPr orthosteric binding sites and did not affect G-protein activation in the presence and absence of DAM GO. Taken together these data suggest that even though the small molecules were unable to inhibit the arrestin-3/MOPr interaction, such a direct modulation of the arrestin-MOPr interaction is possible and specific small molecules can be designed to modulate this interaction of other GPCRs.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available