Dopamine receptors belong to the G protein-coupled receptor (GPCR) superfamily and are classified into two families: D1-like (D1R and D5R) and D2-like (D2R, D3R and D4R), based on their ability to stimulate or inhibit adenylyl cyclase (AC). Classically, GPCRs (including D2R and D3R) are desensitized by the activation of the serine/threonine protein kinase C (PKC) upon phorbol-12-myristate-13-acetate (PMA) treatment. Previous studies demonstrate that while human D5R (hD5R) is also strongly desensitized upon PMA treatment, the human D1R (hD1R) undergo a robust PMA-induced sensitization. The aim of this PhD thesis was to explore how the canonical PKC- or phorbol ester-linked pathway can control the responsiveness of two similar GPCRs like hD1R and hD5R in an opposite fashion. Our data indicate that hD1R sensitization and hD5R desensitization are not mediated by a direct modulation of AC activity by PKC. Using a chimeric approach, we identified the third intracellular loop (IL3) as the key structural determinant controlling in an opposite manner the PMA-mediated regulation of hD1R and hD5R. To delineate the potential PKC phosphorylation sites, a series of mutation of serine (Ser) and threonine (Thr) located into IL3 of hD1R and hD5R were used. No hD1R mutation decreased the PMA-mediated sensitization. This suggests that hD1R phosphorylation is not required for PMA-induced sensitization. In contrast, our results indicate that PMA-mediated hD5R desensitization occurs through a hierarchical phosphorylation of Ser260, Ser261, Ser271 and Ser274. Notably, these hD5R mutants exhibited a PMA-induced sensitization, reminiscent of the PMA-induced hD1R sensitization. Additionally, using short hairpin RNAs (shRNAs), we showed that PKCε is the potentiating PKC while the desensitizing isoform is δ. Overall, our work suggests the presence or absence of specific Ser residues on IL3 of hD1-like receptors dictate if phosphorylation-dependent desensitization (through PKCδ) or phosphorylation-independent potentiation (via PKCε) will occur.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/20577 |
| Date | January 2012 |
| Creators | Plouffe, Bianca |
| Contributors | Albert, Paul R., Tiberi, Mario |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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