Modulation of serotonin receptor signalling by protein kinase activation

I investigated the desensitization mechanisms of the serotonin 1A (5-HT$ sb{ rm 1A}$) and 5-HT$ sb{ rm 1B}$ receptors at the molecular level. Receptor phosphorylation by second messenger kinases and G-protein coupled receptor kinases (GRK) is an initial step in the functional desensitization of G-protein coupled receptor receptors (GPCRs). Evidence suggested that PKC preactivation desensitizes the 5-HT$ sb{ rm 1A}$ receptor. In Ltk-cells, PKC preactivation is pathway-selective, blocks receptor-mediated calcium mobilization but not cAMP inhibition. My first objective was to identify if the prime phosphorylation target of PKC was the receptor itself. Four PKC phosphorylation sites on the 5-HT1A receptor were eliminated using site-directed mutagenesis. The signalling profile of single mutants, and third loop double and triple mutants were examined in Ltk-cells. I determined that the prime target of PKC was the receptor since multiple PKC phosphorylation sites in the third loop mediated pathway-selective uncoupling of the receptor from the calcium mobilization pathway and not the adenylyl cyclase. The PKC site in the second loop of the 5-HT$ sb{ rm 1A}$ receptor was shown to be a critical G-protein-contact site in both neuroendocrine and mesenchymal cells.My second objective was to investigate if GRK-mediated phosphorylation could enhance homologous desensitization of the endogenously expressed 5-H$ sb{ rm 1B}$ receptor in the opossum kidney (OK) cell line. To define the role of endogenous GRK in desensitization, I cloned the OK-GRK$ sb2$ from the OK cell Line and generated a kinase inactive mutant. The GRK$ sb2$ was shown to phosphorylate an epitope tagged 5-HT$ sb{ rm 1B}$ receptor in vitro. However, in intact cells the OK-GRK$ sb2$ did not enhance agonist-induced desensitization of the 5-HT1B receptor, but enhanced the desensitization of the $ alpha$2C receptor. The kinase-inactive mutant or reduction in OK-GRK2 protein levels using antisense GRK2 cDNA both attenuated the desensitization of the $ alpha$2C receptor but not the 5-HT$ sb{ rm 1B}$ receptor. These results suggest that phosphorylation mediated by GRKs in vitro may not mimic in vivo receptor desensitization. Processes other than those mediated by GRKs may be more important for the desensitization of the 5-HT$ sb{ rm 1B}$ receptor in the OK cells.In conclusion, I have identified two possible mechanisms by which two related receptors, 5-HT$ sb{ rm 1A}$ and 5-HT$ sb{ rm 1B}$ receptors, are regulated by protein kinases: receptor phosphorylation by PKC and GRK. My results suggest that receptor phosphorylation by PKC plays a role in pathway selective desensitization of the 5-HT$ sb{ rm 1A}$ receptor, while phosphorylation the 5-HT$ sb{ rm 1B}$ receptor by GRK, observed in vitro, does not play an important role in the homologous desensitization of the 5-HT$ sb{ rm 1B}$ receptor