Beta2-adrenergic receptors (b2AR) activate the stimulatory heterotrimeric G protein (G-s) while type D2 dopamine receptors (which includes D2, D3 and D4 dopamine receptors) activate the inhibitory heterotrimeric G protein (G-i). Activation of G-s and G-i cause the stimulation and inhibition of adenylyl cyclase (AC), respectively. B2AR and D4 dopamine receptors (D4DR) co-immunoprecipitate from HEK 293 cells exogenously expressing both receptors suggesting the existence of a signaling complex containing these receptors. In order to determine if these receptors are closely associate with each other, and with other components involved in G protein-mediated signal transduction b2AR, D4DR, Gi, and AC were genetically tagged so that bioluminescence resonance energy transfer (BRET) could be used to monitor their interactions. All of the tagged proteins retained biological function. BRET experiments revealed that exogenously expressed b2AR, D4DR, G-I and AC were closely associated as a signaling complex in HEK 293 cells. BRET was also used to monitor D4DR activation of heterotrimeric G-i (G-alpha-i/beta-gamma). BRET experiments indicate that activation of G-i results in a conformational change within the protein, but they do not support the idea that activation leads to dissociation of the G-alpha-i-subunit from the Gbeta-gamma-subunit complex. Bipolar disorder is a serious disease that affects 1% of the population. Carbamazepine, lithium and valproate are effective drugs for the treatment of this disorder. The most popular hypothesis is that these mood stabilizing drugs reduce the level of cyclic AMP produced in response to neurotransmitter-mediated activation of AC. Each drug may do so by a different and as yet ill defined mechanism. HEK 293 cells co-expressing b2AR and D4.2DR were treated with therapeutic concentrations of these drugs. Carbamazepine and lithium reduced the isoproterenol-mediated stimulation of AC in these cells by approximately 50% while valproate had no effect on their response to isoproterenol. Initial experiments indicate that carbamazepine attenuates the response to isoproterenol by blocking trafficking of newly synthesized b2AR to the cell surface. Lithium also reduced the number of newly synthesized b2AR on the cell surface, but to a lesser extent than carbamazepine. Additionally, both carbamazepine and lithium interfered with the ability of dopamine to inhibit AC. BRET is being used to determine if these drugs block the dopamine-mediated inhibition of AC by displacing some protein(s) from the signaling complex, alter the conformation of the complex, or prevent some agonist-induced conformational change in the complex.
| Rebois, R Victor; Maki, Karl; Meeks, Julie A et al. (2012) D2-like dopamine and ýý-adrenergic receptors form a signaling complex that integrates Gs- and Gi-mediated regulation of adenylyl cyclase. Cell Signal 24:2051-60 |
| Dupré, Denis J; Robitaille, Mélanie; Rebois, R Victor et al. (2009) The role of Gbetagamma subunits in the organization, assembly, and function of GPCR signaling complexes. Annu Rev Pharmacol Toxicol 49:31-56 |
