Abstract
We identified that activation of the G(q)-linked dopamine D1-D2 receptor hetero-oligomer generates a PLC-dependent intracellular calcium signal. Confocal FRET between endogenous dopamine D1 and D2 receptors in striatal neurons confirmed a physical interaction between them. Pretreatment with SKF 83959, which selectively activates the D1-D2 receptor heteromer, or SKF 83822, which only activates the D1 receptor homo-oligomer, led to rapid desensitization of the D1-D2 receptor heteromer-mediated calcium signal in both heterologous cells and striatal neurons. This desensitization response was mediated through selective occupancy of the D1 receptor binding pocket. Although SKF 83822 was unable to activate the D1-D2 receptor heteromer, it still permitted desensitization of the calcium signal. This suggested that occupancy of the D1 receptor binding pocket by SKF 83822 resulted in conformational changes sufficient for desensitization without heteromer activation. Bioluminescence resonance energy transfer and co-immunoprecipitation studies indicated an agonist-induced physical association between the D1-D2 receptor heteromeric complex and GRK2. Increased expression of GRK2 led to a decrease in the calcium signal with or without prior exposure to either SKF 83959 or SKF 83822. GRK2 knockdown by siRNA led to an increase in the signal after pretreatment with either agonist. Expression of the catalytically inactive and RGS (regulator of G protein signaling)-mutated GRK2 constructs each led to a partial recovery of the GRK2-attenuated calcium signal. These results indicated that desensitization of the dopamine D1-D2 receptor heteromer-mediated signal can occur by agonist occupancy even without activation and is dually regulated by both the catalytic and RGS domains of GRK2.
Highlights
The neurotransmitter dopamine has fundamental roles in regulating a wide variety of neuronal functions such as locomotion, cognition, reward, and emotion
We demonstrated that pretreatment with any of the three agonists resulted in rapid desensitization of the calcium signal both in cells and in primary culture neurons, and this was shown to occur through selective occupancy of the D1 receptor
We have demonstrated the desensitization of the D1-D2 receptor heteromer-mediated calcium signal by agonists with varying efficacies to activate the calcium signal
Summary
The neurotransmitter dopamine has fundamental roles in regulating a wide variety of neuronal functions such as locomotion, cognition, reward, and emotion. We demonstrated that pretreatment with any of the three agonists resulted in rapid desensitization of the calcium signal both in cells and in primary culture neurons, and this was shown to occur through selective occupancy of the D1 receptor.
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