However a two receptor model
However, a two-receptor model presents a mystery of how MBns know whether they are receiving an acquisition or forgetting signal from the PPL1 DAns. The most attractive explanation for this is that the two receptors might have different signaling properties and mobilize distinct signaling cascades. However, prior pharmacological characterization experiments in reconstituted systems classified both receptors as D1-like, stimulating the accumulation of cyclic AMP (cAMP) by activating the heterotrimeric G protein Gs and the effector adenylyl cyclase (Sugamori et al., 1995, Han et al., 1996, Reale et al., 1997). These observations challenge the hypothesis that the two receptors mobilize different intracellular signaling cascades for acquisition and forgetting.
We have revisited the question of whether the two receptors couple to distinct downstream signaling cascades. We show, using a real-time bioluminescence resonance energy transfer (BRET) assay for G protein activation by G protein coupled receptors (GPCRs) (Masuho et al., 2015b), that dDA1 strongly couples to Gαs and increases cAMP accumulation upon stimulation by DA, whereas the Damb receptor couples preferentially to Gαq over Gαs and induces calcium signaling. Consistent with a Damb/Gαq pathway role in forgetting, RNAi knockdown of Gαq in the MBns produces a selective deficit in forgetting. These results reconcile the dilemma of how DA stimulates both acquisition and forgetting, with acquisition occurring through a dDA1/Gαs/cAMP pathway and forgetting through Damb/Gαq/Ca2+.
Discussion Here we provide biochemical and behavioral evidence that the Drosophila DA receptor Damb couples preferentially to Gαq to mediate signaling by Damb for active forgetting. This conclusion offers an interesting Z-DEVD-AFC to the role of the dDA1 receptor in MBns for acquisition, and it resolves the issue of how MBns distinguish DA-mediated instructions to acquire memory versus those to forget. Prior studies (Sugamori et al., 1995, Han et al., 1996, Reale et al., 1997) had classified both dDA1 and Damb as cAMP-stimulating receptors, similar to mammalian D1/D5 DA receptors that work through Gαs/olf. Our results extend prior studies of dDA1 by examining coupling of this receptor with multiple heterotrimeric G proteins to show that the receptor strongly and preferentially couples to Gs proteins. This affirms the receptor’s role in the acquisition of memory (Kim et al., 2007), consistent with the tight link between acquisition and cAMP signaling (Davis, 2005, Tomchik and Davis, 2009). We found that the Damb receptor can weakly couple to Gs proteins but preferentially engages Gq to trigger the Ca2+-signaling pathway, a feature not displayed by dDA1. Comparing the two Gαq paralogs of Drosophila (G and D) with a human ortholog shows that Drosophila GαqG and human Gαq share a conserved C terminus, essential for selective coupling to GPCRs, but quite distinct in sequence compared to the GαqD C terminus (Figure S3). Since GαqD is a photoreceptor-selective G protein that couples with rhodopsin (Lee et al., 1994), we propose that GαqG is the isoform that relays Damb’s signals to spur forgetting. We envision that memory acquisition triggered by strong DA release from electric shock pulses used for aversive conditioning drives both cAMP and Ca2+ signaling through dDA1 and Damb receptors in the MBns (Figure S4). Forgetting occurs from weaker DA release after the acquisition through restricted Damb/Gαq/Ca2+ signaling in the MBns. The coupling of Damb to Gs at high DA concentrations also explains why Damb mutants have a slight acquisition defect after training with a large number of shocks (Berry et al., 2012). Although the model allows the assignment of acquisition and forgetting to two distinct intracellular signaling pathways, it does not preclude the possibility that other differences in signaling distinguish acquisition from forgetting. These include the possible use of different presynaptic signals, such as a co-neurotransmitter released only during acquisition or forgetting.