The guanine-exchange factor Ric8a binds the calcium sensor NCS-1 to regulate synapse number and probability of release

Abstract

The conserved Ca 2+ -binding protein Frequenin (homolog of the mammalian NCS-1, neural calcium sensor) is involved in pathologies that result from abnormal synapse number and probability of neurotransmitter release per synapse. Both synaptic features are likely to be co-regulated but the intervening mechanisms remain poorly understood. We show here that Drosophila Ric8a (a homolog of mammalian synembryn, which is also known as Ric8a), a receptor-independent activator of G protein complexes, binds to Frq2 but not to the virtually identical homolog Frq1. Based on crystallographic data on Frq2 and site-directed mutagenesis on Frq1, the differential amino acids R94 and T138 account for this specificity. Human NCS-1 and Ric8a reproduce the binding and maintain the structural requirements at these key positions. Drosophila Ric8a and Gas regulate synapse number and neurotransmitter release, and both are functionally linked to Frq2. Frq2 negatively regulates Ric8a to control synapse number. However, the regulation of neurotransmitter release by Ric8a is independent of Frq2 binding. Thus, the antagonistic regulation of these two synaptic properties shares a common pathway, Frq2-Ric8a-Gas, which diverges downstream. These mechanisms expose the Frq2-Ric8a interacting surface as a potential pharmacological target for NCS-1-related diseases and provide key data towards the corresponding drug design.

Interference of the complex between NCS-1 and Ric8a with phenothiazines regulates synaptic function and is an approach for fragile X syndrome

“…We reported previously that Drosophila NCS-1 (dNCS-1; also known as Frq2) and Ric8a interact to regulate synapse number and neurotransmitter release and that this interaction is reproduced by the human homologs (19). Although the structure of the NCS-1/Ric8a complex is unknown, our structural data on Frq2 followed by a mutagenesis study shed light on the molecular mechanism of Ric8a recognition by NCS-1 (19). We found that R94, located at the N-terminal edge of a hydrophobic crevice, is essential for the interaction with Ric8a (Fig.…”

“…However, normal neuronal function requires tight control of probability of neurotransmitter release per synapse in addition to control of synapse number. Indeed, both neuronal properties are coregulated in an antagonistic manner (18,19). Neurons with a high number of release sites usually manifest a low probability of release per site and vice versa.…”

“…Thus, a potentially effective approach should target the signaling mechanism of this coregulation. Recently, we described the mechanism by which the Ca 2+ sensor neuronal calcium sensor 1 (NCS-1; originally named Frequenin in Drosophila) interacts with the guanine exchange factor protein Ric8a to activate Gα proteins and coregulate synapse number and activity (18,19). Interestingly, vertebrate NCS-1 mRNA has been identified as one of the FMRP targets (5), and Frequenin mRNA expression is decreased in the Drosophila fmr1 mutant (20).…”

“…The available structural information on NCS-1/Ric8a recognition (19) and the function of the complex in synapse number control and probability of release led us to search for small compounds that could dock into the NCS-1/Ric8a interface to inhibit complex formation. We hypothesized that such compounds will decrease synapse number in FXS animal models and eventually, patients.…”

“…, with the hydrophobic crevice in green; residues R94 and E26 in orange and blue, respectively; and helix H10 in lilac ribbon with D187 in sticks. The Drosophila ligand-free NCS-1 structures [4BY4 and 4BY5 (19)] have been superimposed, and helix H10, with its three different orientations inside or outside the crevice (orange and yellow ribbons, respectively), is shown to indicate its mobility. (B) Sequence alignment of human and Drosophila NCS-1 proteins.…”