The RIM/NIM Family of Neuronal C2 Domain Proteins

RIM1 is a putative effector protein for Rab3s, synaptic GTP-binding proteins. RIM1 is localized close to the active zone at the synapse, where it interacts in a GTP-dependent manner with Rab3 located on synaptic vesicles. We now describe a second RIM protein, called RIM2, that is highly homologous to RIM1 and also expressed primarily in brain. Like RIM1, RIM2 contains an N-terminal zinc finger domain that binds to Rab3 as a function of GTP, a central PDZ domain, and two C-terminal C2 domains that are separated by long alternatively spliced sequences. Unexpectedly, the 3′-end of the RIM2 gene produces an independent mRNA that encodes a smaller protein referred as NIM2. NIM2 is composed of a unique N-terminal sequence followed by the C-terminal part of RIM2. Data bank searches identified a third RIM/NIM-related gene, which encodes a NIM isoform referred to as NIM3; no RIM transcript from this gene was detected. To test if NIMs, like RIMs, may function in secretion, we investigated the effect of NIM3 on calcium-triggered exocytosis in PC12 cells. NIM3 induced a dramatic increase in calcium-evoked exocytosis (50%), with no significant effect on base-line release, suggesting that NIMs, like RIMs, regulate exocytosis The combination of conserved and variable sequences in RIMs and NIMs indicates that the individual domains of these proteins provide binding sites for interacting molecules during exocytosis, as shown for the zinc finger domain of RIM, which binds to GTP-bound Rab3s. To search for additional interacting proteins for RIMs, we employed yeast two-hybrid screens with the C-terminal half of RIM1. Two members of a new family of homologous brain proteins, referred to as RIM-binding proteins (RIM-BPs), were identified. RIM-BPs bind to RIM in yeast two-hybrid and GST pull-down assays, suggesting a specific interaction. In RIMs, the binding site for RIM-BPs consists of a conserved proline-rich sequence between the two C2domains, N-terminal to the beginning of NIMs. RIM-BPs are composed of multiple domains, including three fibronectin type III-domains and three Src homology 3 domains, of which the second Src homology 3 domain binds to RIMs. With the RIM-BPs, we have identified a partner for RIMs that may bind to RIMs at the synapse in addition to Rab3.