Rim1 and Rabphilin-3 Bind Rab3-GTP by Composite Determinants Partially Related through N-terminal α-Helix Motifs

Abstract

Rim1 is a protein of the presynaptic active zone, the area of the plasma membrane specialized for neurotransmitter exocytosis, and interacts with Rab3, a small GTPase implicated in neurotransmitter vesicle dynamics. Here, we have studied the molecular determinants of Rim1 that are responsible for Rab3 binding, employing surface plasmon resonance and recombinant, bacterially expressed Rab3 and Rim1 proteins. A site that binds GTP-but not GDP-saturated Rab3 was localized to a short ␣-helical sequence near the Rim1 N terminus (amino acids 19 -55). Rab3 isoforms A, C, and D were bound with similar affinities (K d ‫؍‬ 1-2 M). Low affinity binding of Rab6A-GTP was also observed (K d ‫؍‬ 16 M), whereas Rab1B, -5, -7, -8, or -11A did not bind. Adjacent sequences up to amino acid 387, encompassing differentially spliced sequences, the zinc finger module, and the SGAWFF motif of Rim1, did not significantly contribute to the strength or the specificity of Rab3 binding, whereas a point mutation within the helix (R33G) abolished binding. This Rab3 binding site of Rim1 is reminiscent of the N-terminal ␣-helix that is part of the Rab3-binding region of rabphilin-3, and indeed we observed low affinity, specific binding of Rab3A (K d on the order of magnitude of 10 -100 M) to this region of rabphilin-3 alone (amino acids 40 -88), whereas additional sequences up to amino acid 178 are needed for high affinity Rab3A binding to rabphilin-3 (K d ‫؍‬ 10 -20 nM). In contrast, an N-terminal ␣-helix motif in aczonin, with sequence similarity to the Rab3-binding site of Rim1, did not bind Rab3A, -C, or -D or several other Rab proteins. These results were qualitatively confirmed in pull-down experiments with native, prenylated Rab3 from brain lysate in Triton X-100. Munc13 bound to the zinc finger domain of Rim1 but not to the rabphilin-3 or aczonin zinc fingers. Pull-down experiments from brain lysate in the presence of cholate as detergent detected binding to downstream Rim1 sequences, between amino acids 56 and 387, of syntaxin and of Rab3. The latter, however, was inhibited rather than stimulated by GTP.

A Protein Interaction Node at the Neurotransmitter Release Site: Domains of Aczonin/Piccolo, Bassoon, CAST, and Rim Converge on the N-Terminal Domain of Munc13-1

“…SSS) precipitated native CAST, Munc13, and Rim. As positive controls, fusion proteins with the zinc-finger domain of Rim1 precipitated Munc13 only (Betz et al, 2001;Wang et al, 2001). Confirming the specificity and avidity of these interactions, no other Aczonin binding partners could be detected in the precipitates with antibodies against numerous additional presynaptic candidate proteins [Aczonin itself, synapsin-1 (Fig.…”

A family of RIM-binding proteins regulated by alternative splicing: Implications for the genesis of synaptic active zones

“…Genetic studies in mice and worms showed that RIMs regulate active zone function (5-7), but their precise action is incompletely understood (33). Important binding partners for the N-terminal Zn 2ϩ -finger domain [Rab3 and Munc13-1 (3,4,6,8,9)] and the C-terminal C 2 B domain of RIMs [␣-liprins and synaptotagmin 1 (6,14)] were isolated, but the significance of their highly conserved central PDZ domains is unclear. In the present study, we have characterized a family of proteins that bind to these PDZ domains, and that we refer to here by the acronym ''ERC'' based on previous separate namings of members of this protein family, in the order in which they were described: ELKS, which is involved in a chromosomal translocation (15); Rab6IP2, which interacts with rab6 (16); and CAST, which is localized to the active zone (17).…”

“…Several binding partners were identified. The Nterminal Zn 2ϩ -finger domain of RIM1␣ directly binds to Rab3 [which led to the initial discovery of RIMs (3,4)] and to the N terminus of Munc13-1, another active zone protein that functions in neurotransmitter release (6,8,9). A proline-rich sequence in the linker between the C 2 A and C 2 B domain interacts with the SH3 domain of brain-specific adaptor proteins called RIM-BPs (4) that have been implicated in Ca 2ϩ -channel regulation (10).…”

Association between regulating synaptic membrane exocytosis 2 gene polymorphisms and degenerative lumbar scoliosis

“…The α-RIMs (RIM1α and RIM2α) contain the full complement of domains which are the N-terminal Zn 2+ -finger domain, the central PDZ and C 2 A domains and the C-terminal C 2 B domain (15). They are able to bind to Rab3 and Munc13 proteins (13, 21,22) and closely interact with α-liprins (13), which in turn regulate the active zone structure (23,24). Therefore, α-RIMs are considered to be essential for regulating neurotransmitter releases (8,13,25,26), some of which are involved in the neurobiology of schizophrenia (27,28).…”