NORMAL GENOMIC

• Pang Z, Zuo J, Morgan JI
• Cbln3, a novel member of the precerebellin family that binds specifically to Cbln1.
• J Neurosci. 2000; 20: 6333-9
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• Precerebellin (Cbln1) is the precursor of the brain-specific hexadecapeptide cerebellin. Although cerebellin has properties of a conventional neuropeptide, its function is controversial because Cbln1 has structural features characteristic of circulating atypical collagens. Cbln1 is related to the three subunits of the complement C1q complex. Therefore, we hypothesized that Cbln1 participated in analogous heteromeric complexes with precerebellin-related proteins. Using LexA-Cbln1 as bait in a yeast two-hybrid screen, we isolated a cDNA encoding a novel Cbln1-related protein, designated Cbln3. The gene encoding cbln3 had the same intron-exon structure as cbln1 but mapped to a different mouse chromosome (14). The deduced amino acid sequence of Cbln3 was 55% identical to Cbln1 and also contained a C1q signature domain and signal sequence for secretion. In addition to binding avidly to Cbln3, Cbln1 also formed homomeric complexes. In contrast, Cbln3 homomeric association was weak. These interactions exhibited specificity because C1qB bound to neither Cbln1 nor Cbln3. Like cbln1, cbln3 was expressed in the cerebellum and dorsal cochlear nucleus in which it was detected in granule neurons. Because Cbln1 and Cbln3 are coexpressed in the brain and interact avidly, they may function as a secreted heteromeric complex in vivo.

• Davidson JD, Riley B, Burright EN, Duvick LA, Zoghbi HY, Orr HT
• Identification and characterization of an ataxin-1-interacting protein: A1Up, a ubiquitin-like nuclear protein.
• Hum Mol Genet. 2000; 9: 2305-12
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• Expansion of a polyglutamine tract within ataxin-1 causes spinocerebellar ataxia type 1 (SCA1). In this study, we used the yeast two-hybrid system to identify an ataxin-1-interacting protein, A1Up. A1Up localized to the nucleus and cytoplasm of transfected COS-1 cells. In the nucleus, A1Up co-localized with mutant ataxin-1, further demonstrating that A1Up interacts with ataxin-1. Expression analyses demonstrated that A1U mRNA is widely expressed as an approximately 4.0 kb transcript and is present in Purkinje cells, the primary site of SCA1 cerebellar pathology. Sequence comparisons revealed that A1Up contains an N-terminal ubiquitin-like (UbL) region, placing it within a large family of similar proteins. In addition, A1Up has substantial homology to human Chap1/Dsk2, a protein that binds the ATPase domain of the HSP70-like Stch protein. These results suggest that A1Up may link ataxin-1 with the chaperone and ubiquitin-proteasome pathways. In addition, these data support the concept that ataxin-1 may function in the formation and regulation of multimeric protein complexes within the nucleus.

• Wang Y, Sugita S, Sudhof TC
• The RIM/NIM family of neuronal C2 domain proteins. Interactions with Rab3 and a new class of Src homology 3 domain proteins.
• J Biol Chem. 2000; 275: 20033-44
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• 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 C(2) 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 C(2) domains, 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.

• Gallagher MJ, Burgess LH, Brunden KR
• Characterization of multiple forms of the human glycine transporter type-2.
• Brain Res Mol Brain Res. 1999; 70: 101-15
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• The human glycine transporter type 2 (hGlyT2) was cloned from a spinal cord cDNA library using PCR-based methodologies. The isolated sequence exhibits 89% homology with the previously isolated rat GlyT2 cDNA (Liu et al., J. Biol. Chem. 268 (1993) 22802-22808) at the nucleotide level, and 93% amino acid sequence identity. The greatest divergence between the human and rat sequences is found at the amino-terminus, where only 74% amino acid identity exists in residues 1-190. Expression of the intact hGlyT2 transporter sequence in COS-7 cells resulted in a 10-fold increase in high-affinity uptake relative to control cells transfected with vector alone. An artificially truncated form of the transporter, missing the NH2-terminal 153 amino acids, was also capable of mediating glycine uptake. However, an identified variant lacking the first 234 amino acids was non-functional. An hGlyT2 transporter containing a 14-residue deletion in the intracellular loop between transmembrane domains 6 and 7 was also identified and expressed, but failed to mediate glycine uptake. Like rat GlyT2, the high-affinity uptake mediated by hGlyT2 was found to be insensitive to the GlyT1 inhibitor sarcosine.

• Dodelet VC, Pazzagli C, Zisch AH, Hauser CA, Pasquale EB
• A novel signaling intermediate, SHEP1, directly couples Eph receptors to R-Ras and Rap1A.
• J Biol Chem. 1999; 274: 31941-6
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• The Eph family of receptor tyrosine kinases has been implicated in many developmental patterning processes, including cell segregation, cell migration, and axon guidance. The cellular components involved in the signaling pathways of the Eph receptors, however, are incompletely characterized. Using a yeast two-hybrid screen, we have identified a novel signaling intermediate, SHEP1 (SH2 domain-containing Eph receptor-binding protein 1), which is expressed in the embryonic and adult brain. SHEP1 contains an Src homology 2 domain that binds to a conserved tyrosine-phosphorylated motif in the juxtamembrane region of the EphB2 receptor and may itself be a target of EphB2 kinase activity, since it becomes heavily tyrosine-phosphorylated in cells expressing activated EphB2. SHEP1 also contains a domain similar to Ras guanine nucleotide exchange factor domains and binds to the GTPases R-Ras and Rap1A, but not Ha-Ras or RalA. Thus, SHEP1 directly links activated, tyrosine-phosphorylated Eph receptors to small Ras superfamily GTPases.

• Neeb A, Koch H, Schurmann A, Brose N
• Direct interaction between the ARF-specific guanine nucleotide exchange factor msec7-1 and presynaptic Munc13-1.
• Eur J Cell Biol. 1999; 78: 533-8
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• Msec7-1, a mammalian homologue of yeast sec7p, is a specific GDP/GTP exchange factor for small G-proteins of the ARF family. Overexpression of msec7-1 in Xenopus neuromuscular junctions leads to an increase in synaptic transmitter release that is most likely caused by an increase in the pool of readily releasable vesicles. However, the molecular mechanisms by which msec7-1 is targeted to presynaptic compartments and enhances neurotransmitter release are not known. In the present study, we demonstrate that msec7-1 interacts directly with Munc13-1, a phorbol ester-dependent enhancer of neurotransmitter release that is specifically localized to presynaptic transmitter release zones. Given that Munc13-1 and msec7-1 participate in very similar presynaptic processes and because Munc13-1 is specifically targeted to presynaptic active zones, we suggest that the msec7-1/Munc13-1 interaction serves to colocalize the two proteins at the active zone, a subcellular compartment with extremely high membrane turnover.

• Barhite S, Thibault C, Miles MF
• Phosducin-like protein (PhLP), a regulator of G beta gamma function, interacts with the proteasomal protein SUG1.
• Biochim Biophys Acta. 1998; 1402: 95-101
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• Phosducin-like protein (PhLP) and phosducin are highly homologous proteins that interact with the beta gamma subunits of guanine nucleotide binding proteins. While phosducin has a well-characterized role in retinal signal transduction, PhLP function remains unclear. To further understand the function of PhLP, we have examined other potential protein:protein interactions with PhLP using the yeast two-hybrid system. PhLP was found to interact with a mouse homologue of the yeast SUG1, a subunit of the 26S proteasome which may also indirectly modulate transcription. This interaction was further confirmed by an in vitro binding assay and co-immunoprecipitation of the two proteins in overexpression studies. Inhibition of proteasome function by lactacystin led to accumulation of high molecular weight, ubiquitin-immunoreactive protein precipitated by PhLP antiserum. We suggest that PhLP/SUG1 interaction may target PhLP for proteasomal degradation.