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Arc, a Growth Factor and Activity-Regulated Gene, Encodes a Novel C Oskeleton-Associated Protein That Is Enriched in Neuronal Dendrites

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Arc, a Growth Factor and Activity-Regulated Gene, Encodes a Novel C Oskeleton-Associated Protein That Is Enriched in Neuronal Dendrites Neuron, Vol. 14, 433-445, February, 1995, Copyright © 1995 by Cell Press Arc, a Growth Factor and Activity-Regulated Gene, Encodes a Novel C oskeleton-Associated Protein That Is Enriched in Neuronal Dendrites Gregory L. Lyford,* 1 Kanato Yamagata,*l sistent with classical studies of learning and memory that Walter E. Kaufmann,t I Carol A. Barnes,~ demonstrate a requirement for protein synthesis in long- Laura K. Sanders,§ Neal G. Copeland,ll term, but not short-term, memory (Flexner et al., 1963; Debra J. Gilbert,II Nancy A. Jenkins, II Agranoff, 1981; Davis and Squire, 1984). Anthony A. Lanahan,* and Paul F. Worley*t Insight into genomic mechanisms that might underlie *Department of Neuroscience long-term plasticity initially came from studies of growth 1"Department of Neurology factor signaling in nonneuronal tissues. Growth factor §Howard Hughes Medical Institute stimulation induces the rapid and transient expression of and Department of Molecular Biology and Genetics a set of genes, termed immediate-early genes (lEG), that Johns Hopkins University School of Medicine encode transcription factors and cytokines, as well as Baltimore, Maryland 21205 other molecules, that are believed to regulate long-term tDepartment of Psychology and Neurology cellular responses (reviewed by Lau and Nathans, 1991). and Division of Neuronal Systems, Memory, and Aging Similar rapid genomic responses are induced in neurons University of Arizona by neurotransmitter stimulation (Greenberg et al., 1986; Tuscon, Arizona 84724 Sheng and Greenberg, 1990). Kandel and coworkers have IIMammalian Genetics Laboratory used a simplified cultured system of invertebrate neurons ABL-Basic Research Program to demonstrate an essential role for both mRNA and pro- NCI-Frederick Cancer Research and Development Center tein synthesis in long-term synaptic plasticity (Montarolo Frederick, Maryland 21702 et al., 1986) and a direct role for the lEG transcription factor CCAAT enhancer-binding protein (Alberini et al., 1994). Summary To identify components of the genetic program underly- ing long-term responses in the vertebrate brain, we and Neuronal activity is an essential stimulus for induction others have used differential cloning techniques to identify of plasticity and normal development of the CNS. We mRNAs that are rapidly induced by excitatory activity have used differential cloning techniques to identify a (Nedivi et al., 1993; Qian et al., 1993; Yamagata et al., novel immediate-early gene (lEG) cDNA that is rapidly 1993, 1994a, 1994b). In addition to transcription factors induced in neurons by activity in models of adult and (Yamagata et al., 1994a), this approach has identified a developmental plasticity. Both the mRNA and the en- number of lEGs that encode enzymes that may directly coded protein are enriched in neuronal dendrites. modify cellular function, including tissue plasminogen acti- Analysis of the deduced amino acid sequence indi- vator (Qian et al., 1993), an inducible form of cyclooxygen- cates a region of homology with ~x-spectrin, and the ase (Yamagata et al., 1993), and a novel homolog of H-Ras full-length protein, prepared by in vitro transcription/ (Yamagata et at., 1994b). These proteins presumably in- translation, coprecipitates with F-actin. Confocal mi- teract with existing cellular proteins to modify properties croscopy of the native protein in hippocampal neurons of the neuron and effect long-term changes in cellular func- demonstrates that the lEG-encoded protein is en- tion. Here, we describe a novel lEG, which encodes a riched in the subplasmalemmal codex of the cell body protein that exhibits homology to the structural protein and dendrites and thus colocalizes with the actin cy- ~-spectrin and is localized to neuronal dendrites. We have toskeletal matrix. Accordingly, we have termed the termed the gene Arc (activity regulated cytoskeleton- gene and encoded protein Arc (activity-regulated cy- associated protein). Because its mRNA and protein ex- toskeleton-associated protein). Our observations sug- pression are tightly coupled to the activity state of the gest that Arc may play a role in activity-dependent neuron, we hypothesize that Arc may play a role in activity- plasticity of dendrites. dependent changes in dendrite function. Introduction Results Recent studies support the concept that neuronal plastic- Arc cDNA Sequence ity involves two temporally distinct components (Goelet et A novel cDNA corresponding to the 3' noncoding region al., 1986; Nguyen et al., 1994). Transmitter stimulation of a - 3.2 kb m RNA was identified by differential screening results in rapid membrane ionic conductance changes and of a subtracted cDNA library prepared from seizure- associated protein phosphorylation events that underlie stimulated hippocampus (Yamagata et al., 1993). A near short-term cellular changes lasting minutes to hours. By full-length cDNA was identified by iterative screening of contrast, long-term plasticity is distinguished from short- a brain cDNA library prepared in our laboratory. The size term plasticity in that it requires the synthesis of new of the cDNA (3032 bp; Figure 1) corresponds closely to mRNA and proteins. This mechanistic framework is con- the estimated size of the mRNA as determined by Northern analysis. The longest open reading frame (ORF) predicts 1These authors made nearly equivalent contributionsto this paper. a 396 amino acid protein. Translation stop sites are pres- Neuron 434 ~T~TGG~GAGTA~T~CT~ccT~A~cc~AGT~7~c~c~T~TT~AG~TTc~ccc~GA~G~A~A~Tcc~G~T 90 ~A~G~A~TT~A~T~T~c~TT~G~c~AG~G~T~TG~T~TTcTG~TTc~c~cAGAGGAGT 180 translated and coding regions. TNT of the full length cDNA 0 10 HELDH~TTGGL~AYpApR TCTTAGCCTGTCcCG~CC~T~CCCC~CGAGCAGAT~GAGCTGGACCATA~ACGACCGGcGGCCTCCAC~CTACCCTGcCCC~ 270 resulted in synthesis of a protein that migrated on SDS- 2O 30 4O ~Gp~AKPNVILOIGKCRAE~V~T*H~ polyacrylamide gel electrophoresis (SDS-PAGE) with an SO ~0 ~0 apparent molecular weight of 55 kDa (see Figure 9). TNT of VPTGDS'QRWKKS*IKACLCRC~ETIANLERW cDNAs restricted at sites within the predicted ORF yielded products of reduced size, consistent with the position of ~TcAAGcGTGA~ATGcAcG~GT~As~A~T~TTcTA~sT~TGGAGAsGT~c~Acc~cTGsAGT~AT~GGc~GTAcccA 63O restriction sites (data not shown). Additionally, TNT of a GTGGG~AG~GA~G~CC~CACACTGTCT~TGTAGGTG~G~T~CAGAG~TA~T~CAGG~T~ATGGCTAC~TA~A~T 720 cDNA fragment lacking the putative translation start site VSPYAITpppAAGELP~QESVGAQQYQS+WV GTTA~ccTAT~A~A~CC~G~A~CT~c~GcAGGA~AG~TG~cTGA~A~GTcAGTTG~G~T~Asc~A~AGTcTTGsGTG 810 but containing other 3' methionines yielded no product. PGEDSQPSPGLDTQXFEDPR~FLSHL~EYL ccAGs~AGGA~G~c~c~=cccAGGTcT~ATAcccA~AT~T~AssAcc~Ae~GAsTTcc~sAGccAcCT~GAsTAc~T~ 900 Further confirmation that the identified ORF is correct was obtained by generating antisera against a bacterial fusion protein that recognizes an inducible protein in hippocam- ~TcGGTG~s~cTGsGTGGAGTTc~G~AGTTzcTscAGTAcA~TGA~GTAc~T~T~c~cG~cAT~AGC~GGA~Ts~8~ DLpQKQGEpLDQFLW~KRDLYQTLYVDAEE pus that is very similar in size to the TNT product (see GAc~TGccAcAG~A~G~GA~ccAcTT~A~cAGT~cTcT~GT~c~GAcc~YaccAGAcAc~GTATGT~Ac~CT~A~As I170 EEIIQYVVGTLQPKFKRFLRHPLFKTLEQL below). GAGGAGA~A~CAGTATGT~TG~cAC~TGcAG~C~GTTC~G~TTT~TGCGCCACCCACTT~Cc~GACCCTCGAGCA~TC 1260 We compared the predicted Arc amino acid sequence IQRGMEVQDGLEQAAEPSVTPLPTEDETEA ATCCAGA~GGCATGG~GTTCAGGAC~CCTGGAGCAG~A~TGAGCCTTCTGTCACCCCTCTGCCCACAGAGGATGAGACTGA~CA]35~ ~0 s~o with the GenBank database to identify homologous pro- CTCACGCCTGCTCTTACCAGCCAGTCAGTAGCCAGTGACAG~ACCCAGCCTG~TAGA~GCCAGCCCAGGGTCCCCAGCCTGCCTGCC 1440 ACACCCAGTCTGTGGCTT¢TGTC~CTACGACTTGATTGAG~T~GGCT~CACCC~ATGCCCTCTCCAGCCAGACACCTTCTCA 1530 teins. The closest homology identified is with ~-spectrin CCTCACCTGT~TG~CCTAGTCCTG~CCTGTCTCCAGT~GCCTCACCCTCTACACTCTCAGACCATCACAG~CACCTTT~CT~CTCA 1710 (Wasenius et al., 1989; Moon and McMahon, 1990) (Fig- T TC T~ATCAGTGTCCAGG~CC CTTTG~ TAGTC~G~TC~CTGTCTG~GGC~G TAG~ACC~C C~GGC~A T C 1800 ure 2). The region of homology spans 156 amino acids in CGCT~C~CTCCT~CCCCCAGCAGTGATTCATACCAGT~G~GCAGACTTCGGCTCCATGACTCAGCCATGCCA~GCGGA~GT 2070 CCCAGA~GCTGAGTCC~AGCCCCA~TGA~CAGCAGCT~AGTCT~AGAGCC~TG~TGACACCA~TCTC~TGCTCAGA 2160 the carboxyl terminal half of Arc (amino acids 155-316), CACTTTC~AGAG~TAGGATTAC~GA~AGGTGCCTTGGCA~G~CAGCACCCAGCTCA~CTCAGA~TG~GGTG~GA 2340 C~GCCA~GTG~CCCC~GTCTG~CACG~T~CC¢CTCCGCTGGCCAC~ACCA~T~CTGCCAC~GCCACT~AGCTTGAGCA243~ where it is 20% identical to the twenty-first and twenty- CACTTCCCTGACCTGCC~G~AT~CCCAGCT~CC~ATCCACACCCCAGCACC~CCACCTCGT~TTGGT~CTGCTCGTGTCTG 2700 second repeats of ~-spectrin. Though this level of identity is generally too low to be predictive of function, we noted that the degree of identity between repeats in c~-spectrin is Figure 1. Nucleotide and Amino Acid Sequences of Arc typically only 20% (Wasenius et al., 1989). Each a-spectrin Numbers to the right refer to last nucleotide in each line. Predicted repeat is believed to form a tri-a-helix bundle that is stabi- amino acid sequence is displayed above corresponding nucleotide lized by hydrophobic, leucine zipper-like interactions with sequence, and numbersfor amino acids are indicatedabove. Asterisk neighboring coils (Yan et al., 1993). Amino acids that are indicates predicted phosphorylation sites for protein kinase C; plus sign indicates predicted phosphorylation sites for calcium/calmodulin conserved between spectrin repeats correspond to sites dependent kinase I1. ~o%T~0% polyadenylation signal and ATIrA of protein-protein interaction between coils. Because mRNA instability signals are underlined. many of the amino acid differences between a-spectrin and Arc represent conservative changes, the overall level of homology in this region is 77%. Accordingly, the de- duced amino acid sequence of Arc suggests that it pos- ent in all three reading frames 5'to the putative translation sesses a spectrin-like domain that may be important in start site, and the nucleotide sequence surrounding the forming intermolecular interactions. A separate search of predicted initiating methionine conforms to the Kozak con- GenBank using only the NH2 terminal half of Arc was unin- sensus for efficient translation, including a 5' GC-rich re- formative.
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