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内容記述 |
The CRMP (collapsin response mediator protein) family, a family of cytoplasmic proteins predominantly expressed in the developing nervous system, is thought to play key roles in the growth cone guidance． CRMP was first isolated as a factor required for collapsin-1 (Semaphorin3A)-mediated signaling． Four members (CRMP1-4) of the family have been demonstrated to form hetero-multimeric structures through mutual associations． In this study, a novel member of this family, CRMP5, was cloned by the yeast two-hybrid method, and characterized from various aspects．<br />A search for molecules topographically expressed in the embryonic chick retina using Restriction Landmark cDNA Scanning (RLCS) was performed to identify molecules implicated in regional specificity in the retina and in the topographic retinotectal projection． Among a number of molecules thus isolated, CRMP3 was identified as being asymmetrically expressed along the nasotemporal (anteroposterior) axis, although this expression pattern was transient during retinal development．<br />To identify CRMP3-interacting molecules, I performed a yeast two-hybrid screen of a mouse brain cDNA library using chick CRMP3 as bait． In this screening, sixty-one clones, mainly containing CRMP1, 3, 4 and dihydropyrimidinase (DHPase), were isolated． Among them, I found a novel clone which is homologous to the CRMP isoforms already known．I termed this molecule CRMP5． This protein consists of 564 amino acids and has a calculated molecular mass of 61,516Da．When poly (A)+ RNA was analyzed, two bands of 4.8 and 5.2 kb, probably derived from a difference in poly (A) addition sites, were detected． CRMP5 bears several consensus sequences for phosphorylation sites that are conserved among the family, indicating that CRMP5 is also a phosphoprotein as are the other CRMP isoforms． CRMP5 shares relatively low amino acid identity with the other CRMPs (49-50%) and also with DHPase (51%), though CRMP1-4 exhibit higher identity with each other (68-75%)． An unrooted phylogenic tree of CRMP isoforms suggested that CRMP5 might be classified to a subfamily distinct from the four other CRMP members． This notion was supported by the genomic structure of CRMP5 because the exon-intron organization is not completely conserved with that of CRMP1 and CRMP2, or DHPase either． To determine the chromosomal location of the mouse CRMP5 gene, fluorescence in situ hybridization was performed using a mouse CRMP5 genomic DNAfragment as a probe． The CRMP5 gene was mapped to chromosome 5 B1, a region that shares homology with human chromosome 7q． Recently, chromosomal locations of the human CRMP family (CRMP1-4) and DHPase genes were determined． These results indicate that CRMPs loci are widely dispersed throughout the human and mouse genome．<br />To reveal the expression profile of mouse CRMP5, I performed Northern blot analysis and in situ hybridization． Northern blot analysis of various mouse tissues indicated that CRMP5 mRNA is expressed in the central nervous system but not in non-neural tissues． The CRMP5 expression profile during development resembled that of both CRMP1 and CRMP4, in that the expression level peaked in the first postnatal week and markedly decreased in adulthood． To reveal in more detail the expression patterns in mice,in situ hybridization analysis was performed on developing embryonic sections． CRMP5 was expressed specifically in the nervous system, and signals were detected from the retina, olfactory epithelium, spinal cord, dorsal root ganglion, sympathetic ganglion, intestinal nerve, and brain with especially strong signals in the neocortex． This expression pattern is almost identical to that of CRMP4．<br />In contrast to the rapid progress in identification and characterization of the axon guidance molecules and their receptors, much remains to be explored about the intracellular mechanism by which signals are transduced into the eventual response of the growth cone． It has been reported that CRMPs form hetero-tetrameric structures． Consistently, I have identified other CRMPs by a two-hybrid screen using CRMP3 as mentioned above． Therefore, I tested the associations between CRMP5 and all CRMP isoforms． In the yeast two-hybrid system, CRMP5 interacted with each CRMP member with high affinity, except for CRMP1 and CRMP5． The same result was obtained from immunoprecipitation assays using an epitope-tagged expression system in COS-7 cells． Next, I tested the interaction between the CRMP isoforms in all combinations by immunoprecipitation． CRMP3 showed very low association with CRMP1, CRMP3 and CRMP4, similar to that between CRMP5 itself． This means that endogenous CRMP tetramers are composed in combinations of 1/2/4, 2/3/5 and 2/4/5．CRMP5 mRNA was upregulated as was CRMP4 mRNA in PC12h cells treated with NGF, suggesting that CRMP4 and CRMP5 are responsible for the neurite extension． Consistent with this observation, CRMP5 expression increases when the neural network forms during development． CRMP complexes with different isoforms may exert distinct intracellular signalings from the extracellular signal, and explain the variegated responses of axons from different origins． |
要旨・審査要旨 / Abstract, Screening Result (286.3 kB)
