Molecular characterization and regional localization of human microtubule-associated protein 2 (MAP-2)

Abstract

Microtubule-associated protein-2 (MAP-2) functions to maintain neuronal morphology by promoting the assembly of microtubules. Through the isolation of overlapping cDNA clones, the full-length transcripts encoding human high-molecular weight (HMW) MAP-2 and MAP-2c were characterized. By the polymerase chain reaction and sequencing, the 6 kb MAP-2c transcript was shown to be generated by alternate splicing of the 9 kb HMW MAP-2 mRNA.;Isolation of genomic clones has demonstrated that the region spliced out of MAP-2c is encoded by two exons specific to HMW MAP-2. Additional overlapping genomic clones define the intron/exon structure of three of the four imperfect amino acid repeats of the microtubule-binding domain. The fourth repeat is encoded in a single, alternatively spliced exon which is located between the exons encoding the first and second repeats. The second repeat of the microtubule-binding domain is encoded in two exons whereas a single exon encodes the third repeat. The end of the coding sequence, the translational stop site and the 3{dollar}\sp\prime{dollar} untranslated region are contained within the final exon.;Using fusion protein constructs, the epitopes of several monoclonal antibodies have been defined to discrete regions of human MAP-2. To determine the cellular localization of MAP-2c, a MAP-2c-specific polyclonal antibody was generated to a thirteen amino acid synthetic peptide of the MAP-2c splice site. The MAP-2c antiserum was depleted of HMW MAP-2 reactivity by absorption with HMW MAP-2 fusion protein. Western blot analysis demonstrates that the antibody is specific for human MAP-2c. To localize MAP-2c to dendrites or axons, double-label confocal microscopy was performed on fetal spinal cord sections. HMW MAP-2 and MAP-2c co-localize in the cell bodies and dendrites of anterior motor neurons and immunoelectron microscopy showed MAP-2c associated with microtubules in these dendrites. MAP-2c and the neurofilament proteins are found in axons of the dorsal and ventral roots. The presence of MAP-2c within axons and dendrites suggests that MAP-2c contributes to neuronal plasticity during human fetal development.