A study on 4.1 proteins in brain.

The 4.1 family consists of a multi-functional group of proteins expressed from four distinct genes. 4.1R, 4.IN, 4.1G and 4.IB have distinct mammalian tissue and developmental expression patterns and each protein exists as multiple isoforms produced by alternative splicing of their respective genes. Functions as diverse as signalling, regulation of pre-mRNA splicing, mitotic spindle rearrangements and anchoring of membrane proteins have been suggested for these proteins based on their tissue and subcellular distribution and the protein binding partners identified. 4.1R was the first family member to be discovered and was identified as a component of the erythrocyte membrane skeleton important for the integrity of these cells.

The role of 4.1 proteins in the brain is of particular interest. All four proteins are expressed but each has a distinct regional localisation. Limited information is available on the subcellular distribution of 4.1 proteins in brain and other tissues. To begin to address this issue, the complement of 4.1 protein isotypes was investigated in postsynaptic density (PSD) fractions of mammalian forebrain. 4.1R was shown to be expressed predominantly in cell bodies and dendrites of primary rat midbrain cultures supporting a postsynaptic distribution for this 4.1 gene product. Antibodies specific to 4.1R, 4. IN and 4.1G and an antibody recognising 4.1R, G and B localised all four 4.1 proteins in PSD fractions. Of these, two 4.IB isoforms (4.1B150kDa and 4.1Bl26kDa) were most abundant while the single 4.1R isoform identified (4.1R80kDa) was most highly enriched.

To investigate the postsynaptic binding activities of 4.1R, recombinant 4.1R CTerminal Region (4.1R-CTR) was produced and shown to mimic characteristic spectrin-actin binding and digestion properties of native 4.1R. One and two dimensional gel overlay analysis indicated binding of 4.1R-CTR with dynamin, actin and the intermediate filament proteins a-internexin and NF-L. The identity of actin, ainternexin and NF-L was confirmed using protein specific antibodies. These interactions were supported by data from 4.1R-CTR affinity chromatography but the insolubility of PSDs made it difficult to study 4.1R-CTR-PSD protein interactions under native conditions.

Localisation of 4.1 at PSDs provides evidence supporting a role for these proteins at specialised membrane domains. This is consistent with the localisation of the Drosophila homologue of 4.1 (coracle) to septate junctions and the recently identified interaction of mammalian 4.1R with the tight junction protein ZO-2. The interaction of 4.1R-CTR with a-internexin and NF-L indicates a structural role for 4.1R at PSDs. The interaction with actin could in turn suggest a function for 4.1R in the dynamic morphological changes that accompany neuronal plasticity and is consistent with the role 4.1R plays in erythrocyte membrane deformation. The data presented gives further insights into the workings and architecture of PSDs and proposes novel interactions for the C-terminal region of 4.1R.