Molecular and Functional Characterization of Protein 4.1B, a Novel Member of the Protein 4.1 Family with High Level, Focal Expression in Brain

Narla Mohandas,Philippe Gascard,Nadine Chan,Trish Berger,Solomon H. Snyder,Gloria Lee,J.Aura Gimm,Marilyn Parra,Joel A. Chasis,Loren D. Walensky,Yuichi Takakuwa,John G. Conboy,Seth Blackshaw

doi:10.1074/jbc.275.5.3247

Narla Mohandas, Philippe Gascard + Show 11 more

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https://doi.org/10.1074/jbc.275.5.3247

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Abstract

Brain-enriched isoforms of skeletal proteins in the spectrin and ankyrin gene families have been described. Here we characterize protein 4.1B, a novel homolog of erythrocyte protein 4.1R that is encoded by a distinct gene. In situ hybridization revealed high level, focal expression of 4.1B mRNA in select neuronal populations within the mouse brain, including Purkinje cells of the cerebellum, pyramidal cells in hippocampal regions CA1-3, thalamic nuclei, and olfactory bulb. Expression was also detected in adrenal gland, kidney, testis, and heart. 4.1B protein exhibits high homology to the membrane binding, spectrin-actin binding, and C-terminal domains of 4.1R, including motifs for interaction with NuMA and FKBP13. cDNA characterization and Western blot analysis revealed multiple spliceoforms of protein 4.1B, with functionally relevant heterogeneity in the spectrin-actin and NuMA binding domains. Regulated alternative splicing events led to expression of unique 4. 1B isoforms in brain and muscle; only the latter possessed a functional spectrin-actin binding domain. By immunofluorescence, 4. 1B was localized specifically at the plasma membrane in regions of cell-cell contact. Together these results indicate that 4.1B transcription is selectively regulated among neuronal populations and that alternative splicing regulates expression of 4.1B isoforms possessing critical functional domains typical of other protein 4.1 family members.

Highlights

The protein 4.1 family comprises a group of skeletal proteins structurally related to the erythroid membrane skeletal protein, 4.1R, that plays a critical role in determining the morphology and mechanical stability of the red cell plasma membrane
4.1R has been localized to specific neuronal populations including granspectrin-actin binding; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-PCR; PBS, phosphate-buffered saline; BSA, bovine serum albumin; kb, kilobase(s); Epb4.1l3, erythrocyte protein band 4.1-like gene 3
The 12-amino acid peptide encoded by this exon (Fig. 4A) is not homologous to any sequence of the 4.1R. Together these results strongly suggest that alternative splicing mediates muscle-specific expression of 4.1B isoforms that can interact with spectrin and actin, whereas 4.1B isoforms expressed in brain and other tissues are likely to exhibit distinct function(s)

Summary

Introduction

The protein 4.1 family comprises a group of skeletal proteins structurally related to the erythroid membrane skeletal protein, 4.1R, that plays a critical role in determining the morphology and mechanical stability of the red cell plasma membrane. These proteins are characterized by the presence of three main conserved structural/functional domains. Transcriptional regulation of three recently discovered 4.1 genes contributes further heterogeneity to the complement of 4.1 isoforms expressed in a given tissue Among these are protein 4.1G, a widely expressed homolog [14, 26]; protein 4.1N, a neuronal homolog [27]; and protein 4.1B, the novel brain-enriched homolog reported here. 4.1R has been localized to specific neuronal populations including granspectrin-actin binding; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-PCR; PBS, phosphate-buffered saline; BSA, bovine serum albumin; kb, kilobase(s); Epb4.1l3, erythrocyte protein band 4.1-like gene 3 (designated as protein 4.1B in this paper)

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