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J Cell Biol. 2000 Nov 27;151(5):985-1002.

betaIV spectrin, a new spectrin localized at axon initial segments and nodes of ranvier in the central and peripheral nervous system.

Berghs S, Aggujaro D, Dirkx R Jr, Maksimova E, Stabach P, Hermel JM, Zhang JP, Philbrick W, Slepnev V, Ort T, Solimena M.

Source

Department of Internal Medicine, Section of Endocrinology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.

Abstract

We report the identification of betaIV spectrin, a novel spectrin isolated as an interactor of the receptor tyrosine phosphatase-like protein ICA512. The betaIV spectrin gene is located on human and mouse chromosomes 19q13.13 and 7b2, respectively. Alternative splicing of betaIV spectrin generates at least four distinct isoforms, numbered betaIVSigma1-betaIVSigma4 spectrin. The longest isoform (betaIVSigma1 spectrin) includes an actin-binding domain, followed by 17 spectrin repeats, a specific domain in which the amino acid sequence ERQES is repeated four times, several putative SH3-binding sites and a pleckstrin homology domain. betaIVSigma2 and betaIVSigma3 spectrin encompass the NH(2)- and COOH-terminal halves of betaIVSigma1 spectrin, respectively, while betaIVSigma4 spectrin lacks the ERQES and the pleckstrin homology domain. Northern blots revealed an abundant expression of betaIV spectrin transcripts in brain and pancreatic islets. By immunoblotting, betaIVSigma1 spectrin is recognized as a protein of 250 kD. Anti-betaIV spectrin antibodies also react with two additional isoforms of 160 and 140 kD. These isoforms differ from betaIVSigma1 spectrin in terms of their distribution on subcellular fractionation, detergent extractability, and phosphorylation. In islets, the immunoreactivity for betaIV spectrin is more prominent in alpha than in beta cells. In brain, betaIV spectrin is enriched in myelinated neurons, where it colocalizes with ankyrin(G) 480/270-kD at axon initial segments and nodes of Ranvier. Likewise, betaIV spectrin is concentrated at the nodes of Ranvier in the rat sciatic nerve. In the rat hippocampus, betaIVSigma1 spectrin is detectable from embryonic day 19, concomitantly with the appearance of immunoreactivity at the initial segments. Thus, we suggest that betaIVSigma1 spectrin interacts with ankyrin(G) 480/270-kD and participates in the clustering of voltage-gated Na(+) channels and cell-adhesion molecules at initial segments and nodes of Ranvier.

PMID:: 11086001; [PubMed - indexed for MEDLINE]
PMCID:: PMC2174349

Free PMC Article

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Figure 1

(A) Schematic representation of human ICA512. The protein includes 979 amino acids, including a signal peptide (SP), a single transmembrane domain (TMD), and a cytoplasmic domain with an inactive PTP domain. Cleavage at a consensus site for furin-like convertases between amino acids 448 and 449 (arrow) gives rise to an NH₂-terminal fragment and a transmembrane fragment of 60–66 kD that includes the cytoplasmic domain. CHO, glycosylation sites. The residues in the PTP domain of ICA512 that distinguish it from active PTPs are indicated (A877, D911). (B–F) Interaction of the partial clone of βIV spectrin, B8, with ICA512cyt. (B and C) Yeast two-hybrid mating assay. L40 cells transformed with the prey plasmid pACTII-B8 were mated with AMR70 cells transformed with bait plasmids pLexA-ICA512cyt, pLexA-ICA512cyt AD/DA, pLexA-PHOGRINcyt, pLexA-lamin, or pLexA-MSS4 and grown on selective media in the absence (B) and presence (C) of 2 mM 3-AT. Cell survival reflects the interaction of the bait with the prey. (D and E) Expression levels of the different baits and B8 in L40-AMR70 mated cells as determined by Western blotting with an antiserum against LexA (D) and HA (E). (F) Coimmunoprecipitation of ICA512 and B8 from cotransfected COS cells. A mouse antibody directed against ICA512cyt or a rabbit antibody directed against the βIV spectrin specific domain (βIV-SD) were used for immunoprecipitation (IP) from Triton X-100 extracts of nontransfected (NT) and ICA512-B8 cotransfected (T) cells. Immunoprecipitates were Western blotted (WB) with affinity purified antibodies directed against the ectodomain of ICA512, βIV-SD, or a mouse antibody directed against HA. The arrows point to the immunoprecipitated ICA512 transmembrane fragment (ICA512 TMF) and B8, while a bracket indicates the position of pro-ICA512.

?iv Spectrin, a New Spectrin Localized at Axon Initial Segments and Nodes of Ranvier in the Central and Peripheral Nervous System

J Cell Biol. 2000 November 27;151(5):985-1002.

Figure 2

(A) Schematic diagram of the overlapping human βIV spectrin cDNAs forming the total contig of 8,789 bp. Additional clones that did not affect the contig have been left out of the alignment. (B) Graphical representation of the genomic organization of βIV spectrin. Black boxes represent the 36 exons encoding the full-length βIV spectrin. Exons from alternative spliced isoforms are boxed in grey. The length of the intervening sequences is still undetermined. Translation initiation and stop codons of the four alternative spliced variants are indicated. (C) Schematic representation of the domain structure of βIVΣ1 spectrin. CH, calponin-homology. (D) Primary amino acid sequence of βIVΣ1 spectrin. The four major domains of βIVΣ1 spectrin, numbered I–IV, are boxed. Domain I: the residues in the calponin-homology domain are in bold. Domain II: the arabic numbers on the left correspond to the spectrin repeats. Within this domain, the predicted A, B, and C helices are boxed separately and their conserved charged and aromatic residues are in bold. The glycine-rich region of 15 residues following the predicted A helix of spectrin repeat 4 is shown as an insert. Domain III: the four ERQES repeats in this specific domain are in bold. Domain IV: the residues in the pleckstrin homology domain are in bold. The proline-rich regions in spectrin repeat 15 and in the specific domain are underlined. (E) An alignment of the third ERQES repeat within the specific domain of βIV spectrin with sequences in the spectrin repeat 1 of βII and βIII spectrin. (F) Schematic representation of the different isoforms of βIV spectrin. (Top inset) The amino acids at the boundary between exons 17 and 18 in βIVΣ1 spectrin. (Middle inset, bold) The residues encoded by exon 17B in two distinct reading frames. The top sequence is in frame with the preceding exon 17 and includes a stop codon. The bottom sequence begins with a methionine and is in frame with the following exon 18. (Bottom inset) The amino acid sequence of exon 30B at the COOH terminus of βIVΣ4 spectrin. The roman numbers I–IV indicate the four major domains of βIV spectrin as defined in Fig. 2 D.

?iv Spectrin, a New Spectrin Localized at Axon Initial Segments and Nodes of Ranvier in the Central and Peripheral Nervous System

J Cell Biol. 2000 November 27;151(5):985-1002.