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. 2000 Nov 27;151(5):985-1002.
doi: 10.1083/jcb.151.5.985.

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

S Berghs  1 , D Aggujaro, R Dirkx Jr, E Maksimova, P Stabach, J M Hermel, J P Zhang, W Philbrick, V Slepnev, T Ort, M Solimena
Affiliations

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

S Berghs et al. J Cell Biol. .

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.

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Figures

Figure 1
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.
Figure 2
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.
Figure 2
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.
Figure 2
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.
Figure 2
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.
Figure 3
Figure 3
Northern blots for βIV spectrin on polyA+ RNA from various human tissues (A) and purified human pancreatic islets (C). (B) Autoradiography on the same filter as in A after hybridization with the control probe for β-actin. Heart and skeletal muscle express two β-actin mRNAs of ∼2 and 1.6–1.8 kb.
Figure 4
Figure 4
(A) Western blotting with the affinity-purified antibody against a COOH-terminal peptide of βIV spectrin (βIV-CT) on 50 μg protein from rat brain PNS (brain, lanes 1 and 3) and homogenates of human pancreatic islets (islets, lanes 2 and 4). In the case of islets, the βIV-CT antibody was incubated with an excess of the immunogenic peptide before its incubation with the nitrocellulose strips. (B) Western blotting with the affinity-purified antibody against a peptide in the specific domain of βIV spectrin (βIV-SD) on 50 μg protein from rat brain PNS (brain, lanes 1, 3, and 4) and homogenates of human pancreatic islets (islets, lanes 2 and 5). (brain + alk. phos., lane 3) Rat brain PNS was treated with calf intestine alkaline phosphatase for 1 h at 37°C. (lanes 4 and 5) Western blotting with the βIV-SD antibody after preincubation with its antigenic peptide.
Figure 5
Figure 5
Equal volumes of rat brain subcellular fractions immunoblotted with βIV-CT (A and D) or βIV-SD (B and C) antibodies. (C) Fractions were treated with alkaline phosphatase before immunoblotting. (D) The PNS was treated with alkaline phosphatase before fractionation. HSP-S, HSP-detergent soluble material. HSP-IS, HSP detergent-insoluble material.
Figure 7
Figure 7
Confocal microscopy for βIV spectrin (pseudo green), and insulin or glucagon (pseudo red) in rat pancreas. (A and B) Staining of a pancreatic islet for βIV spectrin (A) and insulin (B). (C) Merge of A and B. (D and E) Staining of a pancreatic islet for βIV spectrin (D) and glucagon (E). (F) Merge of D and E. (G and H) High magnifications of pancreatic islet α cells stained either for βIV spectrin alone (G) or both βIV spectrin and glucagon (H). (I) The βIV-CT antibody was incubated with an excess of the antigenic peptide before the incubation with the tissue sections. Bars, 100 μm (A–F), 10 μm (G–I).
Figure 6
Figure 6
Double immunolabeling of V5-tagged COOH-terminal domains of human βIΣ2 (A and B), βIIΣ1 (C and D), βIII (E and F), and βIVΣ1 (G and H) spectrins with the V5 (left) and βIV-CT (right) antibodies in transiently transfected CHO cells. Bar, 25 μm.
Figure 8
Figure 8
In situ hybridization for βIV spectrin on rat brain (dark field images). (A) CA3 region of the hippocampus, (B) dentate gyrus, (C) cerebellar cortex. Bars, 100 μm (A and B), 50 μm (C).
Figure 10
Figure 10
Temporal expression of βIV spectrin during brain development. Western blotting with the βIV-CT antibody on PNS (40 μg protein) from rat brain at embryonic day 10 (E10), 15 (E15), 19 (E19), postnatal day 1 (P1), 10 (P10), and adult age.
Figure 9
Figure 9
Confocal microscopy for βIV spectrin (pseudo green) and myelin basic protein or ankyrin G480/270-kD (pseudo red) in rat brain. (A and B) Staining of the cerebellar cortex for βIV spectrin (A) and myelin basic protein (B). (C) Merge of A and B. The arrow points to the boundary between the initial segment and the internodal segment of a Purkinje cell myelinated axon. (D and E) Staining of the hippocampus for βIV spectrin (D) and ankyrinG 480/270-kD (E). (F) Merge of D and E. (G) High power magnification of axon initial segments in the cerebral cortex costained for βIV spectrin and ankyrinG. (H) High power magnification of nodes of Ranvier in the cerebellum costained for βIV spectrin and ankyrinG. (I) Staining of teased nerve preparation with βIV spectrin and ankyrinG 480/270. Bars, 40 μm (A–C), 100 μm (D–F), 5 μm (H–I).
Figure 11
Figure 11
Appearance of βIV spectrin immunoreactivity at axon initial segments during brain development. Confocal microscopy with the βIV-CT antibody (pseudo green) on sections from rat brain at embryonic day 15 (A), 19 (B), postnatal day 10 (C), and adult age (D). Bars, 50 μm.
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