Original Article

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

Correspondence to: Michele Solimena, Department of Internal Medicine, Section of Endocrinology, Yale University School of Medicine, 330 Cedar Street, New Haven, CT 06520. Tel:(203) 737-1037; Fax: (203) 737-2812

	Abstract

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We report the identification of ßIV spectrin, a novel spectrin isolated as an interactor of the receptor tyrosine phosphatase-like protein ICA512. The ßIV spectrin gene is located on human and mouse chromosomes 19q13.13 and 7b2, respectively. Alternative splicing of ßIV spectrin generates at least four distinct isoforms, numbered ßIV1–ßIV4 spectrin. The longest isoform (ßIV1 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. ßIV2 and ßIV3 spectrin encompass the NH₂- and COOH-terminal halves of ßIV1 spectrin, respectively, while ßIV4 spectrin lacks the ERQES and the pleckstrin homology domain. Northern blots revealed an abundant expression of ßIV spectrin transcripts in brain and pancreatic islets. By immunoblotting, ßIV1 spectrin is recognized as a protein of 250 kD. Anti–ßIV spectrin antibodies also react with two additional isoforms of 160 and 140 kD. These isoforms differ from ßIV1 spectrin in terms of their distribution on subcellular fractionation, detergent extractability, and phosphorylation. In islets, the immunoreactivity for ßIV spectrin is more prominent in than in ß cells. In brain, ßIV spectrin is enriched in myelinated neurons, where it colocalizes with ankyrin_G 480/270-kD at axon initial segments and nodes of Ranvier. Likewise, ßIV spectrin is concentrated at the nodes of Ranvier in the rat sciatic nerve. In the rat hippocampus, ßIV1 spectrin is detectable from embryonic day 19, concomitantly with the appearance of immunoreactivity at the initial segments. Thus, we suggest that ßIV1 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.

Key Words: chromosome 19, diabetes, neuropathies, secretion, signal transduction

	Introduction

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The spectrins were originally identified as rod-shaped molecules that are part of the lattice-like cytoskeleton underneath the erythrocyte membrane (Marchesi and Steers 1968 ; Byers and Branton 1985 ; Bennett and Gilligan 1993 ). This subplasmalemmal meshwork is required to maintain the shape and lipid asymmetry of the plasma membrane. The critical role of these proteins is best revealed by the evidence that several human genetic syndromes, such as elliptocytosis and spherocytosis, are caused by mutant spectrin alleles (for review, see Marchesi 1988 ; Gallagher and Forget 1998 ). The spectrins have also been proposed to participate in the trafficking of organelles along the secretory pathways (Beck and Nelson 1998 ; De Matteis and Morrow 1998 ), and to play a role in regulated secretion (Goodman et al. 1995 ; Sikorski et al. 1991 , Sikorski et al. 2000 ).

The spectrin molecule is a tetramer that consists of two and two ß subunits. A tight association between the NH₂-terminal domain of an subunit and the COOH-terminal domain of a ß subunit forms a heterodimer, which assembles into a tetramer through a head-to-head association. The most distinctive feature of both subunits is the presence of multiple repeats, each consisting of 106 residues that assemble to form a three-helix bundle (Speicher and Marchesi 1984 ; Yan et al. 1993 ). spectrins include 20 spectrin repeats, two of which are less conserved, an SH3 motif between spectrin repeats 9 and 11, and a COOH-terminal domain with two EF-hand motifs. ß spectrins, on the other hand, contain 17 spectrin repeats in between an actin-binding domain at the NH₂ terminus and a pleckstrin homology (PH)¹ domain towards the COOH terminus.

Until recently, four genes encoding two spectrins (I and II) and two ß spectrins (ßI and ßII) were known in mammals. I1 and ßI1 spectrin are primarily expressed in erythrocytes, whereas II 1 and ßII1 spectrin have a wide tissue distribution and are particularly abundant in the brain (Goodman et al. 1981 ; Burridge et al. 1982 ; Glenney et al. 1982 ; Ma et al. 1993 ). An alternative spliced isoform of ßI spectrin, termed ßI2 spectrin is highly expressed in muscle and brain (Riederer et al. 1986 ; Zagon et al. 1986 ; Winkelmann et al. 1990b ; Malchiodi-Albedi et al. 1993 ). Similarly, an alternative spliced form has been found for ßII spectrin (Hayes et al. 2000 ). The number of ß spectrins has recently expanded with the discovery of ßIII (Ohara et al. 1998 ) and ßV (Stabach and Morrow 2000 ) spectrin. ßIII spectrin, which is most abundant in brain and kidney, is concentrated at the Golgi apparatus (Stankewich et al. 1998 ), and may therefore represent one of the Golgi spectrins previously reported (Beck et al. 1994 ; for review, see Beck and Nelson 1998 ; De Matteis and Morrow 1998 ). ßV spectrin is an exception among mammalian spectrins, as it includes 30 repeats, similar to the Drosophila ß heavy (ß_H) spectrin (Dubreuil et al. 1990 ). This large spectrin is most prominent in the outer segments of the retina and in gastric epithelial cells, while low levels are found in many tissues.

Besides with spectrins, ß spectrins interact directly with many additional proteins, including actin, protein 4.1, and ankyrins (Bennett and Gilligan 1993 ). Through the association with protein 4.1, ßI spectrin interacts with the membrane proteins glycophorin C and the Cl^-/HCO^-₃ exchanger (for review, see Conboy 1993 ). The binding to ankyrins provides ß spectrins with an additional linkage to integral membrane proteins, such as the voltage-gated Na⁺ channel, CD44, and the Na⁺/K⁺ ATPase (Bennett and Gilligan 1993 ). Finally, ß spectrins can directly associate with membrane proteins via a domain located at the end of spectrin repeat 1 and with phosphoinositides through the PH domain (Davis and Bennett 1994 ; Lombardo et al. 1994 ; Godi et al. 1998 ; Yao et al. 1999 ). Thus, ß spectrins are essential in the generation of organized cytoplasmic and membrane microdomains.

Here we report the cloning and characterization of a novel human spectrin, termed ßIV spectrin, which was isolated in a two-hybrid screening in yeast as an interactor of the autoantigen of type I diabetes ICA512 (Rabin et al. 1992 , Rabin et al. 1994 ; Lan et al. 1994 ). ICA512 (also known as IA-2, PTP-35, PTPLP, and PTPN) is a receptor tyrosine phosphatase–like protein of unknown function, which is associated with the secretory granules of neurons and peptide-secreting endocrine cells (Solimena et al. 1996 ; Lee et al. 1998 ; Solimena 1998 ; Hermel et al. 1999 ). The association of ICA512 with ßIV spectrin is consistent with previous studies suggesting that ICA512 interacts with the cytoskeleton (Hermel et al. 1999 ; Ort et al. 2000 ). Our data indicate that ßIV spectrin is most abundant in brain and pancreatic islet cells and undergoes extensive alternative splicing. In brain, ßIV spectrin is colocalized with ankyrin_G 480/270-kD at initial segments and nodes of Ranvier of myelinated neurons, the axonal compartments responsible for the generation and regeneration of action potentials. Thus, we propose that ßIV spectrin, in concert with ankyrin_G 480/270, is required for the anchoring of voltage-gated Na⁺ channels and cell adhesion molecules to the actin cytoskeleton and may play an important role in the physiology of nerve conduction.

	Materials and Methods

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Antibodies
ßIV spectrin antisera were raised against two synthetic peptides corresponding to amino acids 2237–2256 (specific domain antiserum, SD) and 2542–2559 (COOH-terminal domain antiserum, CT) of human ßIV spectrin, respectively. Both peptides were coupled to keyhole limpet hemocyanin through an added cysteine at their NH₂ terminus and were injected into rabbits to generate polyclonal antisera. Both antisera were affinity purified on their corresponding peptides that had been covalently linked to ECH-Sepharose beads (Amersham Pharmacia Biotech). Fractions of the antisera were eluted with 0.2 M glycine (pH 3.0), collected and dialyzed with Tris buffer, pH 7.5. The specificity of these antisera and affinity purified antibodies was tested by Western blot on Triton X-100 extracts from COS cells expressing a partial fragment of ßIV spectrin (residues 2068–2559), termed B8.

The following antibodies were from commercial sources: rabbit anti–LexA antiserum and mouse anti–V5 antibody (Invitrogen), mouse monoclonal antibody against ankyrin G 480/270-kD (Zymed Laboratories), mouse monoclonal antibody against glucagon (Sigma-Aldrich), mouse monoclonal antibody against myelin basic protein (Sternberger Monoclonal Antibodies), anti–rabbit and anti–mouse goat IgG antibodies conjugated to Alexa 488 or Alexa 568 (Molecular Probes, Inc.). A mouse monoclonal antibody against hemagglutinin (HA) was a kind gift of R. Collins (Cornell University, Ithaca, NY). Affinity-purified antibodies against the cytoplasmic or the ectodomain of ICA512 have been described previously (Solimena et al. 1996 ; Hermel et al. 1999 ).

Two-Hybrid Screening in Yeast
A yeast two-hybrid screening was executed according to the protocol of Vojtek et al. 1993 . Briefly, the cDNAs encoding the cytoplasmic domains of human ICA512 (amino acids 601–979, ICA512cyt), human PHOGRIN (amino acids 640–1015, PHOGRINcyt) and ICA512 mutant allele (ICA512cyt AD/DA) were amplified by PCR and subcloned into the pLexA vector to create fusion proteins with the LexA DNA binding domain. The ICA512cyt AD/DA mutant was constructed using site-directed mutagenesis on human ICA512 in pRC/RSV (Invitrogen) with the QuickChange mutagenesis kit (Stratagene) according to the manufacturer's instructions (A877D, 5' primer: CCTCAGCTGGCCGGACGAGGGCACACCGG, 3' primer: CCGGTGTGCCCTCGTCCGGCCAGCTGAGG; D911A, 5' primer: CGTGCACTGCTCGGCCGGTGCGGGGAG; 3' primer: CTCCCCGCACCGGCCGAGCAGTGCAC). The cytoplasmic domain of human PHOGRIN was amplified by PCR from a human adult brain cDNA library in pACT2 (CLONTECH Laboratories, Inc.). Automated sequencing of all constructs was carried out by the Keck Biotechnology Resource Laboratory at Yale University School of Medicine.

pLexA-ICA512cyt, pLexA-ICA512cyt AD/DA, and pLexA-PHOGRINcyt were independently transformed into the yeast strain L40 [partial genotype MATa trp1 leu2 his3 LYS2::(lexAop)-HIS3 URA3::(lexAop)-lacZ GAL4] and AMR70 [partial genotype: MAT his3 lys2 trp1 leu2 URA3::(lexAop)-lacZ GAL4]. Expression of the LexA-fusion proteins (baits) in yeast cells was verified by Western blotting using the anti-LexA antiserum (1:1,000). L40 cells expressing pLexA-ICA512cyt were cotransformed with 500 µg of a human adult brain cDNA library in pACT2 (Clontech) that included an in-frame HA tag (Clontech). Double transformants were selected for His⁺ and LacZ⁺ phenotypes. After purging of the bait plasmid, L40 His⁺ LacZ⁺ cells were mated separately with AMR70 cells transformed with the following baits: LexA-ICA512cyt, LexA-ICA512cyt AD/DA, LexA-PHOGRINcyt, LexA-lamin, and LexA-MSS4. Since lamin and MSS4 are proteins unrelated to ICA512, they were not expected to share interactors with ICA512. Accordingly, only those pACT2 plasmids that reconstituted His⁺ auxotrophy with LexA-ICA512 cyt, but not LexA-lamin or LexA-MSS4, in the mating assay were isolated and their cDNA inserts characterized by sequence analysis. The stringency conditions of the mating assay were increased by adding 3-amino-1,2,4-triazole (3-AT) (Sigma-Aldrich) to the plate media at concentrations ranging from 0 to 10 mM. To assess expression of bait and prey proteins in the mating assay, diploid cells were lysed by incubation for 10 min in 0.2 N NaOH/0.5% ß-mercaptoethanol on ice. Lysates were incubated with 10% trichloroacetic acid for 30 min on ice, and then spun for 10 min at 10,000 g at 4°C. After precipitation in 100% acetone, the proteins were resuspended in 2% SDS sample buffer (New England Biolabs, Inc.) to a final concentration of 200–400 µg/OD. Protein concentration in tissue extracts was determined using the BCA assay procedure (Pierce Chemical Co.). 100 µg protein from each lysate was run on 12% SDS-PAGE and blotted with anti-HA (1:10) and anti-LexA (1:1,000) antibodies followed by ¹²⁵I-Protein A (10⁵ cpm/ml) and autoradiography.

Cloning of ßIV Spectrin
Cloning of the full length ßIV spectrin cDNA from adult human brain was performed using standard molecular procedures (Sambrook et al. 1989 ). Briefly, a 325-bp fragment (6381–6705 bp in ßIV spectrin) within the partial clone isolated in the two-hybrid screening was amplified by PCR (5' primer: AGCCGGGAGCTGGGCAGCAGCGTG and 3' primer: GGCGCACATACCCCACCCGAGTCC) and labeled with [-³²P-dCTP] (3,000 Ci/mmol; Amersham Pharmacia Biotech) using the Random Prime Labeling Kit (Amersham Pharmacia Biotech). This probe was used to screen 2 x 10⁶ independent phage clones of a gt11 human brain cDNA library (Clontech) on nitrocellulose filters. Three independent phages were isolated whose sequences partially overlapped that of the original clone. Six additional screenings, each of 2 x 10⁶ phages from a gt10 human cerebellum cDNA library (Clontech), yielded 20 independent phages. Contig alignment of their inserts led to the assembly of 7,779-bp cDNA. The remaining 1,010 bp at the 5' end ßIV spectrin were cloned by PCR on a human brain Marathon-Ready cDNA library (Clontech) according to the manufacturer protocol using the following reverse primer: 5'-CTCCCGAGGCACAAAGAGGCGACG.

A 444-bp cDNA fragment of ßIV spectrin was amplified by PCR from mouse pancreatic islet cDNA (gift of Dr. R. Flavell, Yale University) using the following primers: (5' primer: GGCTCAGAATAAGGAGTGGCTGGAGAAGAT; 3' primer: AACGGCTCGAGCAGGCGCACCAGGCGC). Computer-assisted analysis of all cDNAs was performed using the following software programs: BLAST (Altschul et al. 1990 ), Lasergene (DNASTAR Inc.) and PFSCAN (ISREC Bioinformatics Groups). Intron-exon boundaries of the human ßIV spectrin gene were established by comparing the cDNA sequence with the corresponding genomic sequence.

Chromosomal Localization
Human and mouse BAC clones encompassing the ßIV spectrin gene were isolated at Genome Systems according to established protocols (Shizuya et al. 1992 ) using a fragment of the human ßIV spectrin cDNA (nucleotides 4094–4502) as a probe. Automated sequence analysis of these clones confirmed the presence of exon–intron boundaries within the ßIV spectrin gene. The locus of the ßIV spectrin gene on human and mouse chromosomes was also established at Genome Systems by fluorescent in situ hybridization using human and mouse genomic probes (>10 kb) originating from the corresponding BAC clones (Stokke et al. 1995 ).

Expression of ICA512 and ßIV Spectrin in COS Cells and Coimmunoprecipitations
A full-length human ICA512 construct in the mammalian expression vector pRC/RSV (Invitrogen) has been described previously (Hermel et al. 1999 ). A starting methionine was introduced by PCR at the 5' end of the ßIV spectrin partial clone B8. The resulting cDNA, including the NH₂-terminus HA tag derived from the pACT2 cDNA library, was subcloned as a HindIII–XbaI insert into pRC/RSV. Both ICA512 and B8 constructs (5 µg) were transiently transfected into COS cells by the Ca²⁺-phosphate procedure, as described previously (Dirkx et al. 1995 ). 72 h after transfection, transfected or mock-transfected (no cDNA) COS cells were harvested in PBS, pelleted, and then solubilized for 90 min on ice with homogenization buffer (158 mM NaCl, 10 mM HEPES, pH 7.4, 1mM PMSF, 10 mM benzamidine, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 µg/ml antipain) including 2% Triton X-100. One confluent dish (1.5 x 10⁷ cells) was used per immunoprecipitation. Insoluble material was removed by centrifugation at 15,000 g for 10 min at 4°C. Triton X-100 soluble material was precleared with 125 µl of 50% slurry protein G sepharose beads (Amersham Pharmacia Biotech). Next, extracts were incubated overnight at 4°C with 10 µl of a monoclonal antibody against the cytoplasmic domain of ICA512 or 50 µl of the ßIV-SD antibody. 100 µl of a 50% slurry protein G sepharose beads was added to each sample and incubated for 1 h on ice. Beads were pelleted by centrifugation, washed three times with 2% Triton X-100 in homogenization buffer without protease inhibitors, and then resuspended in 100 µl of 1x SDS sample buffer. 20 µl of each immunoprecipitate was separated on a 8% SDS-polyacrylamide gel, transferred on nitrocellulose, and immunoblotted with the following antibodies: anti–ICA512 ectodomain (1:1,000), anti–HA (1:10), and anti–ßIV-SD (1:250). Immunoreactivity was detected with alkaline phosphatase–conjugated goat anti–mouse (Sigma-Aldrich) or anti–rabbit IgG (Roche) (1:5,000).

Northern Blotting
A nitrocellulose filter with 2 µg polyA⁺ RNA from various human tissues (Multi Tissue Northern blot; Clontech) was hybridized with an -³²P-dCTP–labeled probe including nucleotides 6202–6526 within the predicted open reading frame of ßIV spectrin. The hybridization was performed according to the manufacturer's instructions. After x-ray exposure for 5 d, the blot was stripped by immersion in boiling water containing 0.5% SDS and rehybridized with a probe specific for human ß-actin provided by the manufacturer.

10 µg polyA⁺ RNA was purified with an Oligotex Direct mRNA kit (Qiagen) from 4 x 10⁴ human pancreatic islets kindly provided by the JDF Human Islet Distribution Centers at the University of Miami (Miami, FL), and Washington University (St. Louis, MO). 2 µg polyA⁺ RNA was separated on a 1% agarose gel containing 5% formaldehyde, blotted overnight on nitrocellulose by capillarity and hybridized with a probe including nucleotides 1805–2356 in the open reading frame of human ßIV spectrin. The autoradiography was developed after a 5-d exposure.

Transfection of ß-Spectrin Constructs in CHO Cells
The cDNAs encoding the carboxy termini of human ßI2, ßII, ßIII, and ßIV1 spectrin were amplified by PCR using the following templates: a partial clone of human ßI2 spectrin cDNA (Winkelmann et al. 1990b ; gift of Dr. Bertrand Forget, Yale University) (5' primer: ACCATGGAAGAAGAGGGAACGTGG, 3' primer: CTTCTTTTTGGGGAAGAAGCTGAA); the pACT2 human brain cDNA library (Clontech) for ßII1 spectrin (5' primer: GCACCATGCGGATGGCAGAAACGGTGGAC, 3' primer: TTTCTTTTTGCCAAAAAGGCTGAACCGCTT) and ßIII spectrin (5' primer: GCACCATGGGACAACAGAGACTTGAGCAC, 3' primer: CTTGTTCTTCTTAAAGAAGCTGAA); our partial clone B8 for ßIV spectrin (GCACCATGGTGCGGCCACGACCGGAGCGCCAGGAG, 3'primer: CTTCCTGCGCCCGCTGGCCCTGCGATCTCCGCCTTCCC). All cDNAs were subcloned upstream of the V5 epitope tag into pcDNA3.1/V5-His-TOPO/lacZ (Invitrogen) and transfected into CHO cells using the Lipofectin reagent (Life Technologies, Inc.) as described previously (Solimena et al. 1993 ).

Biochemical Procedures on Human Islets and Rat Brain Tissues
Approximately 2.5 x 10⁴ purified human pancreatic islets were sonicated on ice in 200 µl homogenization buffer (HB; 10 mM HEPES, pH 7.4, 5 mM EDTA, 1 mM EGTA, 1 mM NaCl, 1 mM PMSF, 10 mM benzamidine, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 µg/ml antipain). The islet lysates were then solubilized in 2% SDS sample buffer to a final protein concentration of 2.5 mg/ml. SDS insoluble material was removed by centrifugation.

Brain tissues were collected from adult rats, 15- (E15) and 19- (E19) d-old rat embryos and 1- (P1) and 10- (P10) d-old newborn rats. Tissues were homogenized on ice in HB (1:10 wt/vol). Post-nuclear supernatant (PNS) was obtained by centrifugation at 1,000 g for 10 min and solubilized in 2% SDS sample buffer to a final concentration of 1.2 mg/ml. PNS proteins (40 µg/lane) were separated by 6% SDS-PAGE and immunoblotted with ßIV-SD (1:500) or ßIV-CT (1:500) antibodies, followed by peroxidase-conjugated goat anti–rabbit IgG (1:5,000; Sigma-Aldrich) and enhanced chemiluminescence (Amersham Pharmacia Biotech). As controls, PNS proteins from adult rat brain were blotted with the preimmune rabbit sera as well as with ßIV spectrin antibodies that had been preincubated with 100 µg of the corresponding antigenic peptide.

High-speed pellet (HSP) and high-speed supernatant (HSS) from PNS of adult rat brain were obtained by centrifugation at 100,000 g for 1 h at 4°C. The HSS was collected, while the HSP was brought back to the original volume in HB containing 1% Nonidet P-40 (Boehringer) and 0.5% deoxycholic acid (Sigma-Aldrich). The HSP was solubilized for 2 h at 4°C and centrifuged at 100,000 g for 1 h. After the recovery of the soluble fraction, the HSP insoluble fraction was resuspended and sonicated in an equal volume of HB. PNS, HSS, and HSP soluble and insoluble fractions were dissolved in SDS sample buffer, separated by 6% SDS-PAGE, and blotted with the affinity purified ßIV-CT and ßIV-SD antibodies.

For alkaline phosphatase treatment, adult rat brain tissue was homogenized in HB without EDTA and EGTA. 300 µl of the resulting PNS (1.8 mg/ml) was incubated with 50 U of calf-intestine alkaline phosphatase (Roche) in phosphatase buffer for 1 h at 37°C. Alternatively, subcellular fractions of rat brain were prepared as described above, and then treated with alkaline phosphatase before SDS-PAGE and immunoblotting.

In Situ Hybridization
³⁵S-labeled riboprobes were prepared with a transcription kit (Promega) using as a template a partial cDNA of ßIV spectrin (bp's 6384–8792) that had been subcloned into pBluescript SK and KS vectors (Stratagene) as a HindIII-XbaI fragment. The antisense and control sense cRNAs were transcribed in the presence of ³⁵S-UTP (Amersham Pharmacia Biotech) from the XbaI-linearized SK plasmid and the HindIII-linearized KS plasmid, respectively. In situ hybridization on cryosections of rat brain (10 µm) was performed as previously described (Dunbar et al. 1998 ). Dark-field images were acquired with a microscope (BX50; Olympus) connected to an Olympix CCD camera driven by IP-Lab Software 3.0 (Scanalytics).

Immunocytochemistry
Males Sprague-Dawley rats (150–175 g) were fixed by trans-cardiac perfusion with 1% paraformaldehyde in 120 mM sodium phosphate buffer. Tissues of interest were collected, fixed for an additional 3 h, and then infiltrated with 30% sucrose in PBS. Tissues from rats at days E15, E19, and P10 were collected, fixed in 1% paraformaldehyde for 3 h, and then infiltrated with 30% sucrose. Teased fibers from rat sciatic nerve were prepared and fixed with 1% paraformaldehyde as previously described (Fjell et al. 2000 ). Single and double immunolabeling on 12-µm cryostat tissue sections and transfected CHO cells was performed as previously described (De Camilli et al. 1983 ; Cameron et al. 1991 ). Primary antibodies were diluted as follows: anti–ßIV-CT and anti–ßIV-SD, 1:500; anti–ankyrin G 480/270, 1:25; anti–ICA512 ectodomain, 1:10; anti–glucagon, 1:200, anti–myelin basic protein, 1:200; anti–V5, 1:200. As controls, sections were incubated with the preimmune serum of the rabbit immunized with the ßIV spectrin COOH-terminal peptide (1:50) and with anti–ßIV-CT antibody that had been preincubated with the antigenic peptide. Confocal microscopy was performed using an MRC 1024 station (Bio-Rad Laboratories) attached to an Axiovert microscope (Carl Zeiss, Inc.).

	Results

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

The COOH-terminal Region of a Novel ß Spectrin Binds the Cytoplasmic Domain of ICA512
ICA512 belongs to the receptor protein tyrosine phosphatase (RPTP) family, but does not display phosphatase activity towards conventional PTP substrates, because its cytoplasmic PTP homology domain differs in two critical residues from that of active PTPs. Specifically, in ICA512, the catalytic aspartate of conventional PTPs is replaced by an alanine (A877, Fig 1 A), while an aspartate (D911) rather than the obligatory alanine is present at position +2 from the catalytic cysteine of active PTPs. The replacement of A877 and D911 with aspartate and alanine, respectively, is sufficient to confer PTP activity to the corresponding ICA512 mutant (ICA512 AD/DA) (Magistrelli et al. 1996 ). To gain insight into ICA512's function, we searched for proteins interacting with its cytoplasmic domain by performing a two-hybrid screening in yeast.

View larger version (35K): [in this window] [in a new window]   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 ßIV 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 ßIV spectrin specific domain (ßIV-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, ßIV-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.

L40 yeast cells were cotransformed with LexA-ICA512cyt fused to the DNA binding protein LexA and a human brain cDNA library fused to the Gal4 activation domain in pACT2 as a source for preys. Screening of 10⁷ L40 transformants led to the isolation of 540 colonies positive for the expression of both the reporter genes HIS3 and ß-galactosidase. One of the preys that overcame histidine auxotrophy when expressed with LexA-ICA512cyt (Fig 1 B) was a novel peptide of 493 amino acids whose last 149 residues were >40% similar to the COOH-terminal region of various ß spectrins. This polypeptide, termed B8 (Fig 2 A), also bound to a PTP active mutant of ICA512cyt (ICA512cyt AD/DA) and to the cytoplasmic domain of the ICA512-related protein, PHOGRIN (PHOGRINcyt) (Wasmeier and Hutton 1996 ). B8, on the other hand, did not interact with the unrelated control baits lamin and MSS4. When the two-hybrid assay was performed in more stringent conditions (i.e., in the presence of the HIS3 inhibitor 3-AT), B8 still associated with ICA512cyt, but not with ICA512cyt AD/DA or PHOGRINcyt (Fig 1 C). Notably, cells expressed comparable amounts of B8 (Fig 1 E), but less ICA512cyt than ICA512cyt AD/DA or PHOGRINcyt (Fig 1 D). These data indicated that the viability of yeast cells expressing ICA512cyt and B8 in the presence of 3-AT was not accounted for by a higher expression of the bait and suggests that ICA512cyt has a higher affinity for B8 than ICA512cyt AD/DA or PHOGRINcyt. Thus, the replacement of the two residues that prevent ICA512 from acting as a conventional PTP was sufficient to weaken its interaction with B8.

View larger version (113K): [in this window] [in a new window]   Figure 2. (A) Schematic diagram of the overlapping human ßIV 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 ßIV spectrin. Black boxes represent the 36 exons encoding the full-length ßIV 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 ßIV1 spectrin. CH, calponin-homology. (D) Primary amino acid sequence of ßIV1 spectrin. The four major domains of ßIV1 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 ßIV spectrin with sequences in the spectrin repeat 1 of ßII and ßIII spectrin. (F) Schematic representation of the different isoforms of ßIV spectrin. (Top inset) The amino acids at the boundary between exons 17 and 18 in ßIV1 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 ßIV4 spectrin. The roman numbers I–IV indicate the four major domains of ßIV spectrin as defined in Fig 2 D.

The ability of ICA512 to bind B8 was further investigated by coimmunoprecipitations from extracts of COS cells cotransfected with the cDNAs encoding for full-length ICA512 and HA-tagged B8 (Fig 1 F). In these cells, pro-ICA512 accumulates as a triplet of 110 kD, while the ICA512 transmembrane fragment (ICA512 TMF) generated by the cleavage of the protein's ectodomain represents a minor species (Hermel et al. 1999 ). Immunoprecipitation with the ßIV-SD antibody raised against a peptide within B8 followed by Western blotting with a monoclonal anti–HA antibody allowed the detection of a single protein of 60 kD in transfected COS cells, but not in mock-transfected COS cells. The size of this protein corresponds to the predicted size of HA-tagged B8. The same protein was also immunoprecipitated from cotransfected COS cells using a monoclonal antibody directed against the cytoplasmic domain of ICA512. These data confirmed the interaction between the two proteins revealed by the two-hybrid assay.

Cloning of Human ßIV Spectrin
Screenings of human brain cDNA libraries by filter hybridization and PCR allowed the contig assembly of an 8,789-bp cDNA, including a 5'-untranslated region of 179 bp and a 3'-untranslated region of 933 bp with a polyadenylation signal and a polyA tail (Genbank No. AF082075) (Fig 2 A). By fluorescence in situ hybridization, this new gene, termed ßIV spectrin, was localized on human chromosome 19q13.13 and on the synthenic region on mouse chromosome 7b2 (not shown). Through BLAST analysis, we matched the cDNA of ßIV spectrin to a genomic sequence from human chromosome 19 in the High Throughput Genomic Sequence database (Genbank No. AC021625). Comparison of this genomic sequence, which corresponds to a first draft of 34 unordered pieces, with the cDNA of ßIV spectrin revealed that the latter is generated from 36 exons (Fig 2 B). All exons, except for exon 17, are found in this genomic sequence and are flanked by canonical donor/acceptor intron sites. This genomic region still contains many gaps of unknown length, which may account for the apparent absence of the exon 17 (bp's 3819–4021) found in the cDNA sequences.

A high degree of similarity was found between portions of the ßIV spectrin cDNA and another genomic sequence of 100 unordered pieces from chromosome 16 (Genbank No. AC009140.4). This genomic draft contains 18 sequences that are bounded by donor/acceptor intron sites and are very conserved with 18 exons of ßIV spectrin (exons 1, 8–11, 13, 15–17, 19, 25, 26, 28, 29, 32, 34–36) (not shown). This genomic draft, however, did not appear to include the remaining exons of ßIV spectrin. Our cDNA contig for ßIV spectrin and the genomic sequence on chromosome 19 only differed in three base pairs, possibly because of polymorphism or sequencing artifacts. In contrast, six of the ßIV spectrin-related exons on chromosome 16 contained many base substitutions, deletions, or insertions compared with the related exons in ßIV spectrin. These data confirmed that our cDNA contig was entirely derived from the ßIV spectrin gene on chromosome 19. They also suggested, on the other hand, that a gene closely related to ßIV spectrin resides on chromosome 16. Cloning and characterization of this gene, provisionally termed ßVI spectrin, is in progress.

The conceptual translation of the ßIV spectrin cDNA corresponds to a protein of 2,559 amino acids with a predicted molecular weight of 288 kD and an isoelectric point of 5.9. The putative starting methionine of ßIV spectrin is not in the context of a Kozak sequence, but it is preceded in the 5'-untranslated region by an in-frame stop codon. The domain structure of ßIV spectrin closely resembles that of other ß spectrins (Hu et al. 1992 ; Winkelmann et al. 1990a , Winkelmann et al. 1990b ; Ma et al. 1993 ; Ohara et al. 1998 ), with the exception of ßV spectrin (Stabach and Morrow 2000 ). It includes two calponin homology domains, followed by 16 complete spectrin repeats, a partial spectrin repeat (repeat 17), and a PH domain (Fig 2C and Fig d). Calponin homology domains are found at the NH₂ terminus of many proteins, including ß spectrins, and mediate the interaction with actin (Karinch et al. 1990 ; Frappier et al. 1992 ; Banuelos et al. 1998 ). Each of the first 16 spectrin repeats of ßIV spectrin follows the criteria defined for the repeat 14 of Drosophila ß spectrin (Yan et al. 1993 ; Pascual et al. 1997 ), and thus is predicted to assemble into three helices termed A, B, and C (Fig 2 D). The repeat 15 of ßIV spectrin, which in other ß spectrins binds ankyrins (Kennedy et al. 1991 ), is 43.8, 46.7, and 31.4% identical to the corresponding repeats of ßI, ßII, and ßIII spectrin, respectively. Unlike typical spectrin repeats, but similar to repeat 15 of other ß spectrins, it does not include a tryptophan at position 17 in the helix A (Fig 2 D). As in all other ß spectrins, the repeat 17 of ßIV spectrin lacks the segment corresponding to the C helix (Fig 2 D). This is consistent with the possibility that the A and B helices of repeat 17 of ßIV spectrin form a three-helix bundle with the lone C helix at the NH₂ terminus of an spectrin (Kennedy et al. 1994 ; Deng et al. 1995 ). The putative loop between the predicted helices A and B of repeat 4 contains a stretch of 15 residues that is not found in other ß spectrins (Fig 2 D). This insert is rich in glycines, consistent with a role as a helix breaker.

The region of 294 residues between the repeat 17 and the PH domain is specific for ßIV spectrin as it shows no similarity with regions present in other ß spectrins. This domain is rich in prolines (15% of the residues), is very basic (pI: 10.5), and contains a motif of five residues (ERQES) that is repeated four times within a stretch of 90 residues (residues 2220–2309, henceforth defined as ERQES domain). An identical motif was not found in any other protein in public databases. The ERQES domain is highly charged (55.5% charged residues) because of its high content in glutamate (23.3%), glutamine (8.8%), and arginine (26.6%). The presence of only two aspartates (2.2%) and the absence of asparagines and lysines, however, indicates that there is a strong bias toward specific residues in this region. The equal spacing of 13 residues between the ERQES repeats 1–3 further suggests the repetitive nature of this domain. Interestingly, the sequence corresponding to the third ERQES repeat and the four following residues (ERQESAEHE) is 77% identical to the sequence ERLEKAEHE within spectrin repeats 1 of human ßII (residues 397–405) and ßIII (residues 400–408) spectrin (Fig 2 E). The motif ERLEKAEHE is part of a domain that associates ßII spectrin with membranes, presumably via protein–protein interaction (Davis and Bennett 1994 ; Lombardo et al. 1994 ). Prediction of the secondary structure of ßIV spectrin suggests that the ERQES domain is exposed at the protein's surface (not shown) and, as such, might also engage in protein–protein interaction.

ßIV spectrin contains numerous putative SH3 binding sites (Fig 2 D). Two of these proline-rich motifs (residues 1859–1864 and 2405–2411) are adjacent to putative membrane association domains. Specifically, the first of these motifs precedes the putative B helix in repeat 15, which may act as an ankyrin-binding site, while the second motif precedes the PH domain. The PH domain of ßIV spectrin is very conserved with the corresponding domains in ßI2, ßII, and ßIII spectrins and is therefore likely to bind phosphoinositides, such as phosphatidylinositol 4,5 bisphosphate (Hyvonen et al. 1995 ).

ßIV Spectrin Is Alternatively Spliced
Our data indicated that ßIV spectrin undergoes alternative splicing. Specifically, one cDNA clone of 2.4 kb contained an insert of 88 bp between exon 17 and 18, which was not present in overlapping cDNA clones (Fig 2 F). This insert was also found in the ßIV spectrin gene, where it is flanked by canonic donor and acceptor splice sites at its 5' and 3' ends. Insertion of this exon, termed 17B (Fig 2B and Fig f), introduces a stop codon in the open reading frame of ßIV spectrin, thereby causing its termination in spectrin repeat 9. The conceptual translation of this cDNA corresponds to a polypeptide of 1,302 amino acids with a predicted molecular weight of 145 kD. In a different reading frame, on the other hand, exon 17B encodes for a methionine that is preceded by a stop codon in exon 17 and is in frame with the remaining COOH-terminal portion of ßIV spectrin (Fig 2B and Fig f). Recognition of this methionine as a novel initiation site for translation would generate an additional polypeptide of 1,307 amino acids with an expected molecular weight of 149 kD. It is conceivable that the inclusion of exon 17B gives rise to a bicistronic mRNA, whose translation produces two different ßIV spectrin proteins. Based on the established nomenclature for spectrins (Winkelmann and Forget 1993 ), the originally cloned ßIV spectrin has been termed ßIV1 spectrin, and hence we will refer to these two variants as ßIV2 and ßIV3 spectrin (Genbank No. AY004226).

In an additional clone, an insert of 315 bp (exon 30B) followed exon 30 (Fig 2B and Fig f). This insert encodes 42 amino acids and a stop codon, contains a polyadenylation site, and was found in the ßIV spectrin gene, where it is flanked by canonic 5' and 3' splice sites. The isoform including this exon corresponds to a polypeptide of 2,149 amino acids (expected molecular weight: 242 kD), which contains the calponin-homology domain and all 17 spectrin repeats, but lacks the specific domain and the PH domain (Fig 2 F). This isoform has been termed ßIV4 spectrin (Genbank No. AY004227). A search in publicly available databases revealed the presence of a partial cDNA clone (Genbank No. AL133093) of 1,444 bp from human testis that is 100% identical to ßIV spectrin in the region corresponding to nucleotides 4850–6082, but differs from it in the remaining 211 bp at the 3' end. This divergent sequence encodes 30 residues, followed by a stop codon and a polyA stretch of 109 bp. On chromosome 19, however, the 102 bp preceding the polyA stretch are found immediately after exon 27 of ßIV spectrin, where they are not preceded by a canonic 5' splice site. Furthermore, this sequence does not contain a polyadenylation signal. Thus, this clone includes a part of the intron following exon 27.

Expression of ßIV Spectrin mRNAs
The expression of ßIV spectrin was analyzed by Northern blotting on polyA⁺ RNA from various human tissues using a 324 bp probe within the region encoding for the specific domain common to ßIV1 and ßIV3 spectrin. This probe hybridized with three transcripts of 9.0, 5.1, and 3.1 kb, all of which were predominantly expressed in brain (Fig 3 A). The relationship of these transcripts with ßIV spectrin was suggested by their detection with a distinct probe derived from the 3' untranslated region of ßIV spectrin (not shown). A mRNA of 9.0 kb is in good agreement with the size of the ßIV1 spectrin cDNA. Both the 9.0 and the 5.1 kb transcripts were also detected in various regions of the rat brain, including cerebellum, thalamus, midbrain, medulla, frontal, and posterior cortex and hippocampus (data not shown). Weaker signals for the 9.0-kb transcript were detected in human skeletal muscle and kidney, while the 5.1-kb transcript was barely detectable in pancreas (Fig 3 A). Since pancreatic islets account for only 3% of the pancreas weight, we directly assessed the expression of ßIV spectrin on polyA⁺ RNA isolated from purified human pancreatic islets. In pancreatic islets, as in brain, the ßIV spectrin probe hybridized with three transcripts of 8.5, 4.2, and 2.3 kb (Fig 3 C). The expression of ßIV spectrin in pancreatic islets was confirmed by the amplification by PCR from mouse islets of a product whose cDNA (Genbank No. AF203696) was 98% identical to nucleotides 4223–4665 of human ßIV spectrin.

View larger version (51K): [in this window] [in a new window]   Figure 3. Northern blots for ßIV 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 ß-actin. Heart and skeletal muscle express two ß-actin mRNAs of 2 and 1.6–1.8 kb.

ßIV Spectrin Proteins in Brain and Pancreatic Islets
We further analyzed by Western blotting the expression of ßIV spectrin in brain and pancreatic islets, tissues that are enriched in ICA512. To this end, we used two affinity-purified antibodies directed against peptides encoding amino acids 2237–2256 in the specific domain (ßIV-SD) and 2542-2559 at the COOH terminus (ßIV-CT) of ßIV1 spectrin. These peptides were chosen on the basis of their predicted antigenicity and because they share no significant homology with sequences in other and ß spectrins. Both antibodies reacted by Western blotting with the ßIV spectrin fragment B8 transiently expressed in COS cells (Fig 1 F, and not shown). The ßIV-CT antibody recognized two proteins of 250 and 160 kD in rat brain PNS and total extracts of purified human islets (Fig 4 A), while it detected an additional protein of 140 kD in rat brain only (Fig 4A and Fig B). Preincubation of the ßIV-CT antibody with the antigenic peptide blocked its binding to these three proteins, supporting their relationship with ßIV spectrin (Fig 4 A). Taking into account the aberrant electrophoretic mobility displayed by all known spectrins (Zagon et al. 1984 ; Ohara et al. 1998 ; Stankewich et al. 1998 ), the 250-kD protein might correspond to ßIV1 spectrin, which has a predicted molecular weight of 288 kD. The identity of the 160- and 140-kD proteins remains to be established. Accordingly, we will provisionally refer to these proteins as ßIV spectrin 160 and 140. The ßIV-SD antibody produced a similar immunoblot pattern (Fig 4 B), consistent with the possibility that the 250-, 160-, and 140-kD proteins originate from the ßIV spectrin gene. The ßIV-SD antibody, however, recognized much less prominently ßIV spectrin 160 in brain, and even less in islets (Fig 4 B). The peptide used to raise the ßIV-SD antibody is located within the ERQES domain and contains several optimal consensus sites for serine phosphorylation. We asked therefore whether the limited reactivity of the ßIV-SD antibody toward ßIV spectrin 160 could result from the phosphorylation of its epitope within this protein. Evidence that treatment of rat brain PNS with alkaline phosphatase dramatically enhanced the reactivity of the ßIV-SD antibody toward ßIV spectrin 160 (Fig 4 B) corroborated this hypothesis. The labeling of ßIV1 spectrin and ßIV spectrin 140, on the other hand, was unaffected. As expected, the reactivity of the ßIV-SD antibody was completely blocked by the incubation with its immunogenic peptide (Fig 4 B).

View larger version (14K): [in this window] [in a new window]   Figure 4. (A) Western blotting with the affinity-purified antibody against a COOH-terminal peptide of ßIV spectrin (ßIV-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 ßIV-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 ßIV spectrin (ßIV-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 ßIV-SD antibody after preincubation with its antigenic peptide.

ßIV Spectrins Have Different Biochemical Properties
Upon subcellular fractionation of brain homogenates, both ßIV1 spectrin and ßIV spectrin 140 were recovered in the HSP, while ßIV spectrin 160 was distributed between the HSS and the HSP (Fig 5 A). Virtually all ßIV1 spectrin partitioned in the HSP detergent-insoluble material (Fig 5A and Fig B). ßIV spectrin 160 and 140, on the other hand, were found in both the HSP detergent soluble and insoluble fractions (Fig 5A and Fig B). Notably, the pools of ßIV spectrin 160 in the HSS and HSP detergent soluble fraction did not react with the ßIV-SD antibody (Fig 5 B), unless these fractions were preincubated with alkaline phosphatase (Fig 5 C). These data suggest that phosphorylation of the ERQES domain is associated with the solubilization of ßIV spectrin 160 and its dissociation from membranes. Treatment of the PNS with alkaline phosphatase did not affect the subcellular distribution of ßIV1 spectrin, but allowed an increased recovery of ßIV spectrin 140 in the HSP-soluble fraction (Fig 5 D). This treatment also resulted in a shift of most of ßIV spectrin 160 from the HSP to the HSS (Fig 5 D). Taken together, these results suggest that the interaction of ßIV spectrins with cytoskeletal and/or membrane proteins is modulated by phosphorylation.

View larger version (31K): [in this window] [in a new window]   Figure 5. Equal volumes of rat brain subcellular fractions immunoblotted with ßIV-CT (A and D) or ßIV-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.

Localization of ßIV Spectrin in Pancreatic Islets
To rule out the possibility that our anti-ßIV spectrin antibodies would recognize other ß spectrins by immunocytochemistry, both antibodies were employed for the staining of CHO cells transiently transfected with V5-tagged COOH-terminal domains of ßI2 (amino acids 2117–2328), ßII1 (2140–2365), ßIII (amino acids 2154–2390), or ßIV1 spectrin (2222–2559). All four spectrin fragments were recognized by the anti-V5 mouse monoclonal antibody and partitioned, albeit to a different extent, between the cytosol and the nucleus of transfected cells (Fig 6 A). Conversely, the ßIV-CT (Fig 6 A) and ßIV-SD (not shown) antibodies only stained CHO cells expressing the COOH-terminal region of ßIV1 spectrin.

View larger version (54K): [in this window] [in a new window]   Figure 6. Double immunolabeling of V5-tagged COOH-terminal domains of human ßI2 (A and B), ßII1 (C and D), ßIII (E and F), and ßIV1 (G and H) spectrins with the V5 (left) and ßIV-CT (right) antibodies in transiently transfected CHO cells. Bar, 25 µm.

The tissue and intracellular distribution of ßIV spectrin was then investigated by confocal microscopy using the ßIV-CT antibody. In rat pancreas, ßIV spectrin immunoreactivity was restricted to pancreatic islets. Like ICA512, ßIV spectrin was detected in both the insulin-secreting ß cells as well as in the surrounding cells that secrete glucagon (Fig 7, A–F). In ß cells, there was a fine punctate staining throughout the cytoplasm (not shown). A more intense cytoplasmic labeling was observed in the cells (Fig 7, A–F), often in correspondence of rod-like perinuclear structures of several micrometers in length (Fig 7 G). These organelles did not significantly overlap with secretory granules, as shown by double labeling for glucagon (Fig 7 H). The specificity of this staining was suggested by its complete disappearance when the ßIV-CT antibody was preincubated with its antigenic peptide (Fig 7. I). ß Spectrins are known to associate with various intracellular compartments, including the Golgi complex, endosomes, and lysosomes (Devarajan et al. 1997 ; Hoock et al. 1997 ; Beck and Nelson 1998 ). Thus, we compared the distribution of ßIV spectrin with that of these various organelles in cells. There was no significant colocalization of ßIV spectrin with -adaptin and TGN38, two markers of the trans-Golgi network, with the endosomal markers transferrin receptor and early endosomal antigen-1 or with the lysosome marker lgp120 (not shown). Thus, the identity of the ßIV spectrin-immunoreactive structures in pancreatic islets remains to be established.

View larger version (71K): [in this window] [in a new window]   Figure 7. Confocal microscopy for ßIV spectrin (pseudo green), and insulin or glucagon (pseudo red) in rat pancreas. (A and B) Staining of a pancreatic islet for ßIV spectrin (A) and insulin (B). (C) Merge of A and B. (D and E) Staining of a pancreatic islet for ßIV spectrin (D) and glucagon (E). (F) Merge of D and E. (G and H) High magnifications of pancreatic islet cells stained either for ßIV spectrin alone (G) or both ßIV spectrin and glucagon (H). (I) The ßIV-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).

ßIV Spectrin Is Present at the Axon Initial Segments and Nodes of Ranvier in the Central and Peripheral Nervous System
In brain, expression of ßIV spectrin was first examined by in situ hybridization on adult rat brain sections using an antisense riboprobe corresponding to the partial ßIV spectrin clone B8. The highest levels of expression were detected in the cell bodies of large myelinated neurons, such as pyramidal neurons of the hippocampus, granule cells of the dentate gyrus, and Purkinje cells of the cerebellar cortex (Fig 8). No signal was detected in nonneuronal cells, including glial and endothelial cells. Likewise, no signal was detected on sections incubated with the corresponding control sense riboprobe (not shown).

View larger version (100K): [in this window] [in a new window]   Figure 8. In situ hybridization for ßIV 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).

By immunocytochemistry on rat brain sections, the ßIV-CT antibody produced a strong labeling of pyramidal neurons in the cerebral cortex and the hippocampus, granule cells of the dentate gyrus, and Purkinje cells in the cerebellum (Fig 9, and data not shown). This immunolabeling was most prominent in the axon initial segments (Fig 9A, Fig D, and Fig G) and nodes of Ranvier (D and H), while no staining was observed in the myelinated internodal segments, as visualized with an antibody against myelin basic protein (Fig 9B and Fig C). A fine granular staining reminiscent of the labeling observed in pancreatic islets was appreciable in the perikarya of Purkinje cells (Fig 9 A). No immunoreactivity was detected in dendrites, axon terminals, glial cells, or blood vessels. The specificity of this labeling was supported by the absence of staining in sections incubated with the corresponding rabbit preimmune serum (not shown) and by the ability of the antigenic peptide to compete with endogenous ßIV spectrin for the binding of the ßIV-CT antibody (not shown). Immunocytochemistry on dissociated myelinated fibers from the rat sciatic nerve showed that, like in the central nervous system, ßIV spectrin is concentrated at the nodes of Ranvier in the peripheral nervous system (Fig 9 I).

View larger version (73K): [in this window] [in a new window]   Figure 9. Confocal microscopy for ßIV 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 ßIV 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 ßIV 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 ßIV spectrin and ankyrinG. (H) High power magnification of nodes of Ranvier in the cerebellum costained for ßIV spectrin and ankyrinG. (I) Staining of teased nerve preparation with ßIV spectrin and ankyrinG 480/270. Bars, 40 µm (A–C), 100 µm (D–F), 5 µm (H–I).

Previous studies demonstrated that, in neurons, the ankyrin_G 480/270-kD isoform (also known as ankyrin3) is selectively concentrated at initial segments and nodes of Ranvier (Kordeli et al. 1990 , Kordeli et al. 1995 ; Peters et al. 1995 ). Here, it is involved in the clustering of the voltage-gated Na⁺ channels that generate the saltatory action potentials (Zhou et al. 1998 ). Double immunostaining for ßIV spectrin and ankyrin_G 480/270 showed a perfect overlap of the two proteins at initial segments (Fig 9, D–F) and central (H) and peripheral (I) nodes of Ranvier. These data raise the possibility that ßIV spectrin and ankyrin_G 480/270 bind each other in the brain as well as the peripheral nervous system.

The ßIV Spectrin Isoform Localized at the Axon Initial Segments Is ßIV1 Spectrin
Next, we investigated the expression of ßIV spectrin during brain development. By Western blotting, ßIV1 spectrin was first detected at embryonic day 19 and its expression level progressively increased until adult age (Fig 10). ßIV spectrin 140 appeared only after birth and, similar to ßIV1 spectrin, it was maximally expressed in the adult. ßIV spectrin 160, instead, was already present at embryonic day 10, and its level appeared constant throughout development. These data further suggested that ßIV spectrin 160 is not a proteolytic fragment of ßIV1 spectrin.

View larger version (48K): [in this window] [in a new window]   Figure 10. Temporal expression of ßIV spectrin during brain development. Western blotting with the ßIV-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.

The expression of ßIV spectrin during brain development was also investigated by immunocytochemistry (Fig 11). A weak immunoreactivity for ßIV spectrin was detectable in the neurons of the developing hippocampus at embryonic day 15, but there was no staining reminiscent of axon initial segments (Fig 11 A). By embryonic day 19, however, the initial segments were clearly labeled both in the cortex and the hippocampus (Fig 11 B). Many more initial segments positive for ßIV spectrin were present by postnatal day 10 in both regions (Fig 11 C, and data not shown), with maximum immunoreactivity in the adult brain (D). Taken together, these data suggested that ßIV1 spectrin is the specific isoform localized at axon initial segments.

View larger version (54K): [in this window] [in a new window]   Figure 11. Appearance of ßIV spectrin immunoreactivity at axon initial segments during brain development. Confocal microscopy with the ßIV-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.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

In this study, we have identified a novel spectrin gene in human, termed ßIV spectrin, that is localized on chromosome 19q13.13. The longest product of this gene, termed ßIV1 spectrin, includes 36 exons and corresponds to a protein of 2,559 amino acids, whose domain structure closely resembles that of other ß spectrins (for review, see Bennett and Gilligan 1993 ; Goodman et al. 1995 ; Viel and Branton 1996 ). Specifically, ßIV1 spectrin contains two calponin-homology domains at its NH₂ terminus and may therefore associate with F-actin and protein 4.1. Similar to other ß spectrins, it may also interact with ankyrin through spectrin repeat 15 and with an spectrin via its partial spectrin repeat 17, whereas its COOH-terminal PH domain could bind phospholipids. ßIV1 spectrin also contains a specific domain between its partial spectrin repeat 17 and the PH domain. This very basic domain includes several putative SH3 binding sites and a unique ERQES domain in which there is a sequence of significant similarity with a motif within the membrane association domain of ßII and ßIII spectrin (Davis and Bennett 1994 ; Lombardo et al. 1994 ). Thus, this specific domain could mediate the interaction of ßIV1 with other proteins.

The apparent size of the ßIV1 spectrin transcript was 9.0 kb in brain and 8.5 kb in pancreatic islets. In both tissues, however, ßIV1 spectrin was detected as a protein of 250 kD. The small discrepancy in the size of the transcripts may result therefore from tissue-specific differences in the untranslated region or from variations in the laboratory procedures, as the blot of human islets was prepared in our laboratory, while the blot including the brain sample was acquired from a commercial source.

Similar to other ß spectrins, the ßIV spectrin gene undergoes alternative splicing. Specifically, we have identified three ßIV spectrin splice variants in addition to ßIV1 spectrin. Two of these isoforms, ßIV2 and ßIV3 spectrin, originate from one messenger that contains an additional exon (exon 17B) between exons 17 and 18 of ßIV1 spectrin. The predicted amino acid sequences of ßIV2 and ßIV1 spect