In this study, we have identified a novel spectrin gene in human, termed βIV spectrin, that is localized on chromosome 19q13.13. The longest product of this gene, termed βIVΣ1 spectrin, includes 36 exons and corresponds to a protein of 2,559 amino acids, whose domain structure closely resembles that of other β spectrins (for review, see Bennett and Gilligan 1993
; Goodman et al. 1995
; Viel and Branton 1996
). Specifically, βIVΣ1 spectrin contains two calponin-homology domains at its NH2
terminus and may therefore associate with F-actin and protein 4.1. Similar to other β spectrins, it may also interact with ankyrin through spectrin repeat 15 and with an α spectrin via its partial spectrin repeat 17, whereas its COOH-terminal PH domain could bind phospholipids. βIVΣ1 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 βII and βIII spectrin (Davis and Bennett 1994
; Lombardo et al. 1994
). Thus, this specific domain could mediate the interaction of βIVΣ1 with other proteins.
The apparent size of the βIVΣ1 spectrin transcript was ~9.0 kb in brain and ~8.5 kb in pancreatic islets. In both tissues, however, βIVΣ1 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 β spectrins, the βIV spectrin gene undergoes alternative splicing. Specifically, we have identified three βIV spectrin splice variants in addition to βIVΣ1 spectrin. Two of these isoforms, βIVΣ2 and βIVΣ3 spectrin, originate from one messenger that contains an additional exon (exon 17B) between exons 17 and 18 of βIVΣ1 spectrin. The predicted amino acid sequences of βIVΣ2 and βIVΣ1 spectrin are identical up to the middle of the C helix in repeat 9. The sequence corresponding to the last 23 residues of βIVΣ2 spectrin is unique and does not follow the consensus for spectrin repeats, as it is very rich in prolines (6/23 amino acids). Accordingly, the last repeat of βIVΣ2 spectrin (repeat 9) is not complete, and contains only helices A and B. This observation raises the intriguing possibility that this partial repeat, similar to the partial repeat 17 of other β spectrins, including βIV spectrin, interacts with the lone C helix at the NH2
terminus of an α spectrin. An alternative possibility is that this partial repeat interacts in a head-to-tail fashion with the NH2
-terminal domain of βIVΣ3 spectrin. Specifically, the first 19 amino acids of βIVΣ3 encoded by exon 17 cannot be part of an α helix because they include six prolines. The next 10 amino acids together with the following 15 residues encoded by exon 18, however, are predicted to form a coiled-coil domain (Lupas et al. 1991
). Similar to the partial NH2
-terminal repeat of α spectrin, this domain contains an arginine at position 8, a residue that is thought to interact with residues 7 and 29 in helices A and B, respectively (Yan et al. 1993
). This domain of βIVΣ3 spectrin could therefore be equivalent to the single C helix at the NH2
terminus of an α spectrin and pair with the predicted A and B helices at the COOH terminus of βIVΣ2 spectrin. The fourth βIV spectrin isoform, termed βIVΣ4 spectrin, extends only 42 residues beyond the partial repeat 17, because of the insertion of an additional exon (exon 30B) that contains an in-frame stop codon. Thus, βIVΣ4 spectrin, similar to βIΣ1 (Winkelmann et al. 1990b
) and βIIΣ2 (Hayes et al. 2000
) spectrin, does not include a PH domain.
Both the βIV-SD and βIV-CT antibodies are directed against peptides that are not found in βIVΣ2 and βIVΣ4 spectrin. Thus, the biochemical properties and localization of these isoforms remain to be determined. Both antibodies, on the other hand, recognize two proteins of 160 and 140 kD. βIV spectrin 160 and 140 show significantly different properties than βIVΣ1 spectrin. For instance, treatment of the PNS with alkaline phosphatase increased the pool of βIV spectrin 160 recovered in the HSS, and enhanced the detergent extractability of βIV spectrin 140. Conversely, βIVΣ1 spectrin partitioned in all conditions in the HSP detergent insoluble material. Furthermore, βIV spectrin 140 was detected in brain, but not in islets. Finally, the temporal expression of βIV spectrins 160 and 140 during brain development was different from that of βIVΣ1 spectrin. These data suggest that βIV spectrin 160 and 140 are not proteolytic fragments of βIVΣ1 spectrin.
We have shown that the reactivity of the βIV-SD antibody with βIV spectrin 160 is significantly enhanced upon alkaline phosphatase treatment. Specifically, upon subcellular fractionation, this antibody reacts with the pool of βIV spectrin 160 recovered in the HSP detergent-insoluble material, but not with the pool present in the HSS and HSP detergent-soluble material, unless these fractions are incubated with alkaline phosphatase before immunoblotting. Since the antigenic peptide of the βIV-SD antibody contains an optimal consensus sequence for phosphorylation, these data suggest that the antigenic epitope within the ERQES domain is phosphorylated in vivo and that phosphorylation at this site affects the association of βIV spectrin 160 with membranes. This possibility is consistent with previous studies indicating that an increased phosphorylation of β spectrin during mitosis is associated with its redistribution from the detergent-insoluble to the detergent-soluble fraction and its dissociation from membranes (Fowler and Adam 1992
Like ICA512, βIV spectrin is enriched in pancreatic islets and brain. In the α cells, βIV spectrin's immunoreactivity is concentrated in the cytosol, most often in perinuclear, rod-like structures. This compartment did not significantly overlap with the Golgi, endosomes, or lysosomes, three organelles along the secretory pathway where β spectrins have been found (Beck and Nelson 1998
; De Matteis and Morrow 1998
). Thus, its identity remains to be determined. In rat brain and teased fibers of the sciatic nerve, βIV spectrin is concentrated at the axon initial segments and the nodes of Ranvier. βIV spectrin is the first spectrin that is selectively enriched in these specialized domains, in both the central and the peripheral nervous tissue. βII spectrin is present both in axons and synaptic terminals (Riederer et al. 1986
; Zagon et al. 1986
; Trapp et al. 1989
), while βIΣ2 spectrin is found in neuronal cell bodies, dendrites (Riederer et al. 1986
; Zagon et al. 1986
; Malchiodi-Albedi et al. 1993
), and post-synaptic densities (Malchiodi-Albedi et al. 1993
). βIII spectrin is also expressed in brain, where it is enriched in dendrites (Ohara et al. 1998
; Stankewich et al. 1998
). Taken together, these data suggest a role for β spectrins as potential organizers of different neuronal microdomains.
The axon initial segments and the nodes of Ranvier share a common molecular organization and may have evolved from a common precursor (Davis et al. 1996
). Both compartments are characterized by an electron-dense cytoskeletal matrix beneath the axolemma (Matsumoto and Rosenbluth 1985
) and by a 40-fold enrichment of voltage-gated sodium channels (Ritchie and Rogart 1977
), whose opening triggers the generation of action potentials and their conduction from one node to the next. Other proteins enriched at these sites are the cell adhesion molecules neurofascin and NrCAM (Davis et al. 1996
480/270-kD (Kordeli et al. 1995
; Lambert et al. 1997
), and an amphiphysin II–like protein (Butler et al. 1997
). The repeat 15 of βIV spectrin is similar to the corresponding ankyrin-binding repeat in βI and βII spectrins (Kennedy et al. 1991
). In view of their colocalization at initial segments and nodes of Ranvier, it seems plausible that βIV spectrin and ankyrinG
are associated and together mediate the anchoring of nodal membrane proteins to the actin cytoskeleton.
In the developing rat hippocampus, the immunoreactivity for βIV spectrin at the initial segments first appears at embryonic day 19 and progressively increases thereafter. This pattern closely resembles the developmental expression profile of βIVΣ1 spectrin in rat brain. A similar correlation may exist between the appearance of βIV spectrin 140 at postnatal day 10 and the progressive development of nodes of Ranvier during myelination after birth. The initial segments and the nodes of Ranvier play a similar role in the initiation and propagation of action potentials. Initial segments, in addition, act as barriers that prevent lateral diffusion of membrane proteins and entry of various organelles, including rough endoplasmic reticulum, Golgi complex, and lysosomes into axons, while allowing the progression of others, such as mitochondria and secretory vesicles. Although the molecular foundation of this selective diffusion barrier remains unknown, there is evidence that the actin cytoskeleton is essential in its establishment and maintenance (Winckler et al. 1999
). Thus, the localization of βIVΣ1 spectrin and βIV spectrin 140 at initial segments and nodes of Ranvier, respectively, could correlate with the related and yet distinct functions of these axonal compartments. It would be interesting to determine whether a similar different localization occurs in the case of the ankyrinG
isoforms 480 and 270 kD.
We have identified βIV spectrin as an interactor of the RPTP-like protein ICA512 in a two-hybrid screening in yeast. The binding of ICA512 to the COOH-terminal domain of βIV spectrin has been confirmed by coimmunoprecipitation from transfected fibroblasts. βIV spectrin and ICA512 are both enriched in neurons and pancreatic islets, but their intracellular localization is different. This observation, however, does not preclude the possibility that ICA512, at one stage of its intracellular route, interacts with βIV spectrin. In steady state conditions, virtually all ICA512 (Lee et al. 1998
; Hermel et al. 1999
; our unpublished observations) is found on secretory granules (Solimena et al. 1996
), while it is not detectable in other compartments associated with the secretory and endocytic pathways. Taking into account the fact that ICA512 has a very short half life (Lee et al. 1998
; Hermel et al. 1999
; our unpublished observations), it would be very difficult to detect by immunocytochemistry a transient colocalization of the two proteins in a compartment other than the secretory granules. More sensitive assays for protein–protein interaction in vivo, such as coimmunoprecipitation, were precluded by the fact that virtually all βIVΣ1 spectrin and most of βIV spectrin 140 and 160 partitioned in the HSP detergent insoluble fraction. We have recently shown that, in insulinoma cells, ICA512 is associated with β2 syntrophin (Ort et al. 2000
). β2 syntrophin, in turn, interacts with the spectrin-related protein utrophin. These data, consistent with our previous studies (Hermel et al. 1999
), suggest that ICA512 is associated with the actin cytoskeleton.
Besides ICA512, the COOH-terminal domain of βIV spectrin bound the cytoplasmic domain of the RPTP-like molecule PHOGRIN (Wasmeier and Hutton 1996
). PHOGRIN (also known as IA-2β, ICAAR, IAR, PTP-NP, and PTPX) has many properties in common with ICA512. Its cytoplasmic domain is 74% identical to the corresponding region of ICA512, and also contains an aspartate instead of an alanine in its inactive PTP “signature motif.” Moreover, PHOGRIN is also enriched in the secretory granules of neurons and peptide-secreting endocrine cells (Wasmeier and Hutton 1996
) and is a major target of autoimmunity in type I diabetes (Lu et al. 1996
). ICA512 and PHOGRIN are not the only members of the RPTP family that interact with spectrin-related molecules. LAR, an RPTP expressed in numerous cell types including neurons, interacts through its distal, “non-catalytic” PTP domain with Trio, a protein that includes numerous spectrin repeats and two PH domains (Debant et al. 1996
). CD45, an RPTP involved in T and B cell signaling, binds βII spectrin through its “inactive,” distal domain. Notably, binding of spectrin increases the catalytic activity of its proximal PTP domain (Lokeshwar and Bourguignon 1992
; Iida et al. 1994
). Like the “inactive” PTP domains of ICA512 and PHOGRIN, the distal PTP domain of CD45 includes an aspartate (D) rather than the obligatory alanine (A) at position +2 from the catalytic cysteine of canonic PTP domains. Overall, these data point to spectrin-related proteins as common binding partners of noncatalytic PTP domains of RPTPs.