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J. Biol. Chem., Vol. 275, Issue 49, 38182-38189, December 8, 2000

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Structural and Functional Differences between 3-Repeat and 4-Repeat Tau Isoforms

IMPLICATIONS FOR NORMAL TAU FUNCTION AND THE ONSET OF NEURODEGENERATIVE DISEASE^*

Bruce L. Goode, Miu Chau^§, Paul E. Denis^¶, and Stuart C. Feinstein

From the Neuroscience Research Institute and Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, California 93106

Received for publication, August 16, 2000, and in revised form, September 7, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Tau, MAP2, and MAP4 are members of a microtubule-associated protein (MAP) family that are each expressed as "3-repeat" and "4-repeat" isoforms. These isoforms arise from tightly controlled tissue-specific and/or developmentally regulated alternative splicing of a 31-amino acid long "inter-repeat:repeat module," raising the possibility that different MAP isoforms may possess some distinct functional capabilities. Consistent with this hypothesis, regulatory mutations in the human tau gene that disrupt the normal balance between 3-repeat and 4-repeat tau isoform expression lead to a collection of neurodegenerative diseases known as FTDP-17 (fronto-temporal dementias and Parkinsonism linked to chromosome 17), which are characterized by the formation of pathological tau filaments and neuronal cell death. Unfortunately, very little is known regarding structural and functional differences between the isoforms. In our previous analyses, we focused on 4-repeat tau structure and function. Here, we investigate 3-repeat tau, generating a series of truncations, amino acid substitutions, and internal deletions and examining the functional consequences. 3-Repeat tau possesses a "core microtubule binding domain" composed of its first two repeats and the intervening inter-repeat. This observation is in marked contrast to the widely held notion that tau possesses multiple independent tubulin-binding sites aligned in sequence along the length of the protein. In addition, we observed that the carboxyl-terminal sequences downstream of the repeat region make a strong but indirect contribution to microtubule binding activity in 3-repeat tau, which is in contrast to the negligible effect of these same sequences in 4-repeat tau. Taken together with previous work, these data suggest that 3-repeat and 4-repeat tau assume complex and distinct structures that are regulated differentially, which in turn suggests that they may possess isoform-specific functional capabilities. The relevance of isoform-specific structure and function to normal tau action and the onset of neurodegenerative disease are discussed.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Regulation of microtubule function is mediated in large part by the activities of microtubule-associated proteins (MAPs).¹ The tau/MAP2/MAP4 family of MAPs is involved in promoting microtubule assembly, stabilizing microtubule dynamics, and bundling microtubules (1-3). These MAPs are related by a common carboxyl-terminal region that is ~200 amino acids long, 60-70% identical in sequence, and possesses microtubule binding and assembly activities (4, 5). This region is characterized by the presence of either three or four 18-amino acid long imperfect repeats, separated by 13-14-amino acid long inter-repeat (IR) sequences. Tau and MAP2 are expressed primarily in neural cells, whereas MAP4 is more ubiquitous.

Both phosphorylation and alternative RNA splicing regulate the microtubule binding properties of these MAPs. Whereas the effects of phosphorylation upon MAP function have been investigated intensively (reviewed in Refs. 1, 6, and 7), the consequences of alternative RNA splicing of MAP RNAs remains a poorly understood but critical aspect of MAP biochemistry and cell biology. Alternative splicing of tau, MAP2, and MAP4 can be regulated developmentally and/or in a tissue-specific manner (2, 3, 8-10), raising the possibility of functional differences among the different isoforms. In the microtubule binding region (i.e. the repeat:inter-repeat region), splicing leads to either the presence or absence of a 31-amino acid sequence that contains the first inter-repeat ("R1-R2 IR") and repeat 2, thereby generating 4-repeat and 3-repeat MAP, respectively. In tau, there are two additional alternatively spliced exons leading to the presence of 0, 1, or 2 insertions of 29 amino acids each in the amino terminus. Thus, differential utilization of these three exons leads to the generation of six different tau isoforms in the central nervous system (Fig. 1). Whereas only the shortest of the six tau isoforms is expressed in the fetal human brain, all six isoforms are expressed in the adult human brain (11). Finally, there is one more alternatively spliced exon encoding 237 amino acids located in the middle of the tau protein (12, 13). This exon is expressed primarily in the peripheral nervous system but is also expressed in a small subset of central nervous system neurons. However, it is not detectable in neurons of the cerebral cortices (14, 15).

Considerable effort has been aimed at understanding the tau structure-function relationship and the biochemical nature of the tau-microtubule interaction. Early biophysical and electron microscopic investigations suggested that purified tau had little secondary or tertiary structure in solution (16-19). The subsequent discovery of the 18-amino acid long imperfect repeats led to the hypothesis that each repeat might function as an independent tubulin binding domain, aligned sequentially along the length of the molecule (4). It was hypothesized that tau might stabilize tubulin polymers by stretching out across the microtubule lattice, with each repeat forming an independent association with a different tubulin subunit. Thus, tau might cross-link multiple subunits together non-covalently. Shortly thereafter, several studies on 4-repeat tau provided evidence consistent with this "linear" view of tau action (4, 20-22).

Building upon this linear perspective, the functional differences between 3-repeat and 4-repeat tau have been viewed generally as quantitative differences, with 4-repeat molecules binding to microtubules with greater affinity than 3-repeat molecules (20, 22). Similarly, 4-repeat tau assembles microtubules more efficiently than does 3-repeat tau (23). However, competition experiments also suggest that there may be qualitative differences in how 3-repeat and 4-repeat tau interact with microtubules (21). Additionally, two sets of observations emphasize the importance of understanding how 3-repeat tau and 4-repeat tau may interact differently with microtubules. First, as noted above, the 3-repeat tau:4-repeat tau ratio is tightly regulated developmentally (10), suggesting that different isoforms may possess distinct capabilities. Second, recent genetic data demonstrate that mutations affecting the ratio of 3-repeat tau:4-repeat tau (without affecting the amino acid sequence of either protein) cause severe neurological disease (known as fronto-temporal dementia and Parkinsonism linked to chromosome 17 or simply as FTDP-17). This collection of disorders is characterized by abnormal filamentous tau pathology similar to that seen in the neurofibrillary tangles of Alzheimer's disease and neuronal cell death (24-26).

In previous work comparing truncated fragments of tau differing from one another only by the presence or absence of the 31-amino acid sequence by which 4-repeat tau differs from 3-repeat tau, we observed dramatic effects of these sequences upon microtubule binding affinity (~20-fold effect; Ref. 21). Since 3-repeat tau is, in effect, an internal deletion of these same exact sequences from 4-repeat tau, the linear view of tau structure-function predicts that the difference in microtubule binding affinity between 3-repeat and 4-repeat tau should be of a similar magnitude. However, it has been shown that the difference in binding affinity between these two isoforms is only ~3-fold (see Refs. 20 and 22; see also Fig. 1B). In the present study, we have investigated the molecular basis of this inconsistency. Our data suggest that 3-repeat tau possesses a core of microtubule binding activity composed of its first two repeats and the intervening inter-repeat. Furthermore, the carboxyl-terminal sequences strongly enhance 3-repeat tau core unit microtubule binding activity. Comparing and contrasting these observations with previous investigations of 4-repeat tau leads to the conclusion that tau does not bind microtubules through a linear array of multiple independent binding sites. Rather, the data suggest that both 3-repeat and 4-repeat tau possess core microtubule binding domains that have both common and unique mechanistic features. Furthermore, there appears to be isoform-specific regulation of 3-repeat and 4-repeat tau by sequences flanking the repeat region. Thus, the isoforms differ from one another both quantitatively and qualitatively. The implications of these observations to development and neurological disease are discussed.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Tau DNA Constructs-- The cloning and/or reconstruction of rat full-length cDNAs encoding 4-repeat tau and 3-repeat tau containing both amino end inserts were described previously (10, 21). To generate the other four isoforms, DNA sequences encoding the 29-amino acid amino-terminal inserts from the longest 4-repeat and 3-repeat tau isoforms were internally deleted using site-directed mutagenesis strategies ("Transformer" mutagenesis kit: CLONTECH; Palo Alto, CA). Similar methods were used to generate all of the single amino acid substitutions, amino-terminal deletions, and internal deletions described in this study. All mutations were verified by DNA sequence analysis.

The 3-repeat tau carboxyl-terminal truncations shown in Fig. 2 were generated as described previously for 4-repeat tau (21). A primer located 50 bases upstream of the T7 promoter in pGEM3-3-repeat tau was used in combination with specific downstream tau primers to amplify fragments of the 3-repeat tau cDNA by PCR. The PCR products were blunt-ended using the Klenow fragment of DNA polymerase and gel-purified prior to use in a coupled in vitro transcription/translation reaction. Downstream tau primers for the PCRs were 18 nucleotides in length and initiated at DNA sequences encoding the following amino acids (according to the numbering of rat 4-repeat tau; see Ref. 10): Gly²⁶⁴, Lys³⁰⁸, Ile³¹⁹, Gly³²⁶, Arg³⁴⁰, and Gly³⁵⁸.

In Vitro Transcription and Translation of Tau Polypeptides-- In vitro transcription/translation reactions were performed according to the specifications of the Promega TnT rabbit reticulocyte lysate kit in the presence of ³⁵S-labeled methionine (>1000 Ci/mmol, 10 µCi/µl, PerkinElmer Life Sciences). Translation products were analyzed prior to use in microtubule binding assays by fractionation on polyacrylamide gels, which were fixed in 30% ethanol, 10% glacial acetic acid, enhanced with Apex (55% glacial acetic acid, 15% xylenes, 30% ethanol, 0.5% 2,5-diphenyloxazole), rehydrated in H₂O for 5 min, dried onto filter paper, and autoradiographed. Translation product yields were determined as described in the Promega Protocols and Applications Guide. Routinely, specific activities of 10⁶ cpm/ng tau were obtained, which corresponds to ~0.1 ng of tau/µl of TnT reaction product. TnT products were stored at 20 °C and used in microtubule binding assays within 1 week of synthesis. All tau polypeptides compared in a given microtubule binding assay were synthesized in parallel.

Bovine Tubulin Purification-- MAP-free tubulin was purified from bovine brain by two cycles of temperature-controlled polymerization and depolymerization as described (27), followed by phosphocellulose chromatography in PEM buffer (50 mM Pipes (pH 6.8), 1 mM EGTA, 1 mM MgCl₂) supplemented with 1 mM GTP. Aliquots were drop-frozen in liquid nitrogen and stored at 70 °C. Protein concentrations were determined by the method of Bradford (28).

Microtubule Binding Assays-- Microtubule binding assays were performed as described previously (21) with the following modifications. Microtubules were assembled from 3.5 mg/ml MAP-free tubulin at 35 °C for 30 min in the presence of 1 mM GTP and 30 µM taxol (Calbiochem). The integrity of the taxol-stabilized microtubules was verified by uranyl fixation and negative staining electron microscopy. Dilutions of the microtubules were then made in BRB-80 buffer (80 mM Pipes (pH 6.8), 1 mM MgCl₂, 1 mM EGTA) supplemented with 1 mM GTP and 10 µM taxol. In each binding reaction, 9 µl of polymerized tubulin (0.01-40 µM) was mixed with 1 µl of in vitro translated tau product (~0.1 ng of tau) and incubated at room temperature for 15 min. Reactions were then layered over an 80-µl sucrose cushion (50% sucrose in BRB-80 buffer supplemented with 10 µM taxol) in 5 × 20-mm ultraclear centrifuge tubes (Beckman Instruments, Palo Alto, CA) and centrifuged in a Beckman SW50.1 swinging bucket rotor with adaptors for 15 min at 40,000 rpm at room temperature. Supernatants were harvested, sucrose cushions aspirated, and pellets re-solubilized in SDS sample buffer. The pellets and supernatants were fractionated on 10 or 15% polyacrylamide gels, processed as described above, and autoradiographed. The relative amounts of tau in the supernatants and pellets were determined by laser densitometry. The concentration of microtubules required to co-sediment 50% of the tau is defined as the apparent K_d (20, 21). Each tau construct was tested multiple times, always using full-length tau as an internal control to ensure that experiment-to-experiment comparisons were valid. This accounts for the differences in "n" reported for 4RL tau and 3RL tau in different figures. It also accounts for the slight differences in the binding constants for these constructs in different figures.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

3-Repeat Tau and 4-Repeat Tau Differ in Microtubule Binding Affinity by Only ~3-Fold-- To gain initial insight into possible functional differences among the six different central nervous system tau isoforms, we first compared their microtubule binding affinities. Each tau isoform was synthesized by coupled in vitro transcription/translation of its corresponding tau cDNA in the presence of [³⁵S]methionine (Fig. 1A). The resulting radiolabeled tau polypeptides were tested for microtubule binding capability in a quantitative co-sedimentation assay with taxol-stabilized microtubules. As shown in Fig. 1B, all three of the 4-repeat tau isoforms bind to microtubules with comparable affinities, and all three of the 3-repeat tau isoforms bind with comparable affinities. However, the 4-repeat tau isoforms bind to microtubules with ~3-fold stronger affinity than the 3-repeat tau isoforms. Thus, the presence of the developmentally regulated 31-amino acid insert (containing the R1-R2 inter-repeat and repeat 2) results in an ~3-fold increase in microtubule binding affinity. On the other hand, the presence or absence of 0, 1, or 2 of the 29-amino acid insertions in the amino portion of tau has little effect on microtubule binding affinity.

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Microtubule binding affinities of the six tau isoforms. A, autoradiograph of in vitro translated polypeptides corresponding to the six rat brain tau isoforms. Tau cDNAs were in vitro transcribed and translated in a rabbit reticulocyte lysate in the presence of 35S-labeled methionine. Translation products were fractionated on a 10% polyacrylamide gel and visualized by autoradiography. B, tau isoforms are designated as either 3-repeat or 4-repeat, which differ by the presence or absence of a 31-amino acid insertion that contains repeat 2 and the R1-R2 inter-repeat. Isoforms also are designated as either S (short), M (medium), or L (long), depending on whether they contain 0, 1, or 2 of the 29-amino acid inserts (shaded boxes) in their amino termini. Microtubule binding affinities were determined by a microtubule co-sedimentation assay. The Kd value of each isoform shown is an average from multiple experiments (n = the number of times each polypeptide was tested), with the standard error of the mean. The P box corresponds to the proline-rich region that can regulate microtubule binding activity indirectly (20, 32).

These results are intriguing because they reveal an inconsistency. On one hand, previous binding analyses performed on 4-repeat tau mutants with successively larger carboxyl or amino end truncations demonstrated that loss of the R1-R2 inter-repeat and repeat 2 leads to a very large loss of microtubule binding affinity (~20-fold; see Ref. 21). On the other hand, when wild type 3-repeat tau and 4-repeat tau are compared directly (in effect, an internal deletion of the same sequences), the data in Fig. 1 demonstrate that the effects on binding affinities are modest (~3-fold).

In order to test if differential phosphorylation of 3-repeat and 4-repeat tau in the reticulocyte lysate might be responsible for these observations (phosphorylation is well known to affect tau binding activity; for review see Ref. 7 and references therein), we compared the microtubule binding affinities of in vitro translated 3-repeat tau and 4-repeat tau both before and after treatment with alkaline phosphatase. No changes in binding affinity were observed (data not shown), suggesting that other mechanism(s) are likely involved.

These conclusions are in agreement with previous studies of microtubule binding and assembly activities (20, 22, 23). Based on these data and the genetic analyses implicating aberrant splicing of exon 10 (encoding the R1-R2 inter-repeat and repeat 2) with neurodegenerative disease (24-26), we focused our further comparisons on 3-repeat and 4-repeat tau isoforms containing both amino end insertions, which will be referred to here simply as 3-repeat and 4-repeat tau.

3-Repeat Tau Structure and Function, a Core Microtubule Binding Unit Regulated by Carboxyl-terminal Flanking Sequences-- To begin the molecular dissection of 3-repeat tau structure and function, we next performed a carboxyl end truncation analysis. Comparison of full-length 3Rtau with 3Rtau358 reveals that the carboxyl terminus makes a strong (~10-fold) contribution to microtubule binding affinity (Fig. 2). Comparison of 3Rtau358 with 3Rtau340 and 3Rtau326 shows that additional truncations removing repeat 4 and the R3-R4 inter-repeat have little effect. On the other hand, the data for 3Rtau 308 and 3Rtau264 demonstrate that further truncation into repeat 3 and beyond abolishes the remaining detectable microtubule binding activity.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Binding affinities of full-length and carboxyl end truncated 3-repeat tau polypeptides. The microtubule binding affinities (Kd values) of in vitro translated tau polypeptides were compared by a microtubule co-sedimentation assay. The average Kd value and standard error of the mean is shown for each tau polypeptide, where n = the number of times each polypeptide was assayed. In each experiment, truncated tau polypeptides were directly compared with full-length tau as an internal control. The P box corresponds to the proline-rich region that can regulate microtubule binding activity indirectly (20, 32). (Note, the numbering nomenclature is based upon the positions of each amino acid in a 4RL context.)

To test directly whether or not the carboxyl-terminal sequences have inherent microtubule binding activity, we constructed tau247-358, which is an internal deletion of the entire repeat:inter-repeat region. As seen in Fig. 2, this construct exhibits no detectable microtubule binding activity.

Together, these data suggest that 3-repeat tau interacts with microtubules via a core microtubule binding domain composed of repeat 1, the R1-R3 inter-repeat, and repeat 3. Furthermore, the data suggest that the carboxyl-terminal flanking sequences regulate the microtubule binding activity of the core unit but are not themselves sufficient to bind microtubules. However, since we have not yet tested an internal deletion lacking repeat 4 but containing the carboxyl-terminal sequences, our data do not exclude the formal possibility that microtubule binding activity may be regulated by the presence of either the carboxyl-terminal sequences or repeat 4. Nevertheless, we favor the simpler model, i.e. that the carboxyl-terminal sequences regulate core microtubule binding activity by an indirect molecular mechanism.

Lys-265 Is Essential for the Microtubule Binding Activity of the R1-R3 Inter-repeat-- Comparison of 3Rtau308 with 3Rtau264 in Fig. 2 reveals that the R1-R3 inter-repeat plays a significant role in microtubule binding in 3-repeat tau, although not as strong as the contribution of the R1-R2 inter-repeat in 4-repeat tau (21). We noted that there is a significant sequence conservation between the two inter-repeats in the sub-region of the R1-R2 inter-repeat known to interact with microtubules (Fig. 3A). Given the sequence conservation and their analogous positions in tau adjacent to repeat 1, we tested the possibility that these two inter-repeats might promote microtubule binding by similar molecular mechanisms.

View larger version (45K): [in this window] [in a new window]   Fig. 3.   Site-directed mutagenesis of the R1-R3 inter-repeat of 3-repeat tau. A, sequence of relevant inter-repeat regions (from Lee et al. (4)). B, wild type 3-repeat tau and substitution mutants K265A, P272K, and Y270N/P2727K were in vitro translated and compared directly in microtubule binding affinity assays. The microtubule binding affinity of each polypeptide is expressed relative to the wild type 3-repeat tau microtubule binding affinity (Kd). n = the number of times each polypeptide was assayed. Error bars represent the standard error of the mean.

In our earlier work (21), we determined that Lys²⁶⁵ contributes 2-3-fold and Lys²⁷² contributes 3-4-fold to microtubule binding affinity in 4-repeat tau. Since Lys²⁶⁵ is conserved in the R1-R2 inter-repeat and the R1-R3 inter-repeat, we tested whether mutation of this residue would have a similar effect on microtubule binding activity in 3-repeat tau. As shown in Fig. 3B, substitution of an alanine for Lys²⁶⁵ in 3-repeat tau (K265A) causes a 2.3-fold reduction in 3-repeat tau microtubule binding affinity, similar to the effect of a K265A mutation in 4-repeat tau (21).

It is thus possible that the R1-R3 inter-repeat might function in 3-repeat tau similarly to the way in which the R1-R2 inter-repeat functions in 4-repeat tau. However, subsequent mutations suggested that there are at least some unique aspects of R1-R3 inter-repeat function relative to R1-R2 inter-repeat function. For example, the position occupied by Lys²⁷² makes a strong (~3-4-fold) contribution to microtubule binding affinity in 4-repeat tau. In the R1-R3 inter-repeat in 3-repeat tau, this position is occupied by a proline. If the two inter-repeats function by a similar mechanism, one would predict that mutation of that proline to lysine in 3-repeat tau might increase microtubule affinity by ~3-4-fold. However, this mutation (P272K) has no detectable effect upon the microtubule binding affinity of 3-repeat tau (Fig. 3B). The only other difference between the two inter-repeat sequences that is a likely candidate to have functional significance is the asparagine at position 270 in the R1-R2 inter-repeat, which is a tyrosine in the R1-R3 inter-repeat. Therefore, we generated a double mutant Y270N/P272K 3-repeat tau. However, the resulting molecule was actually less active than wild type 3-repeat tau (Fig. 3B). Therefore, based upon these data, we conclude that the two inter-repeats function, at least in part, by distinct molecular mechanisms.

The R1-R3 Inter-repeat Sequence and Spacing Relationships within the Core Microtubule Binding Unit Are Important for 3-Repeat Tau Function-- To examine further the molecular basis by which the 3-repeat tau core microtubule binding unit functions, we next generated several internal deletion mutations within the core unit and determined their effects on microtubule binding activity. Initially, we tested the effect of deleting repeat 3 from 3-repeat tau (3RR3), which led to an ~80-fold reduction in microtubule binding activity (Fig. 4A). This is consistent with the earlier conclusion (based upon truncation data; see Fig. 2) that repeat 3 plays an important role in 3-repeat tau function.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Effects of internal deletions in the repeat region on the microtubule binding affinity of tau. The microtubule binding affinities (Kd values) of in vitro translated full-length and internally deleted tau polypeptides were compared directly in a microtubule co-sedimentation assay. The average Kd and the standard error of the mean are shown for each tau polypeptide, where n = the number of times each polypeptide was tested. The P box corresponds to the proline-rich region that can regulate microtubule binding activity indirectly (20, 32).

Since this internal deletion did not include deletion of an adjacent inter-repeat along with repeat 3, comparison of this mutant with full-length 3-repeat tau involved loss of the repeat as well as disruption of the normal spacing relationships between repeats and inter-repeats. Therefore, we next tested each of these variables individually.

First, to eliminate the effects of disrupted spacing, we tested 3RR3-IR, which deletes repeat 3 and the R3-R4 inter-repeat, effectively replacing repeat 3 with repeat 4 while keeping repeat 1 and the R1-R3 inter-repeat in place. In contrast to the 80-fold loss of activity observed with 3RR3, 3RR3-IR binds microtubules only 2.9-fold less effectively than does full-length 3-repeat tau. These comparisons indicate that spacing is critical for optimal core unit function. In addition, these data demonstrate that repeat 4 can substitute, at least in large part, for repeat 3 within the core microtubule binding unit.

Next, in order to test the importance of the R1-R3 inter-repeat to core unit function, we compared 3RIR-R3 with 3RR3-IR; these two constructs differ only by the presence of either the R1-R3 inter-repeat or the R3-R4 inter-repeat within their core microtubule binding unit. As seen in Fig. 4, 3RIR-R3 is severely compromised in microtubule binding affinity, demonstrating that the R1-R3 inter-repeat is critical for core unit function in 3-repeat tau.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The major findings of this study can be summarized as follows. First, 3-repeat tau possesses a core of microtubule binding activity residing within the first two repeats and their intervening inter-repeat. Second, within this core, specific sequence elements and spacing are essential for proper function. Third, the carboxyl-terminal sequences flanking the core microtubule binding unit in 3-repeat tau strongly enhance the microtubule binding affinity of the core unit.

The "Linear View" of Tau Structure and Function, Origins and Shortcomings-- Early biophysical and microscopic data suggested that tau has very little secondary or tertiary structure in solution (16, 19) and that it may be best described as a highly elongated, even "rod-like" molecule (17, 18). Minimal secondary structure was predicted from the derived amino acid sequence of the cloned tau cDNA (4). This conclusion was consistent with earlier observations demonstrating that tau retains its solubility and microtubule assembly and stabilizing activities even after exposure to harsh conditions such as treatment with perchloric acid or boiling (29, 30). These observations led many investigators to regard tau as a highly flexible and extended protein. Schweers et al. (19) suggested that tau in solution may be as much as 77% random coil and behave like "Gaussian coils" or "worm-like chains," in which the direction of the polypeptide backbone chain varies in a more-or-less random fashion.

This view of tau structure has influenced strongly models of tau binding to microtubules. Observations that the saturating molar ratio of tau binding to polymerized tubulin dimers is ~1:2.5 (16, 17), together with the identification of 3-4 repeated sequences in the carboxyl-terminal microtubule-binding half of tau (4, 8-11, 31), led to a model involving a "multiplicity of binding" mechanism. This model depicted a single tau molecule stretched across 3-4 tubulin subunits in the microtubule lattice with each 18-amino acid repeat forming an independent association with a separate tubulin subunit. This idea was supported by truncation analyses in which sequentially larger deletions from the carboxyl end of tau led to a processive loss of microtubule binding affinity (20, 21). These results were interpreted to suggest that tau could assume multiple conformations, pivot, and perhaps even migrate on the surface of the microtubule.

On the other hand, considerable data inconsistent with a simple linear view of sequentially arranged, independent tubulin binding domains exist. Gustke et al. (22) showed that intact 3-repeat tau, 4-repeat tau, and various constructs with multiple repeats deleted have similar saturation stoichiometries (one tau per two tubulin dimers). In addition, the data here suggest that repeat 4 has no effect on the microtubule binding affinity of 3-repeat tau, and our previous work (21) showed that repeats 3 and 4 make minimal contributions in 4-repeat tau. Furthermore, we have shown previously that sequentially larger amino-terminal deletions through the proline-rich and repeat regions of 3-repeat tau and 4-repeat tau lead to distinct patterns of loss and gain of microtubule binding activity (32), suggesting structural and functional complexity in the microtubule binding domain. None of these observations would be predicted if tau behaved like a string of 3-4 independent tubulin-binding sites.

The First Two Repeats and Their Intervening Inter-repeat Function as a Core Microtubule Binding Domain in Both 3-Repeat Tau and 4-Repeat Tau-- Many investigators (21, 32-34) have demonstrated that the microtubule binding activity in tau and MAP2 is restricted to the repeat/inter-repeat region of the each protein. In the case of 3-repeat tau, the data in Fig. 2 demonstrate that significant losses of microtubule binding activity within this region are not observed until the truncations reach repeat 3, i.e. repeat 4 and the R3-R4 inter-repeat are of minimal significance. Complete loss of microtubule binding activity occurs when truncations extend to include repeat 1. Analogous truncation analyses of 4-repeat tau revealed a similar pattern (21). In this case, major losses of microtubule binding activity were not observed until the truncations reached repeat 2, i.e. repeat 4, the R3-R4 inter-repeat, repeat 3, and the R2-R3 inter-repeat were of only minor significance. In considering all of these data, the key observation is that the critical sequences occupy the same relative positions downstream of repeat 1 (i.e. the R1-R3 inter-repeat and repeat 3 sequences in 3-repeat tau and the R1-R2 inter-repeat and repeat 2 in 4-repeat tau). Thus, in both 3-repeat and 4-repeat tau, the first two repeats and their intervening inter-repeat constitute a core microtubule binding domain. This conclusion is especially apparent when the relative contribution to microtubule binding affinity for each segment of each tau isoform is viewed graphically (Fig. 5).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Relative contributions made to microtubule binding affinity by different regions in 3-repeat tau and 4-repeat tau, as determined by truncation analyses of mutants with end points located on either side of the sequence being assessed. Regions possessing inherent microtubule binding activity are shaded light gray, and sequences affecting microtubule binding activity indirectly are shaded dark gray. The contributions for 3-repeat tau sequences were obtained from the data in Fig. 2A. The contributions for 4-repeat tau sequences are from Ref. 21. The contributions of sequences amino-terminal to repeat 1 in both 3-repeat and 4-repeat tau are from Ref. 32. For technical reasons, it is not possible to calculate a precise magnitude for the contribution of the R1-R3 IR in 3-repeat tau, but a reasonable approximation is provided. The black bar below the R1-R2 inter-repeat and repeat 2 in the 4-repeat tau figure marks the 31-amino acid region by which 3-repeat and 4-repeat tau differ, i.e. the sequences encoded by exon 10.

In our earlier work on 4-repeat tau (21), we did not recognize explicitly that repeat 1, the R1-R2 inter-repeat, and repeat 2 constitute a core microtubule binding domain. This followed from the fact that slight losses of microtubule binding affinity were observed with truncation of repeat 3 and repeat 4, although these losses were minor (<2-fold in each case; see Fig. 5). However, when viewed together with the present data for 3-repeat tau (Fig. 5), we suggest that the total body of data supports the conclusion that both 3-repeat and 4-repeat tau possess a core microtubule binding domain at the corresponding locations in their primary structures. Consistent with this interpretation, the vast majority of the tau point mutations causing neurodegenerative disease maps to the sequences contained within these proposed core units (for reviews, see Refs. 6 and 35).

Isoform-specific Differences in Tau Structure-Function and Regulation-- Despite the similarity between the 3-repeat and 4-repeat tau core microtubule binding domains, important differences exist. The data in Fig. 1 demonstrate an ~3-fold quantitative difference in the strength of microtubule binding affinity between 3-repeat and 4-repeat tau. However, it should be emphasized that there are also qualitative differences between 3-repeat and 4-repeat tau action. For example, the site-directed mutagenesis data in Fig. 3 demonstrate that the R1-R2 inter-repeat and the R1-R3 inter-repeat interact with microtubules, at least in part, via different mechanisms. This is consistent with earlier experiments demonstrating that a synthetic peptide corresponding to the R1-R2 inter-repeat could compete with 4-repeat tau but not 3-repeat tau for binding to microtubules (i.e. 4-repeat tau interacts with microtubules with at least some unique features relative to 3-repeat tau; see Ref. 21). Since structure leads to function, it follows that 3-repeat and 4-repeat tau may possess at least some distinct functional capabilities. Indeed, isoform-specific qualitative differences have been observed in tau-transfected cultured cells (36, 37).

In addition, both the present work and previous work (21) indicate that flanking sequences affect the microtubule binding ability of tau in an isoform-specific manner, thereby introducing additional qualitative regulatory differences between the different isoforms. Here, our data suggest a strong effect of the carboxyl-terminal sequences on 3-repeat tau binding affinity, whereas previous work has shown that these same sequences confer almost no effect upon microtubule binding affinity in 4-repeat tau. Previously, we and others (20, 32) have observed that the sequences flanking the repeat region on the amino side have a more pronounced effect upon microtubule binding affinity in 3-repeat tau than they do in 4-repeat tau. Consistent with these observations, we have shown similar isoform-specific effects of the flanking sequences in microtubule assembly assays (data not shown). Taken together, these data suggest the existence of isoform-specific, intramolecular regulatory folding interactions. This conclusion is consistent with immunological epitope mapping data suggesting that there are intramolecular interactions between sequences located on either side of the repeat region (38). Finally, recalling that the vast majority of the phosphorylation sites in tau reside in the sequences flanking the repeat:inter-repeat region (reviewed in Refs. 1, 35, and 39), it follows that there is vast capacity for isoform-specific regulation through the flanking sequences.

"Induced Fit" Model for Isoform-specific Tau-Microtubule Binding Interactions-- In Fig. 6, we present a schematic view by which tau-microtubule interactions may occur. This view integrates biophysical observations suggesting that tau has little structure in solution with data presented here suggesting that the microtubule binding conformation of tau is complex and isoform-specific. Our model is based on the induced fit mechanism that occurs between many enzymes and their substrates (40, 41). In this model, tau has a highly flexible and extended conformation in solution. Upon contact with microtubules, tau structure becomes more ordered. Initial interactions form between cognate binding sites on tau and the microtubules, which in turn lead to stabilizing intramolecular interactions within tau that help guide it into a stable, folded microtubule binding conformation. This model accommodates the observation that tubulin is a highly polymorphic protein that changes conformation readily in response to binding interactions (42). Taken together with the proposed highly flexible and extended nature of tau in solution, these properties are ideally suited for an induced fit mechanism, since they would allow subtle changes in tubulin conformation and larger changes in tau conformation.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Isoform-specific tau-microtubule interaction hypothesis. This model integrates an induced fit perspective along with isoform-specific action by 3-repeat and 4-repeat tau (see "Discussion"). In this model, tau has little higher ordered structure when it is in solution, consistent with biophysical studies (16, 18, 19). Upon interaction with microtubules, each tau isoform adopts a more complex and isoform-specific folded structure involving intramolecular interactions between the core microtubule binding domains and flanking regions, which in turn could differentially influence microtubule behavior. Each sphere represents an - or -tubulin monomer, as labeled. The P box corresponds to the proline-rich region that can regulate microtubule binding activity indirectly (20, 32).

In our model, we suggest that the initial binding between tau and microtubules might be mediated by the microtubule binding cores composed of the first two repeats and the inter-repeat located between them, both in 3-repeat and 4-repeat tau. Subsequent intramolecular folding interactions, driven both by isoform-specific sequences within the core microtubule binding unit and isoform-specific influences of the flanking sequences, would lead each tau isoform to assume distinct microtubule binding conformations. These different conformations, in turn, could lead to isoform-specific functional differences in terms of the tau-microtubule interaction, which would in turn lead to differential regulation of microtubule behavior. The details of these isoform-specific differences remain to be determined and will likely require the acquisition of atomic level structural information.

Although this model is speculative, it is more consistent with all of the available data than the widely held linear view of tau structure and function and makes several important testable predictions for future investigations.

Relevance to Pathological Tau Filament Formation and Neurodegenerative Disease-- Recent work (6, 24-26, 35) has shown that point mutations in the tau gene coding region, as well as mutations that alter splicing efficiency and thereby alter the ratio of wild type 4-repeat tau:wild type 3-repeat tau, segregate genetically with pathological tau filament formation, cell death, and a variety of neurodegenerative disorders collectively termed FTDP-17.

The mechanistic cause of cell death from tau dysfunction/deregulation is an especially important but poorly understood issue. Since all of the tau/FTDP-17 mutations thus far described are dominant, it has been suggested that the mutations may lead to a "gain of toxic function" (43). One frequently discussed mechanism for this toxic function is the formation of abnormal tau fibers, which in turn could activate the apoptotic pathway. However, there are alternative mechanisms also worth considering. For example, another mechanism consistent with a dominant phenotype is oligomerization. Indeed, recent data suggest that tau can form clusters in a microtubule-dependent manner, at least in vitro (44). However, whereas this mechanism could easily explain the coding region tau mutations, it is less obvious how it would explain the splicing mutations since the resulting proteins are wild type in sequence.

It is also possible that alterations in the level of different tau activities, or the balance among them, might trigger apoptosis. For example, tau is well known to suppress microtubule dynamics (45-47). It is also well established that drugs that alter normal microtubule dynamics can send cells down an apoptotic pathway even at very low concentrations (for example see Ref. 48). It follows, therefore, that the FTDP-17 tau mutations may lead to altered regulation of microtubule dynamics, which in turn leads to apoptosis. Although treatment of cells with these drugs does not lead to aberrant tau fiber formation, this event may require more time than is possible in these cell culture experiments.

Finally, if there are indeed qualitative differences between 3-repeat tau and 4-repeat tau action as suggested here, this will be an essential factor to consider in assessing the precise molecular mechanisms underlying neuronal cell death caused by structural and regulatory mutations in the tau gene.

Conclusion-- Taken together with previous work, we suggest that 3-repeat and 4-repeat tau assume complex and distinct microtubule binding structures that are regulated differentially, which suggests that they may possess isoform-specific functional capabilities. Such isoform-specific functions are likely to be extremely important to both normal tau action as well as the onset of neurodegenerative disease.

	ACKNOWLEDGEMENTS

We are enormously grateful to Herb Miller and Les Wilson for their insightful suggestions and their many tubulin contributions. We also thank Virginia Lee and Ken Kosik for several valuable conversations and Kathy Foltz, Beth Hinkle, Maryann Jordan, Vicky Makrides, Monte Radeke, and Avi Rodal for helpful comments on the manuscript.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant RO1 NS35010 (to S. C. F.), the California Department of Health Services, Alzheimer's Disease Program Grant 15716, and a National Research Service award fellowship (to M. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Biology, Brandeis University, Waltham, MA.

^§ Present address: DAKO Corp., Carpinteria, CA.

^¶ Present address: AMGEN, Inc., Thousand Oaks, CA.

To whom correspondence should be addressed: Neuroscience Research Institute and Dept. of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA 93106. Tel.: 805-893-2659; Fax: 805-893-2005; E-mail: feinstei@lifesci.ucsb.edu.

Published, JBC Papers in Press, September 12, 2000, DOI 10.1074/jbc.M007489200

	ABBREVIATIONS

The abbreviations used are: MAP(S), microtubule associated protein(s); IR, inter-repeat; 3R, 3-repeat; 4R, 4-repeat; PCR, polymerase chain reaction; Pipes, 1,4-piperazinediethanesulfonic acid.

	REFERENCES

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