BACKGROUND
the ca2+-dependent activator protein for secretion  family consists of two members, caps1/cadps <dig>  <cit>  and caps2/cadps <dig>  <cit> .
caps <dig> was shown to play a role in the atp-dependent priming step of ca2+-triggered dense-core vesicle  exocytosis by binding to both phosphatidyl inositol  <dig> -bisphosphate and dcvs in response to ca2+ increase  <cit> ; it also interacts with the dopamine receptor  <cit> .
a study of caps1-deficient mice suggested that caps <dig> is involved in catecholamine loading of dcvs in embryonic chromaffin cells  <cit> .
on the other hand, caps <dig> is associated with secretory vesicles containing the neurotrophins bdnf and nt- <dig> in the parallel fiber terminals of cerebellar granule cells, and is involved in the release of bdnf and nt- <dig>  <cit> .
caps <dig> is widely localized across various brain regions, and shows overlapping distribution patterns with bdnf in many areas  <cit> .
our recent studies of caps2-deficient mice indicated that caps <dig> plays pivotal roles in bdnf release, cellular phenotypes , and behavioral phenotypes   <cit> .
the expression of an aberrant splicing variant of caps <dig> in some patients with autism has also been reported  <cit> .
the autistic isoform, caps2Δexon <dig>  is missing exon  <dig>  which encodes the dynactin 1-interacting domain   <cit> .
exogenously expressed caps2Δexon <dig> failed to localize to axons but accumulated in somato-dendritic areas in cultured cerebellar and cortical neurons, suggesting a possible disturbance of local caps2-mediated neurotrophin release  <cit> .
in this study, we further explore splicing variations in caps <dig> and identified five additional splice isoforms.
we characterized the expression patterns and neurotrophin release properties of these isoforms.
RESULTS
using cdna probes comprising the sequence of both exons  <dig> and  <dig> of caps <dig>  we screened a p <dig> mouse cerebellar cdna library for splicing variants of caps <dig>  in addition to the original clone designated caps2a , which was identical to the one that we previously reported  <cit> , we isolated five additional variants, probably derived from full-length transcripts, designated caps2b , 2c , 2d , 2e , and 2f .
sequence analyses against genome databases showed that these sequences could be produced by the different usage of the  <dig> exons of the caps <dig> gene .
all splicing acceptor and donor sequences of these clones were consistent with the ag/gt rule.
the full-length caps <dig> protein consists of the following functional domains, as shown in figure 2: a dynactin 1-interacting domain , a c <dig> domain, a pleckstrin homology  domain and a munc13-1-homologous domain .
caps2b lacks only exon  <dig>  which encodes a part of the mhd.
caps2a lacks two exons,  <dig> and  <dig>  which encode part of the mhd sequence.
caps2c lacks two exons, exon  <dig> encoding a c-terminal part of the ph domain, and exon  <dig>  in contrast to these three long isoforms , caps2d, 2e and 2f have large c-terminal large truncations from exon  <dig>   <dig>  and  <dig>  respectively; thus, these isoforms have neither the mhd nor the c-tail domain, which mediates the association of caps with dcvs  <cit> .
these three short isoforms  have an additional unique exon at their 3' end.
the shortest isoform, caps2f, has only the first four authentic exons and none of the functional domains identified so far.
all isoforms  have exon  <dig>  which contains the domain interacting with p150glued  <cit> .
the mouse caps <dig> exon  <dig> coding sequence is identical to the human caps <dig> exon  <dig> coding sequence, which is absent in some autistic patients.
as shown in figure 3a, no exon  <dig> skipping was observed during the development of the cerebellum, from e <dig> to p <dig>  provided there were sufficient pcr cycles.
moreover, no exon  <dig> skipping was observed in whole brain, thymus, lung, heart, liver, spleen, kidney, or testis from p <dig> or p <dig> mice .
these results suggest that exon  <dig> skipping does not normally occur in mouse caps <dig>
we examined the patterns of caps <dig> splicing during cerebellar development, by rt-pcr using specific primer sets for each isoform.
caps2b is the only isoform that contains exon  <dig>  therefore, rt-pcr with a primer pair spanning exon 21-exon  <dig> resulted in a longer band  for caps2b in addition to a shorter band  for caps2a and 2c.
interestingly, there was a peak in the expression of caps2a and/or 2c at p <dig>  whereas caps2b was up-regulated throughout development .
the expression of caps2d reached a peak at p <dig> and decreased thereafter, whereas that of both caps2e and 2f reached a peak between p <dig> and p <dig>
among the eight mouse tissues tested, the brain, lung, kidney, and testis showed expression of all isoforms except for caps2b .
interestingly, expression of caps2b was detectable only in the brain.
among these isoforms, the expression level of caps2f was the lowest as shown by its requiring the highest cycle number of pcr cycles to detect it .
we next examined the cellular distribution of caps <dig> isoform mrnas in p <dig> and p <dig> mouse brains by in situ hybridization analyses.
exon  <dig>  which contains a part of the ph-domain coding sequence, is skipped in caps2c, but is included in caps2a, 2b, 2d, and 2e .
the probe for exon  <dig> showed expression in the hippocampal formation, neocortex, midbrain, and cerebellar granule cells .
exon  <dig>  which contains a part of the mhd coding sequence, is skipped in caps2a and 2c, but is included in caps2b .
the probe for exon  <dig> showed an expression pattern with the most prominent level in cerebellar granule cells .
expression of caps2d, 2e and 2f was also observed mainly in cerebellar granule cells .
moreover, there was no apparent difference in the expression patterns of these isoforms in the p <dig> cerebellar cortex , as shown in figure 4k.
caps family proteins are essentially soluble proteins, but a fraction of them is capable of being associated with membrane  <cit> .
therefore, we next examined the subcellular fractionation patterns of these splicing variant proteins, exogenously expressed in pc <dig> cells.
all isoforms were stably expressed in transfected pc <dig> cells .
caps <dig> isoforms containing an intact ph domain  were recovered from the membrane fraction, although a major portion of each isoform was included in the soluble fraction.
on the other hand, caps <dig> isoforms lacking a part of the ph domain  or the entire ph domain  were not detectable in the membrane fraction, suggesting that the ph domain has a role in the membrane association of caps <dig> protein, as previously shown  <cit> .
we next examined the expression of the endogenous proteins produced from these splice isoform mrnas in the mouse brain.
immunoreactive bands corresponding to short isoforms  were observed at low to undetectable protein levels in the mouse cerebellum .
in addition, the previous report showed that in non-neural tissue such as pituitary, lung, pancreas and kidney, only a  <dig> kda band immunoreactive for caps <dig> was detected  <cit> .
at the mrna level, more reaction cycles and time were needed for the amplification of transcripts encoding the short isoforms by rt-pcr and for the colorimetric detection of transcripts by in situ hybridization, respectively, indicating that the expression levels of the genes encoding the short isoforms are very low.
these results indicate that the function of caps <dig> in vivo is apparently due to the long isoforms  rather than the short isoforms .
however, the functional role of the short isoforms remain to be studied.
to analyze functional differences between splicing variants, we carried out a bdnf secretion assay .
to investigate the effect of these isoforms on bdnf releasing activity, we exogenously expressed the three long isoforms  together with bdnf in pc <dig> cells.
short isoforms  were not analyzed because of their large c-terminal truncation  and very low expression levels in mouse brain .
as shown in figure  <dig>  the levels of bdnf released into the culture medium in response to high-kcl stimulation were increased approximately two-fold by the expression of caps2a or 2b compared with cells without exogenous caps <dig> expression.
caps2c-expressing cells showed lower bdnf-releasing activity than caps2a- or 2b-expressing cells; however, caps2c-expressing cells also showed an increase in bdnf release in response to high-kcl stimulation .
moreover, there was no significant difference in basal bdnf release between caps2c-expressing cells and cells expressing either of the other two isoforms .
there was a partial deletion of the ph domain in caps2c .
the partial ph domain of plc-gamma  <dig> is known to interact with a complementary partial ph-like domain of another protein and elicit its function  <cit> .
therefore, it is possible that caps2c has some bdnf-releasing activity, although its activity is lower than that of intact ph domain-containing isoforms.
interestingly, expression of the exon 22-containing isoform was up-regulated in the developing mouse cerebellum, whereas exon 22-skipped isoforms were expressed transiently, peaking at p <dig>  moreover, the exon 22-containing isoform showed higher bdnf-releasing activity than other long isoforms .
on the other hand, as described in our previous report, caps <dig> immunoreactivities are detected in both the soma and axon of cerebellar granule cells at p <dig>  but almost exclusively in the axon at p <dig>  <cit> .
these results suggest that the exon  <dig> splicing patterns are related to high releasing activity and axonal localization in cerebellar granule cells.
caps2-mediated regulation of neurotrophin actions in terms of their releasing dose and subcellular localization may be important for normal cerebellar development.
the splicing of exon  <dig> is interesting for reasons other than the developmental differences in expression; that is, the exon 22-containing isoform was detectable only in the brain, whereas exon 22-skipped isoforms were widely expressed in various tissues.
exon  <dig> encodes part of the mhd, specifically a proline-rich domain .
proline-rich motifs occurred often in many protein-protein interaction sites, including one recognized by the src homology  <dig>  domain.
our preliminary screening of protein candidates that interact with the exon 22-encoded region, by yeast two-hybrid, isolated five candidates; however, none of these had an sh <dig> domain .
future analyses of caps <dig> proline-rich domain-interacting proteins will clarify the significance of the alternative splicing of exon  <dig>
CONCLUSIONS
we explored the alternative splicing diversity of caps <dig> and identified six caps <dig> isoforms.
the alternative splicing of exon  <dig>  was associated with a difference in tissue distribution and a developmental expression pattern.
our study showed that an intact ph domain is required for the association of caps <dig> with the membrane and efficient bdnf release activity.
on the other hand, exon 3-skipping, found in autistic patients, was not observed in the mouse tissues tested.

 the ca2+-dependent activator protein for secretion family consists of two members, caps1/cadps and caps2/cadps. caps was shown to play a role in the atp-dependent priming step of ca2+-triggered dense-core vesicle exocytosis by binding to both phosphatidyl inositol -bisphosphate and dcvs in response to ca2+ increase; it also interacts with the dopamine receptor. a study of caps1-deficient mice suggested that caps is involved in catecholamine loading of dcvs in embryonic chromaffin cells. on the other hand, caps is associated with secretory vesicles containing the neurotrophins bdnf and nt- in the parallel fiber terminals of cerebellar granule cells, and is involved in the release of bdnf and nt-. caps is widely localized across various brain regions, and shows overlapping distribution patterns with bdnf in many areas. our recent studies of caps2-deficient mice indicated that caps plays pivotal roles in bdnf release, cellular phenotypes, and behavioral phenotypes. the expression of an aberrant splicing variant of caps in some patients with autism has also been reported. the autistic isoform, caps2Δexon is missing exon which encodes the dynactin 1-interacting domain. exogenously expressed caps2Δexon failed to localize to axons but accumulated in somato-dendritic areas in cultured cerebellar and cortical neurons, suggesting a possible disturbance of local caps2-mediated neurotrophin release. in this study, we further explore splicing variations in caps and identified five additional splice isoforms. we characterized the expression patterns and neurotrophin release properties of these isoforms.  using cdna probes comprising the sequence of both exons and of caps we screened a p mouse cerebellar cdna library for splicing variants of caps in addition to the original clone designated caps2a, which was identical to the one that we previously reported, we isolated five additional variants, probably derived from full-length transcripts, designated caps2b, 2c, 2d, 2e, and 2f. sequence analyses against genome databases showed that these sequences could be produced by the different usage of the exons of the caps gene. all splicing acceptor and donor sequences of these clones were consistent with the ag/gt rule. the full-length caps protein consists of the following functional domains, as shown in figure 2: a dynactin 1-interacting domain, a c domain, a pleckstrin homology domain and a munc13-1-homologous domain. caps2b lacks only exon which encodes a part of the mhd. caps2a lacks two exons, and which encode part of the mhd sequence. caps2c lacks two exons, exon encoding a c-terminal part of the ph domain, and exon in contrast to these three long isoforms, caps2d, 2e and 2f have large c-terminal large truncations from exon and respectively; thus, these isoforms have neither the mhd nor the c-tail domain, which mediates the association of caps with dcvs. these three short isoforms have an additional unique exon at their 3' end. the shortest isoform, caps2f, has only the first four authentic exons and none of the functional domains identified so far. all isoforms have exon which contains the domain interacting with p150glued. the mouse caps exon coding sequence is identical to the human caps exon coding sequence, which is absent in some autistic patients. as shown in figure 3a, no exon skipping was observed during the development of the cerebellum, from e to p provided there were sufficient pcr cycles. moreover, no exon skipping was observed in whole brain, thymus, lung, heart, liver, spleen, kidney, or testis from p or p mice. these results suggest that exon skipping does not normally occur in mouse caps we examined the patterns of caps splicing during cerebellar development, by rt-pcr using specific primer sets for each isoform. caps2b is the only isoform that contains exon therefore, rt-pcr with a primer pair spanning exon 21-exon resulted in a longer band for caps2b in addition to a shorter band for caps2a and 2c. interestingly, there was a peak in the expression of caps2a and/or 2c at p whereas caps2b was up-regulated throughout development. the expression of caps2d reached a peak at p and decreased thereafter, whereas that of both caps2e and 2f reached a peak between p and p among the eight mouse tissues tested, the brain, lung, kidney, and testis showed expression of all isoforms except for caps2b. interestingly, expression of caps2b was detectable only in the brain. among these isoforms, the expression level of caps2f was the lowest as shown by its requiring the highest cycle number of pcr cycles to detect it. we next examined the cellular distribution of caps isoform mrnas in p and p mouse brains by in situ hybridization analyses. exon which contains a part of the ph-domain coding sequence, is skipped in caps2c, but is included in caps2a, 2b, 2d, and 2e. the probe for exon showed expression in the hippocampal formation, neocortex, midbrain, and cerebellar granule cells. exon which contains a part of the mhd coding sequence, is skipped in caps2a and 2c, but is included in caps2b. the probe for exon showed an expression pattern with the most prominent level in cerebellar granule cells. expression of caps2d, 2e and 2f was also observed mainly in cerebellar granule cells. moreover, there was no apparent difference in the expression patterns of these isoforms in the p cerebellar cortex, as shown in figure 4k. caps family proteins are essentially soluble proteins, but a fraction of them is capable of being associated with membrane. therefore, we next examined the subcellular fractionation patterns of these splicing variant proteins, exogenously expressed in pc cells. all isoforms were stably expressed in transfected pc cells. caps isoforms containing an intact ph domain were recovered from the membrane fraction, although a major portion of each isoform was included in the soluble fraction. on the other hand, caps isoforms lacking a part of the ph domain or the entire ph domain were not detectable in the membrane fraction, suggesting that the ph domain has a role in the membrane association of caps protein, as previously shown. we next examined the expression of the endogenous proteins produced from these splice isoform mrnas in the mouse brain. immunoreactive bands corresponding to short isoforms were observed at low to undetectable protein levels in the mouse cerebellum. in addition, the previous report showed that in non-neural tissue such as pituitary, lung, pancreas and kidney, only a kda band immunoreactive for caps was detected. at the mrna level, more reaction cycles and time were needed for the amplification of transcripts encoding the short isoforms by rt-pcr and for the colorimetric detection of transcripts by in situ hybridization, respectively, indicating that the expression levels of the genes encoding the short isoforms are very low. these results indicate that the function of caps in vivo is apparently due to the long isoforms rather than the short isoforms. however, the functional role of the short isoforms remain to be studied. to analyze functional differences between splicing variants, we carried out a bdnf secretion assay. to investigate the effect of these isoforms on bdnf releasing activity, we exogenously expressed the three long isoforms together with bdnf in pc cells. short isoforms were not analyzed because of their large c-terminal truncation and very low expression levels in mouse brain. as shown in figure the levels of bdnf released into the culture medium in response to high-kcl stimulation were increased approximately two-fold by the expression of caps2a or 2b compared with cells without exogenous caps expression. caps2c-expressing cells showed lower bdnf-releasing activity than caps2a- or 2b-expressing cells; however, caps2c-expressing cells also showed an increase in bdnf release in response to high-kcl stimulation. moreover, there was no significant difference in basal bdnf release between caps2c-expressing cells and cells expressing either of the other two isoforms. there was a partial deletion of the ph domain in caps2c. the partial ph domain of plc-gamma is known to interact with a complementary partial ph-like domain of another protein and elicit its function. therefore, it is possible that caps2c has some bdnf-releasing activity, although its activity is lower than that of intact ph domain-containing isoforms. interestingly, expression of the exon 22-containing isoform was up-regulated in the developing mouse cerebellum, whereas exon 22-skipped isoforms were expressed transiently, peaking at p moreover, the exon 22-containing isoform showed higher bdnf-releasing activity than other long isoforms. on the other hand, as described in our previous report, caps immunoreactivities are detected in both the soma and axon of cerebellar granule cells at p but almost exclusively in the axon at p. these results suggest that the exon splicing patterns are related to high releasing activity and axonal localization in cerebellar granule cells. caps2-mediated regulation of neurotrophin actions in terms of their releasing dose and subcellular localization may be important for normal cerebellar development. the splicing of exon is interesting for reasons other than the developmental differences in expression; that is, the exon 22-containing isoform was detectable only in the brain, whereas exon 22-skipped isoforms were widely expressed in various tissues. exon encodes part of the mhd, specifically a proline-rich domain. proline-rich motifs occurred often in many protein-protein interaction sites, including one recognized by the src homology domain. our preliminary screening of protein candidates that interact with the exon 22-encoded region, by yeast two-hybrid, isolated five candidates; however, none of these had an sh domain. future analyses of caps proline-rich domain-interacting proteins will clarify the significance of the alternative splicing of exon  we explored the alternative splicing diversity of caps and identified six caps isoforms. the alternative splicing of exon was associated with a difference in tissue distribution and a developmental expression pattern. our study showed that an intact ph domain is required for the association of caps with the membrane and efficient bdnf release activity. on the other hand, exon 3-skipping, found in autistic patients, was not observed in the mouse tissues tested. 
caps proteins were mostly recovered in soluble fractions, but some were present in membrane fractions, except for caps2c and 2f, both of which lack the ph domain, suggesting that the ph domain is important for membrane association. in contrast to caps2a and 2b, caps2c showed slightly decreased bdnf-releasing activity, which is likely due to the c-terminal truncation of the ph domain in caps2c. in the brain, all isoforms showed predominant expression patterns in the cerebellum. in the developing cerebellum, caps2b showed an up-regulated expression pattern, whereas the other isoforms exhibited transiently peaked expression patterns. these results demonstrate that the splicing variations correlate with their expression patterns and intracellular distribution, and affect bdnf release; however, whether or not the short forms possess activities other than bdnf release, for example as natural dominant-negative isoforms, remains to be determined. in this study, we defined exons in the mouse caps gene and identified six alternative splicing variants, caps2a-f. caps2a is an isoform lacking exons and which encode part of the munc13-1-homologous domain. on the other hand, a mouse counterpart of caps2Δexon was not detected in the mouse tissues tested.
 ca2+-dependent activator protein is a secretory vesicle-associated protein involved in the release of neurotrophin. we recently reported that an aberrant, alternatively spliced caps mrna that lacks exon is detected in some patients with autism. splicing variations in mouse caps and their expression and functions remain unclear.  in this study, we defined exons in the mouse caps gene and identified six alternative splicing variants, caps2a-f. caps2a is an isoform lacking exons and which encode part of the munc13-1-homologous domain. caps2b lacks exon caps2c lacks exons and caps2d, 2e, and 2f have c-terminal deletions from exon exon and exon respectively. on the other hand, a mouse counterpart of caps2Δexon was not detected in the mouse tissues tested. caps2b was expressed exclusively in the brain, and the other isoforms were highly expressed in the brain, but also in some non-neural tissues. in the brain, all isoforms showed predominant expression patterns in the cerebellum. in the developing cerebellum, caps2b showed an up-regulated expression pattern, whereas the other isoforms exhibited transiently peaked expression patterns. caps proteins were mostly recovered in soluble fractions, but some were present in membrane fractions, except for caps2c and 2f, both of which lack the ph domain, suggesting that the ph domain is important for membrane association. in contrast to caps2a and 2b, caps2c showed slightly decreased bdnf-releasing activity, which is likely due to the c-terminal truncation of the ph domain in caps2c.  this study indicates that, in mouse, there are six splicing variants of caps, and that these are subdivided into two groups: a long form containing the c-terminal mhd and a short form lacking the c-terminal mhd. these results demonstrate that the splicing variations correlate with their expression patterns and intracellular distribution, and affect bdnf release; however, whether or not the short forms possess activities other than bdnf release, for example as natural dominant-negative isoforms, remains to be determined. 