(Received for publication, December 20, 1995; and in revised form, February 27, 1996)
From the
We have isolated cDNA clones encoding novel proteins belonging
to the cadherin family. These novel proteins are encoded by two
distinct mRNA species generated by alternative splicing from a single
gene, and based on preferential expression in the pituitary gland and
brain, we named it PB-cadherin. One mRNA species encodes long type
PB-cadherin composed of 803 amino acid residues with a longer
cytoplasmic domain, whereas the other species encodes short type
PB-cadherin composed of 694 amino acid residues with a shorter
cytoplasmic domain. Both long and short type PB-cadherin contain five
repeats of a cadherin motif in the extracellular domain, the
transmembrane domain, and the cytoplasmic domain, and the deduced amino
acid sequences have a 30% homology to those of E-, N-, and P-cadherins.
Although the primary structure of N-terminal amino acids is identical
between long and short type PB-cadherin, the following structures in
the cytoplasmic regions are completely different. The long type
PB-cadherin but not the short type contains the putative
catenin-binding domain. When these two distinct forms of PB-cadherins
were stably expressed in L cells, L cells expressing long type
PB-cadherin or short type PB-cadherin both acquired a
Ca-dependent cell adhesion property, thereby
indicating that both types of PB-cadherin are responsible for
Ca
-dependent cell adhesion. Persistent expression of
PB-cadherin mRNA was found in the brain of rat embryos at least from
embryonic day 15 to the postnatal period. In situ localization
of PB-cadherin mRNA in the adult rat brain indicated that PB-cadherin
mRNA is expressed in the inner granular layer of the olfactory bulb,
Purkinje cell layer of the cerebellum, and in the pineal gland.
PB-cadherin may play an important role in morphogenesis and tissue
formation in neural and non-neural cells for the development and
maintenance of the brain and neuroendocrine organs by regulating
cell-cell adhesion.
The morphogenetic process involved in cellular aggregation,
segregation, and migration is mediated and controlled by a large and
complex number of cell adhesion molecules that exhibit a well-regulated
spatiotemporal pattern of expression during development and
regeneration. Cadherins are cell adhesion molecules originally
identified as a cell surface molecule responsible for
Ca-dependent cell adhesion(1) . The
homophilic interaction of cadherin confers cell-cell binding
interaction and adhesion specificity on cells that relate to
segregation, morphogenesis, neural network formation, and tumor
metastasis(2, 3, 4) .
Early
characterization and molecular cloning revealed the presence of three
distinct cadherin molecules E-, ()N-, and P-cadherin, in
which their cell and tissue specificity and temporal expression are
quite
different(5, 6, 7, 8, 9) .
Cadherins are transmembrane proteins consisting of an extracellular
domain that confers homophilic Ca
-dependent cell-cell
binding, a transmembrane domain, and a cytoplasmic domain. The
extracellular domain contains five cadherin repeat motifs and mediates
calcium-dependent cell-cell interaction. The cytoplasmic domain of
cadherin interacts with intracellular proteins,
-,
-, and
-catenins(10) .
-Catenin interacts with cytoskeletal
proteins, whereas
-catenin is considered to regulate the function
of cell-cell adhesion by tyrosine
phosphorylation(11, 12, 13) .
In addition to classical E-, N-, and P-cadherins, recent work(14, 15, 16, 17) revealed that cadherin-related molecules are structurally diverse and that they constitute a cadherin superfamily. R-cadherin, B-cadherin, OB-cadherin, and cadherin 4-11 conserve a membrane spanning structure in classic cadherins. In contrast, T-cadherin lacks both the transmembrane domain and the conserved cytoplasmic domain but is attached to the plasma membrane anchored with a glycosyl phosphatidylinositol(18) . Protocadherins contain 6 or 7 extracellular repeats of the cadherin motif and the cytoplasmic domain not homologous to that of other cadherins(19) . Furthermore, the Drosophila fat gene was described to be a tumor suppressor gene and contains 34 cadherin motifs in the extracellular domain; its cytoplasmic domain has no homology with that of vertebrate cadherin(20) . Desmogleins, pemphigus vulgaris antigen, and desmocollins were identified as the adhesion molecule localized at the desmosome(21, 22, 23, 24, 25) . The extracellular domain of these molecules has homology with classical cadherins, but cytoplasmic domains differ from those of classical cadherins. These diverse cadherin family molecules are thought to confer diverse cell and tissue specificities.
Involvement of cadherins in complex morphogenetic processes has been well noted, for example in neural tissue development. At the stage of neural tube closure, neural precursor cells express N- and E-cadherin, but the neural crest cells express c-cad6B, a homolog of cadherin 6, without expressing N- and E-cadherins(26) . When the neural crest cells migrate outward from the neural tube, expression of c-cad6B disappears, while the cells begin to express c-cad7. Thus, in addition to a diverse repertoire of cadherin molecules, temporal expression of these cadherin superfamily molecules in a cell- and tissue-specific manner is likely to regulate cellular aggregation and segregation in a cell- and tissue-specific manner during complex morphogenic processes.
During efforts to molecularly clone hepatocyte growth factor (HGF)-related genes(27) , we isolated the cDNA clone encoding a novel protein belonging to the cadherin superfamily. The deduced amino acid sequence indicated that this cadherin has 24-51% homology with other cadherins and consists of an extracellular domain that includes five cadherin repeat motifs, a transmembrane domain, and two forms of the cytoplasmic domain. Here we report the primary structure and the localization of this novel cadherin-related molecule. Because of its predominant expression in pituitary gland and brain, we named it ``PB-cadherin.''
Rat brain cDNA synthesis was performed according to
the ZAP Express cDNA synthesis kit (Stratagene) and
screened using
P- labeled extracellular domain of short
type PB-cadherin as a probe. Eleven positive clones were identified
from 7.5
10
plaques, and the cDNA fragment was
subcloned into pBK-CMV phagemid vector (Stratagene). Determination of
the DNA sequence was carried out according to the dideoxynucleotide
chain termination method described above.
L cells were transfected with the resultant expression plasmid by the calcium phosphate method using a mammalian transfection kit (Stratagene), and stable transfectants were isolated by G418 selection. Individual clones were screened by RNA dot blot analysis, and clones exhibiting high expression of PB-cadherins were used for the cell aggregation assay.
Figure 1: Nucleotide sequence and deduced amino acid sequence of PB-cadherin short type (A) and long type (B). Nucleotides and amino acid residues are numbered on the right. The putative signal peptide is underlined. Possible N-glycosylation sites are denoted with closed triangles. The transmembrane domain is double underlined. A potential polyadenylation site is boxed.
In addition to a tissue-specific
expression, Northern hybridization revealed that two distinct mRNAs
were transcribed in both the pituitary gland and the brain (Fig. 2A) when cDNA for the extracellular region of
PB-cadherin was used as the hybridization probe (see below). We
therefore constructed a rat brain cDNA library, and re-screened the
library, using a cDNA fragment for the extracellular region of
PB-cadherin as a probe. Eleven positive clones were obtained from 7.5
10
phage plaques. Sequence analysis revealed the
presence of a distinct cDNA from the originally isolated cDNA obtained
from the pituitary cDNA library.
Figure 2:
Northern blot analyses of PB-cadherin
expression in rat tissues. A, 1 µg of poly(A) RNA prepared from adult rat pituitary and brain; B, 30
µg of total RNA prepared from various tissues of adult rats; C, 30 µg of total RNA prepared from fetal and postnatal
rat brain were electrophoresed, transferred, and hybridized with the
P-labeled extracellular domain of PB-cadherin cDNA. The
same blot was rehybridized as in A with
P-labeled
cDNA for rat glyceraldehyde phosphate dehydrogenase (GAPDH),as
a control. The lower panels of B and C show
the 18 and 28 S rRNA bands, as visualized by ethidium bromide
staining.
The open
reading frame begins with an ATG initiation codon at position 409 of
long type PB-cadherin and at position 520 of short type PB-cadherin, in
which both ATG codons are in agreement with the Kozak
criteria(33) . Long type PB-cadherin terminates with a stop
codon at position 2848, while short type PB-cadherin at position 2602.
The nucleotide sequences of 2333 bp (nucleotide 78-2410 in long
type PB-cadherin and nucleotide 189-2521 in short type PB-cadherin)
were completely identical in both long and short types PB-cadherin, but
the following sequences (nucleotide 2411-3502 in long type
PB-cadherin and 2522-4153 in short type PB-cadherin) were
different. Thus, the same amino acid sequences that encode the
extracellular domain, transmembrane domain, and a part of the
cytoplasmic region of 23 amino acids are identical in both long and
short types PB-cadherin. The poly(A) tail was not
found in short type PB-cadherin, but the coding region of PB-cadherin
long type is followed by 655 bp of 3`-untranslated region that contains
polyadenylation signal sequences upstream of the poly(A)
tail.
PB-cadherin contains the signal sequence and postulated proteolytic cleavage site of cadherin precursor polypeptides. Cleavage of the peptide at the endogenous protease cleavage site RXKR is one of the posttranslational modifications common to cadherins, and the RVKR site is contained in PB-cadherin(34) . The deduced amino acid sequence of mature PB-cadherin exhibits structural homology with the cadherin family (Fig. 3). The extracellular domain consists of five repeats of a cadherin-specific motif, and one putative transmembrane domain is located between the extracellular domain and the cytoplasmic domain. Long type PB-cadherin has a large cytoplasmic domain and short type PB-cadherin has a small one. There are three possible N-linked glycosylation sites in the extracellular domain.
Figure 3: Alignment of deduced amino acid sequence with cadherins. Rat PB-cadherin and the members of the cadherin family are aligned and numbered on the right. Residues found in all of the cadherins are marked with a dot. The boxed amino acid sequences are the cadherin motifs in the repeated extracellular domain of the cadherin family. The shaded boxes are the cysteine residues conserved among the cadherin family. EC1-5, extracellular domain; TM, transmembrane domain; CP, cytoplasmic domain; N-cad, mouse N-cadherin; E-cad, mouse E-cadherin; P-cad, mouse P-cadherin; OB-cad, mouse OB-cadherin; T-cad, chicken T-cadherin.
In the extracellular fifth cadherin motif (EC5), all four cysteine residues are conserved. In EC1 the N-terminal WV is conserved in these cadherins, except for T-cadherin. On the other hand, the HAV sequence in the EC1 domain, which is considered to confer adhesion specificity (36) , is replaced with QAR in PB-cadherin, as well as OB-cadherin.
The size of the cytoplasmic domain of long type PB-cadherin is similar to those of typical cadherins. The cytoplasmic domain of long type PB-cadherin has relatively low homology with those of cadherins (<40.1%) (Table 1). However, the 70-amino acid stretch at the C terminus of long type PB-cadherin has higher homology (46%) than those of other cytoplasmic regions (Fig. 3). These homologous domains are known to be catenin-binding domains in other cadherins. In contrast, the short type PB-cadherin has only 50 amino acid residues in the cytoplasmic region, but 120 amino acid residues at the C terminus that contained catenin-binding sites are deleted. The cytoplasmic domain of the short type PB-cadherin has no significant homology with other members of the cadherin family.
Based on the structural characteristics of long and short type PB-cadherin, we hypothesized that these distinct mRNAs might be generated by alternative splicing. For elucidation, the same blot was hybridized with specific probes corresponding to respective cytoplasmic domain. The result indicated that the long type PB-cadherin mRNA was 4 kb, whereas the short type was 9 kb in the brain and 10 kb in the pituitary gland, respectively (data not shown). Fig. 2B shows tissue distribution of a 4-kb long type PB-cadherin mRNA. Long type PB-cadherin mRNA was strongly expressed in the brain and to a lesser extent in the pituitary gland. Very low levels of mRNA expression were noted in lung and heart with no expression in other organs, including submandibular gland, thymus, liver, spleen, adrenal, and kidney.
To examine developmental changes in PB-cadherin mRNA expression, RNA was prepared from rat brain at embryonic days 15 and 19 and postnatal day 1, and Northern hybridization was performed using a probe corresponding to the consensus extracellular domain. The mRNA level was persistently expressed in the brain, at a high level during late fetal to neonatal developmental stages (Fig. 2C).
Figure 4:
Genomic Southern blot analysis. Rat
genomic DNA was digested with an excess of PstI (lane
1), EcoRI (lane 2), and EcoRV (lane
3), electrophoresed, transferred, and hybridized with a P-labeled fragment of PB-cadherin cDNA. Size markers are
in kb.
Figure 5:
The
expression of PB-cadherin mRNAs and cell aggregation assay of
PB-cadherin transfectant L cells. A, the expression of
PB-cadherin mRNAs in stable transformant of L cells. 20 µg of total
RNA prepared from long type, short type, and mock transfected cells
were electrophoresed, transferred, and hybridized with the P-labeled extracellular domain of PB-cadherin cDNA. The lower photograph shows the 18 and 28 S rRNA bands, as
visualized by ethidium bromide staining. B, cell aggregation
assay. Long type, Short type, and Mock transfectant cells were treated with 0.01% trypsin in the presence
of 1 mM CaCl
and allowed to aggregate in the
Hepes-buffered Ca
-/Mg
-free
Hanks' solution with 1 mM CaCl
. Aggregation
did not occur when mock transfectants were used. Long type PB-cadherin
transfectants showed stronger adhesive activity than short type
PB-cadherin transfectants. Bars represent 100
µm.
Each transfectant was
morphologically similar to the parental cells. To examine
Ca-dependent cell adhesion, a cell aggregation assay
was done. Single cells treated with trypsin reaggregated in the
presence of 1 mM CaCl
, but these cells did not
aggregate with trypsinization without CaCl
(Fig. 6).
Long type PB-cadherin-transfected cells were more aggregate than the
short type PB-cadherin transfectants (Fig. 5B and Fig. 6). However, the activity was not seen in the parental
cells and L cells transfected with the plasmid only. These results
suggest that PB-cadherin has Ca
-dependent adhesive
activity, which is typical for the cadherin family.
Figure 6:
Ca-dependent cell
aggregation of PB-cadherin transfectants. Aggregation of transfectants
and parental cells (L cells) is shown. Aggregation index (N15/N0) represents the ratio of the total particle
number in the cell suspension after 15 min incubation (N15) to
the initial particle number (N0), with or without 1 mM CaCl
.
Figure 7: In situ localization of PB-cadherin mRNA in adult rat brain. A and B, olfactory bulb; IGr, inner granular layer; Gl, glomerular layer; AON, anterior olfactory nucleus; C, primary olfactory cortex: Pir, piriform cortex; D and E, cerebellum: Pur, Purkinje cell layer; F, Pi, pineal gland; G, pineal gland obtained using a sense probe for PB-cadherin. Bars represents 500 µm in B, C,and D and 250 µm in A, E, F,and G, respectively.
In the olfactory bulb, intense hybridization was observed in the inner granular layer and the glomerular layer, whereas external and internal plexiform layers and the mitral cell layer were devoid of any signal. In the cerebral cortex, labeling was observed in the olfactory cortex, whereas specific hybridization signals were not detected in other cortices. Strong hybridization signals were located in the pineal gland. In the cerebellum, dense signals were observed in the Purkinje cell layer, but the granule cell layer, molecular layer, and deep cerebellar nuclei were not labeled. No significant signals were observed in the diencephalon, mesencephalon, pons, and oblongata.
Cadherins are involved in the morphogenesis and maintenance
of tissue architecture by regulating Ca-dependent
cell-cell adhesion. Distinct expression patterns of cadherins
coexpressed in varying combinations in a cell- and tissue-specific
manner confer segregation, segmentation, and homeostasis of the tissue
architecture. In developing neural retina, early embryo retinas
incubated with antibody to N-cadherin tended to dissociate and could
not be maintained as a tissue formation (37) . The presence and
expression of multiple types of cadherins thus enable specification of
diverse tissue specificities. We cloned and characterized a novel
member of cadherin, PB-cadherin, which is predominantly expressed in
the pituitary gland and the brain. We also found that two types of
PB-cadherin generated by alternative splicing from a single gene are
functional in Ca
-dependent cell adhesion.
Cadherins bind cells by means of homophilic interaction, but cadherins have a binding preference for their own type. A stretch of N-terminal 113 amino acids located in the cadherin repeat motif determines the specificity of cadherins(38) . Synthetic peptides with an amino acid sequence corresponding to that of the specific binding site containing the HAV sequence can inhibit the cadherin-mediated cell-cell interactions(36) . However, even though the HAV sequence is conserved in E, N, P-cadherin, heterotypic adhesion between these cadherins was nil, thus cooperation with other sites is necessary for complete binding specificity. In the EC1 domain of PB-cadherin, the HAV motif is replaced by a QAR sequence; thus, the QAR motif may be involved in the adhesive function and binding specificities of PB-cadherins.
The intracellular domain of cadherin
plays a key role in cell-cell binding function through association with
cytoplasmic components -,
-, and
-catenins(10) .
-Catenin directly binds to E-, N-, and P-cadherins and
intermediates the connection of cadherins and cytoskeletal proteins.
Tyrosine phosphorylation of
-catenin has been found to affect the
intracellular adhesion system. A specific recognition site for
-catenin is located in the C-terminal stretch comprising the
72-amino acid domain(39, 40) . A comparison of the
amino acid sequences of the cytoplasmic domain of long type PB-cadherin
with those of the classical cadherins revealed a 33-38% sequence
identity. Even in the amino acid sequence of the C-terminal 70-amino
acid residues, sequence homology between long type PB-cadherin and
classical cadherin was no more than 44%. These values are significantly
lower than those seen within classical cadherins. Cytoplasmic domains
of mouse E-, N-, and P-cadherins share a 57-80% homology in amino
acid sequences. Recently, OB-cadherin, a new member of the cadherin
family, was isolated, and the cytoplasmic domain of OB-cadherin showed
only a 44-50% similarity to the classical cadherins as well as to
long type PB-cadherin(16) . We have yet to determine if
identical catenins that associate with classical cadherins interact
with PB-cadherins, but distinct homologous catenin molecules may
possibly do so.
Among the cadherin superfamily, desmocollins and
OB-cadherin have two distinct isoforms generated by alternative
splicing(24, 25) . Desmocollin III differs from
desmocollin II by additional 46-bp sequences located in the cytoplasmic
domain. The truncated form of OB-cadherin lacking the cytoplasmic
region is generated as a splice variant, but expression in tissues and
whether it is functional in cell-cell interaction are unknown. It is
notable that short type PB-cadherin that lacks the distinct
catenin-binding domain is synthesized as an alternative splice variant
from a single gene and that this short type PB-cadherin is expressed in
the pituitary gland and in the brain, at significant levels. Moreover,
even though short type PB-cadherin lacks the catenin-binding domain, it
is functional in the Ca-dependent interaction.
Therefore, the lack of the catenin-binding domain in short type
PB-cadherin suggests that its expression may result in a constitutive
homophilic binding of cells, without association of catenins by which
homophilic cadherin-cadherin interaction and cytoskeletal rearrangement
are regulated. Alternatively, short type PB-cadherin associates
specific cytoplasmic molecules distinct from catenins. In this context,
T-cadherin lacking the classical transmembrane and cytoplasmic domain
is attached to the plasma membrane through a glycosyl
phosphatidylinositol glycan, but T-cadherin remains functional with
regard to Ca
-dependent adhesion properties (18) .
In situ mRNA localization analysis showed that PB-cadherin is expressed predominantly in neurons of various regions in the adult rat brain. Localized distribution of PB-cadherin mRNA in the brain overlaps with that of N-cadherin, but cellular distribution is distinct(41) . PB-cadherin mRNA expression is confined to neuronal populations in the adult rat brain. Prominent hybridization signals were detected in the olfactory bulb, primary olfactory cortex, pineal gland, and Purkinje cells of the cerebellum. The Purkinje cell is one neuron that expresses only low levels of N-cadherin, but PB-cadherin mRNA was strongly expressed. It seems to be noteworthy that PB-cadherin is also expressed in tissues responsible for neuroendocrine functions, including pineal gland and pituitary gland. PB-cadherin is also expressed in PC12 rat pheochromocytoma cells originally derived from the adrenal medulla (data not shown). The mammalian pineal gland is an endocrine component in the regulation of photoperiodic responses. The endocrine function of the pineal gland that secretes melatonin is regulated by light via the nervous system (42) . In addition to the localization of PB-cadherin, PB-cadherin is expressed at high levels in fetal rat brains.
In conclusion, we cloned a novel type of cadherin, which we termed PB-cadherin. This cadherin is highly unique in its exclusive expression in the pituitary gland and in the brain, plus the presence of splicing variants. We predict that long and short types of PB-cadherin may have distinct roles, and both types of PB-cadherin may play a role in morphogenesis and tissue formation in neural and non-neural cells for the development and maintenance of the brain and neuroendocrine organs, through the potential to regulate cell-cell adhesion, and other functions such as signal transduction. Our ongoing studies directed at spatiotemporal expression of PB-cadherin and targeted disruption of the PB-cadherin gene may elucidate biological functions of this novel member of cadherin.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D83348 [GenBank]and D83349[GenBank].