From the Department of Neurology, Yale University School of Medicine, New Haven, Connecticut 06520
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ABSTRACT |
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M-SemF is a membrane-associated, neurally
enriched member of the semaphorin family of axon guidance signals. We
considered whether the cytoplasmic domain of M-SemF might possess a
signaling function and/or might control the distribution of M-SemF on
the cell surface. We identify a PDZ-containing neural protein as an M-SemF cytoplasmic
domain-associated protein (SEMCAP-1). SEMCAP-2 is a closely related nonneuronal protein. SEMCAP-1 has recently also
been identified as GIPC, by virtue of its interaction with the RGS
protein GAIP in vitro (De Vries, L., Lou, X., Zhao, G., Zheng, B., and Farquhar, M. G. (1998) Proc. Natl. Acad. Sci.
U. S. A. 95, 12340-12345). Expression studies support the
notion that SEMCAP-1(GIPC) interacts with M-SemF, but not GAIP, in
brain. Lung SEMCAP-2 and SEMCAP-1(GIPC) are potential partners for both GAIP and M-SemF. The protein interaction requires the single PDZ domain
of SEMCAP-1(GIPC) and the carboxyl-terminal four residues of M-SemF,
ESSV. While SEMCAP-1(GIPC) also interacts with SemC, it does not
interact with other proteins containing a class I PDZ binding motif,
nor does M-SemF interact with other class I PDZ proteins. Co-expression
of SEMCAP-1(GIPC) induces the redistribution of dispersed M-SemF into
detergent-resistant aggregates in HEK293 cells. Thus, SEMCAP-1(GIPC)
appears to regulate the subcellular distribution of M-SemF in brain,
and SEMCAPs could link M-SemF to G protein signal transduction pathways.
The creation and refinement of highly specific neuronal
connections is essential for the function of vertebrate nervous
systems. The molecular mechanisms guiding axonal extension and synapse formation are becoming better understood. Pioneering studies
demonstrated the general importance of neurotrophic factors and cell
adhesion molecules in supporting neurite outgrowth (1, 2). More recent studies have defined additional pathway-specific roles for the netrins,
ephrins, and semaphorins (3).
Semaphorins are an expanding family of glycoproteins expressed in the
nervous system and in many somatic tissues. There are more than 20 semaphorins reported at the present time (4). While they may have
diverse functions ranging from vasculogenesis (5, 6) to neoplastic
transformation (7) to immune modulation (8), their role in axon
repulsion is best documented. Members of the semaphorin family feature
an extracellular Sema domain of 500 amino acid residues with multiple
conserved cysteine residues (9). Collapsin-1 (semaphorin III/D), a
secreted semaphorin, was initially purified from chick brain as a
potent inducer of dorsal root ganglion neuronal growth cone collapse
(10). SemIII/D binds to neuropilin-1 on the axonal growth cone surface
(11, 12) and inhibits growth cone motility through a signal
transduction cascade, which may involve heterotrimeric G proteins,
collapsin response mediator proteins, and Rac1 (13-16). Analyses of
mice with targeted SemIII(D) gene deletions have verified the role of
the protein as a repulsive signal for selected sensory neurons in the
peripheral nervous system (5, 17).
Phylogenetic analysis has divided semaphorins into at least six groups.
Group II and group III are secreted semaphorins. Group VI semaphorins
associate with plasma membrane via a GPI linkage (18). Groups I, IV,
and V, which represent about half of known semaphorins, contain
transmembrane and cytoplasmic domains. Since they are integral membrane
proteins, these transmembrane semaphorins are thought to provide
contact-mediated guidance cues to developing neurons. There is only
limited evidence concerning specific physiologic roles for
transmembrane semaphorins. Antibody blockade of G-Sema I, a grasshopper
transmembrane semaphorin, interferes with Ti1 neuron axonal pathfinding
in the limb bud (19). Genetic analysis of its Drosophila
homologue, D-Sema I, suggests that it may also function as a repulsive
signal for selected motor and central nervous system axons (20).
Although expression of a secreted derivative of D-Sema I with a
truncated tail partially complements a loss of function mutation of
D-Sema I (20), several recent reports suggest that the cytoplasmic domains of transmembrane semaphorins may contribute to their biological functions. M-SemaG (h-CD100), which regulates B lymphocyte
differentiation, associates with a serine kinase through its
cytoplasmic domain (21). Mouse SemaVIb forms a complex with c-Src
through a proline-rich intracellular domain (22). These data raise the
possibility that the transmembrane semaphorins may function as
receptors, as well as ligands, and transduce extracellular binding
events to cytoplasmic changes. Alternatively, the cytoplasmic domains may control the spatial distribution and hence ligand activity of
transmembrane semaphorins. The activity of secreted collapsin (SemIII/D) requires dimerization and proteolytic activation (23, 24).
The clustering of ephrins due to membrane anchorage is critical for the
activation of Eph receptors and their biological activities (25).
M-SemF is a transmembrane semaphorin of unknown function identified by
its homology in the Sema domain (26). The predicted cytoplasmic portion
of M-SemF, 146 amino acids in length, contains a proline-rich region
that includes the consensus sequence for interaction with Src homology
3 domains as well as cytoskeletal proteins. In this report, we
demonstrate that the cytoplasmic domain of M-SemF associates
specifically with two related intracellular proteins containing single
PDZ domains. We isolated these proteins as M-SemF
cytoplasmic domain-associated
proteins (SEMCAP-1 and SEMCAP-2).1 SEMCAP-1 is
identical to GIPC, a protein interacting with the RGS protein GAIP
(27). Thus, SEMCAP(GIPC) may regulate the distribution of M-SemF and
link a semaphorin to G protein signaling cascades.
The MATCHMAKER two-hybrid assay system and mouse E17 cDNA
pGAD10 library were purchased from CLONTECH
Laboratories, Inc. (Palo Alto, CA). General chemicals of molecular
biology grade were obtained from Sigma, J. T. Baker Inc., or
American Bioanalytical (Natick, MA). Reagents for tissue culture were
from Life Technologies, Inc. Reagents used in yeast and bacteria
culture were from Difco. Restriction enzymes were obtained from New
England Biolabs (Beverly, MA), and thermophilic DNA polymerase
(Taq) was from Promega (Madison, WI). 9E10 ascites (M-5546),
anti-bIII tubulin monoclonal antibody (T-8660), fluorescein
isothiocyanate-conjugated IgG fraction of goat anti-rabbit IgG
(F-6005), and tetramethylrhodamine isothiocyanate-conjugated IgG
fraction of goat anti-mouse IgG (T-7782) were from Sigma. The IMAGE
Consortium expressed sequence tag cDNA clones were from Genome
Systems Inc. (St. Louis, MO).
Construction of Expression Vectors--
The fusion protein
expression vectors, pGBT-SEMF, pGBT-SEMF, and pGAD-SEMF, contain
sequence for amino acid residues 689-834 of mouse M-SemF in pGEX5X-3,
pGBT9, and pGAD424, respectively. pGBT-SEMB contains sequence for amino
acid residues 705-760 of mouse SemB in pGBT9 and was prepared with a
PCR fragment amplified from a plasmid containing SemB cDNA
using primers GCGAATTCCCACTGGGGGCGCTG and CCTCGAGTCACCACAGAATCCC.
pGBT-SEMC contains sequence for 95 amino acids from the carboxyl end of
mouse SemC in pGBT9 and was prepared with a PCR fragment amplified from
a mouse E17 cDNA library using primers CCCGGGCATGAAACTCTTCCTAAAGC
and CCCGGGTCATACCACAGAGTCTCGG. pGAD-NR2C contains amino acid residues
1046-1250 of rat NMDA glutamate receptor subunit 2C (NR2C) in pGAD10
and was derived from a XhoI-EcoRI cDNA
fragment of plasmid pNR2C4 (U08259). pGBT-PDZ1-3 and pGAD-PDZ1-3 contain sequences for amino acid residues 1-460 of rat postsynaptic density (PSD)-95 in pGBT9 and pGAD424 and were prepared with a PCR
fragment amplified from the PSD-95 cDNA (28) using primers GCGAATTCATGGACTGTCTCTGTATAG and CCTCGAGCACATCCCCAAGCG. The M-SEMF cytoplasmic domain mutant constructs, pGEX-SEMF-831, pGEX-SEMF-S832A, pGEX-SEMF-S832D, and pGEX-SEMF-V834A, were prepared with PCR fragments amplified from pGAD-SEMF using a forward primer based on the vector sequence and the reverse primers GCGAATTCACTCCTCTGGGTTGGAGTC, GCGAATTCATACTGAAGCCTCCTCTGG, GCGAATTCATGCTGAAGACTCCTCTGG, and GCGAATTCATACTGAATCCTCCTCTGG, respectively. The mutations have been
confirmed by DNA sequencing. pGBT-SEMCAP-1(GIPC), pGAD-SEMCAP-1(GIPC), and pCMVMycSEMCAP-1(GIPC) contain the full-length coding region of
SEMCAP-1(GIPC) plus an additional 16 amino acid residues before the
initiation methionine in pGBT9, pGAD10, and pCMVMyc, respectively. pGBT-SEMCAP-2 and pGAD-SEMCAP-2 contain the full-length coding region
of SEMCAP-2 with an additional 2 amino acid residues before the
initiation methionine in pGBT9 and pGAD424. The expression vector
pGBT-GAIP containing full-length coding region of GAIP was prepared
with a PCR fragment amplified from a mouse E17 cDNA library using
primers GAATTCATGCCCACCCCACATGAGG and GGATCCCTAGGCCTCAGAGGACTGTGG. The mammalian expression vectors pcDNA- M-SEMF,
pcDNA-SEMCAP-1(GIPC), and pcDNA-SEMCAP-2 consist of the
full-length coding region of the indicated proteins in pcDNA I (Invitrogen).
Yeast Two-hybrid Analysis--
The procedure for yeast
two-hybrid analysis has been described previously (29). In brief, yeast
reporter strain Hf7C was sequentially transformed with pGBT-SEMF and a
mouse E17 cDNA library cloned in the pGAD10 vector. Colonies that
grew under triple selection (Leu RNA Isolation and Northern Analysis--
Cellular RNA was
extracted from indicated murine tissues using the acid guanidium
thiocyanate/phenol/chloroform method (30). Twenty micrograms of total
RNA were separated on 1% agarose-formaldehyde gel and transferred to
Hybond N+ nylon membrane (Amersham Pharmacia Biotech) by
capillary elution with 20× SSC. SEMCAPs and M-SemF cDNA probes
were prepared by random priming with the Klenow fragment of DNA
polymerase in the presence of [ In Vitro Binding--
Glutathione S-transferase
(GST), GST-SEMF, and GST-SEMF mutant fusion proteins were expressed in
bacteria and purified on glutathione-agarose beads as described (32).
Recombinant SEMCAP proteins were transiently expressed in HEK293 cells
by calcium phosphate transfection using 20 µg each of
pCMVMycSEMCAP-1(GIPC), pcDNA-SEMCAP-1(GIPC), and
pcDNA-SEMCAP-2, respectively. Forty eight hours following
transfection, cells were resuspended in 1-3 ml of Dulbecco's
phosphate-buffered saline (DPBS, pH 7.4) plus 1 mM
phenylmethylsulfonyl fluoride and 10 µM leupeptin. The cytosol was collected after three freeze-thaw cycles in liquid nitrogen
and centrifuged at 13,000 rpm (30,000 × g) for 20 min at 4 °C. Aliquots (300 µl) of cytosol were incubated with 100 µg
of the designated fusion proteins immobilized on 20-µl
glutathione-agarose beads for 4 h at 4 °C. The beads were
washed five times with 1 ml of DPBS containing 0.1% Triton X-100. The
bound proteins were eluted in 100 µl of SDS sample buffer, and
20-µl aliquots were used for immunoblot analyses. Brain cytosol was
prepared by homogenizing mouse brain in 2.5 ml of DPBS/g of tissue
followed by centrifuging at 100,000 × g for 1 h
at 4 °C. The supernatant brain cytosol was collected and utilized
for the in vitro binding assay as described above.
Antisera Preparation--
The antisera against M-SemF were
prepared using GST-SEMF as the immunogen. Rabbits were initially
immunized with 100 µg of GST-SEMF fusion protein mixed with complete
Freund's adjuvant and were boosted with 100 µg of GST-SEMF in
incomplete Freund's adjuvant at 3-6-week intervals. For
anti-SEMCAP-1(GIPC) antisera, the rabbits were immunized with purified
MycSEMCAP-1(GIPC) fusion proteins from transfected HEK293 cells with
high level expression of the fusion protein. With high level
expression, the fusion protein forms a 1 M NaCl and 1%
Triton X-100-insoluble complex. The pellet from centrifugation in
NaCl/Triton X-100 at 30,000 × g for 15 min contained
MycSEMCAP-1(GIPC) fusion protein of >95% purity as judged by
Coomassie Blue staining of SDS-PAGE. Such pellets were sonicated in
DPBS and used for the immunization using the same protocol as for
GST-M-SEMF. The immunization service was performed at the Yale Animal
Resource Center.
Immunoblotting--
The immunoblotting procedure was carried out
as described by Wang and Strittmatter (29). The anti-M-SemF antisera
were used at a 1:2500 dilution. The anti-SEMCAP-1(GIPC) antisera were
used at a 1:2000 dilution. Immunoreactive proteins were visualized using TMB peroxidase substrate reagents (Vector), after incubation with
horseradish peroxidase-conjugated goat anti-rabbit IgG and horseradish
peroxidase-conjugated rabbit anti-mouse IgG.
Immunoprecipitation--
HEK293T cells were cotransfected by the
calcium phosphate method with the pCMVMycSEMCAP-1(GIPC) and
pcDNA-M-SEMF plasmids, 10 µg of each plasmid/100-mm plate. Twenty
four hours following transfection, cells from one 100-mm plate were
solubilized in 5 ml of lysis buffer (DPBS containing 1% Triton X-100,
0.2% SDS, 0.5% deoxycholate, and 1 mM
phenylmethylsulfonyl fluoride) for 1 h at 4 °C. One-ml aliquots
were centrifuged for 20 min at 13,000 rpm (30,000 × g)
at 4 °C. The supernatant was incubated overnight at 4 °C with 25 µl of undiluted anti-M-SemF or anti-SEMCAP-1(GIPC) or preimmune sera.
The samples were then incubated for an additional 2 h with 20 µl
of Protein G-agarose beads at 4 °C. After extensive washing of the
beads with lysis buffer, the bound proteins were solubilized in SDS
sample buffer and analyzed by immunoblot with the anti-M-SemF antiserum
and 9E10 monoclonal antibody.
Indirect Immunofluorescence Microscopy--
Transfected HEK293T
cells or rat E15 cortical cells (33) were cultured on coverslips for
48 h and then fixed with 4% paraformaldehyde in phosphate buffer
(100 mM sodium phosphate, pH 7.4, 150 mM NaCl) for 1 h at room temperature. Fixed cells were treated sequentially with permeabilization solution (0.1% Triton X-100 in phosphate buffer)
and blocking solution (1% bovine serum albumin in phosphate buffer)
and then were incubated with primary antibodies in blocking solution
overnight at 4 °C, followed by a 1-h incubation with secondary
antibodies at room temperature. After each antibody incubation, cells
were washed four times in phosphate buffer containing 0.1% bovine
serum albumin for 15 min. Anti-M-SemF serum, anti-SEMCAP-1(GIPC) serum,
and 9E10 ascites fluid were each used at 1:1000 dilution. The secondary
antibodies, fluorescein isothiocyanate-conjugated IgG fraction of goat
anti-rabbit IgG and tetramethylrhodamine isothiocyanate-conjugated IgG
fraction of goat-anti-mouse IgG, were diluted 1:100. After antibody
incubations, coverslips were rinsed in distilled water and mounted on
glass slides. Immunofluorescence was captured using either a CCD camera
(Astrocam TE3) or a laser scanning confocal image system (MRC600,
Bio-Rad), and digital images were processed with Adobe Photoshop software.
Triton X-100 Extraction of Membranes--
Brains from 2-day-old
rats (1.25 g) were homogenized in a glass homogenizer with 3 ml of
DPBS. Membrane fractions were collected by centrifuging at 100,000 × g for 1 h at 4 °C. Brain membranes were
rehomogenized in 3 ml of DPBS and collected by centrifugation. Washed
membranes were homogenized again in 12 ml of DPBS, and Triton X-100
(TX-100) was added slowly to a final concentration of 1%, followed by
a 1-h incubation at 4 °C. Insoluble membrane fractions were
collected by centrifugation and re-extracted with 1% TX-100 in DPBS.
The final membrane pellet was resuspended in DPBS and frozen at
SEMCAP-1(GIPC) Overlay Assay--
Brain membrane proteins and
293 cell lysates were separated by SDS-PAGE and transferred to
nitrocellulose membrane. The membrane was incubated with 5% dry milk
in TBS (20 mM Tris, pH 7.6, 137 mM NaCl, and
0.1% Tween 20) for 1 h at room temperature and then incubated
with purified MycSEMCAP-1 fusion protein (8 µg/ml) that had been
preincubated with a 1:2500 dilution of anti-SEMCAP antiserum for 2 h at room temperature. The bound MycSEMCAP-1-antibody complexes were
detected using horseradish peroxidase-conjugated goat anti-rabbit IgG
and visualized with TMB peroxidase substrate reagents.
Expression of M-SemF--
To explore possible functions of M-SemF,
we analyzed its mRNA and protein expression pattern in the mouse.
Northern analysis reveals that M-SemF mRNA is expressed primarily
in the nervous system. The mRNA is detected in lung at much lower
levels, whereas expression in kidney, liver, heart, and skeletal muscle
is almost undetectable (Fig.
1A). The expression of M-SemF
is first apparent at E12 and persists through adulthood at nearly
constant levels. This raises the possibility that M-SemF participates
both in axon guidance during development and in synaptic plasticity of
the adult central nervous system.
An antiserum generated against a GST/M-SemF cytoplasmic tail fusion
protein specifically recognizes an Mr 100,000 protein in M-SemF-expressing HEK293T cell membranes but not in control 293T cell membranes (Fig. 1B). The same protein species is
detected by immunoblot analysis of brain membranes. Coincident with the mRNA distribution, the expression of M-SemF protein is neurally restricted and continues into adulthood. Any protein expected to
interact with M-SemF should exhibit an overlapping pattern of expression.
Isolation of SEMCAPs--
To search for cellular proteins that may
interact with the cytoplasmic domain of M-SemF, we employed a yeast
two-hybrid screen of a E17 mouse cDNA library with a fusion protein
expressing the GAL4 DNA binding domain and the cytoplasmic tail of
M-SemF (residues 689-834) as bait. From 8 × 106
colonies, 22 clones were obtained that activate the expression of a
A BLAST search of nucleic acid data bases using the predicted amino
acid sequence identified a matching partial sequence derived from a
human cDNA that interacts with the carboxyl-terminal portion of the
transactivator (Tax) protein of human T cell leukemia virus type 1. Data base searches after this manuscript was in preparation revealed
that SEMCAP-1 is identical to GIPC, a protein identified by virtue of
its interaction with the RGS protein, GAIP (27). The search identified
two predicted open reading frames of Caenorhabditis elegans
genes, C35D10.2 and F44D12.6, with significant homology. We also found
several overlapping clones of mouse expressed sequence tags whose
sequences were highly homologous to SEMCAP-1(GIPC). From two of those
expressed sequence tag clones (IMAGE consortium, AA266091 and
AA107535), we obtained a 1.35-kb cDNA. The predicted 314-amino acid
residue polypeptide interacts specifically with the cytoplasmic tail of
M-SemF in yeast two-hybrid analysis (Fig. 2A). Thus, this
protein is designated SEMCAP-2.
The amino acid sequences of SEMCAP-1(GIPC) and SEMCAP-2 share 62%
identity, whereas those of C35D10.2 and F44D12.6 share 86% identity
with one another and 52% similarity with SEMCAP-1(GIPC) (Fig.
2B). Homology is distributed throughout these four proteins, but the most variable region is the amino terminus, and the two C. elegans gene products have extended carboxyl-terminal
regions of about 20 amino acids. In addition, an internal segment of
about 70 amino acids displays homology with sequences from a variety of
proteins including CASK, PSD-95, syntrophin, and ZO-1. Further analysis
reveals that this 70-amino acid segment aligns with the PDZ domains of
those proteins. PDZ domains are known to interact with the cytoplasmic
tail of transmembrane proteins and localize such proteins to sites of
specialized cell-cell contacts such as PSD and zona occludens (34, 35).
The interaction requires conserved amino acid residues at the carboxyl
terminus of the transmembrane protein. We examined the sequence of
M-SemF and found that the last four amino acid residues are
Glu-Ser-Ser-Val, which agrees with the consensus sequence of the class
I PDZ domain binding motif: X-(Ser/Thr)-X-Val
(36). This suggests that the cytoplasmic tail of M-SemF may associate
with cellular protein(s) through a PDZ domain. When the PDZ domains
from the four predicted SEMCAP proteins were compared with those from
PSD-95 and CASK, they displayed only 10-20% sequence identity.
However, conservation is high for residues most critical in the
formation of the carboxylate binding loop (hydrophobic
residue-Gly-hydrophobic residue) and for the histidine residue in helix
B (Fig. 2C) (37, 38).
SEMCAP-1(GIPC) also possesses weak sequence similarity to acyl carrier
proteins (residues 264-320) (27), but no other protein modules are
identifiable in the SEMCAPs. There is no recognizable transmembrane
domain or nuclear localization signals; therefore, SEMCAPs are
predicted to be cytosolic proteins. The partial human cDNA of Tax
interacting protein clone 2 (TIP-2) is 98% identical to SEMCAP-1(GIPC)
from amino acid 78 to the end (39). Thus, TIP-2 is a human homolog of
SEMCAP-1(GIPC). TIP-2 binding to Tax requires the class I PDZ binding
motif, Glu-Thr-Glu-Val, at the carboxyl terminus of Tax.
Characterization of SEMCAP Expression--
If the SEMCAPs are to
interact with M-SemF in vivo, then they must be co-expressed
in the same cells. To examine the potential of SEMCAPs for
physiological interaction with M-SemF, the mRNA and protein
distribution of SEMCAP-1(GIPC) and SEMCAP-2 were determined. Northern
analysis of SEMCAP-1(GIPC) reveals a single mRNA species of 1.8 kb,
whereas SEMCAP-2 Northern blotting shows the presence of a single
1.5-kb transcript (Fig. 3A).
Thus, it is very likely that we have obtained clones containing
full-length cDNAs for both SEMCAP-1(GIPC) and SEMCAP-2.
SEMCAP-1(GIPC) mRNA is most abundant in the brain; less prominent
in kidney and lung; and essentially undetectable in heart, liver, and
skeletal muscle of adult mice. In contrast, the SEMCAP-2 transcript is
present in kidney and lung but is undetectable in the brain, heart,
liver, and skeletal muscle of adult mice. SEMCAP-1(GIPC) mRNA is
expressed as early as E8, and expression persists throughout embryonic
and adult stages (Fig. 3B).
To characterize the SEMCAP proteins, three rabbit antisera against
full-length SEMCAP-1(GIPC) protein were prepared. Each antiserum
recognizes a 39-kDa protein in HEK293 cells transfected with
SEMCAP-1(GIPC) cDNA. The antisera also detect a 37-kDa protein in
SEMCAP-2 cDNA-transfected cells, while no immunoreactive protein is
detected in control cells (Fig. 3C). In P1 mouse tissue
extracts, a 39-kDa protein is detected by the anti-SEMCAP sera. The
39-kDa SEMCAP-immunoreactive protein, likely to be SEMCAP-1(GIPC), is most abundant in brain, kidney, and lung with less expression in heart,
liver, and skeletal muscle (Fig. 3C). In addition, a 37-kDa
protein is found in kidney and lung. The apparent molecular weight of
this protein suggests that it is SEMCAP-2. In adult mice, the
expression of 39-kDa SEMCAP-1(GIPC) is restricted exclusively to brain
and lung (Fig. 3C). In adult lung, the 37-kDa immunoreactive protein is likely to be SEMCAP-2. In adult kidney, the sera recognize a
major 35-kDa protein. Since SEMCAP-2 but not SEMCAP-1(GIPC) mRNA is
highly expressed in the adult kidney, the 35-kDa species might reflect
an adult kidney-selective post-translational modification of
SEMCAP-2.
We conclude that the SEMCAP-1(GIPC) and SEMCAP-2 mRNAs are
full-length and account for the distribution of SEMCAP proteins. Furthermore, the SEMCAP-1(GIPC) protein is expressed in a pattern consistent with an in vivo interaction with M-SemF in the
brain. In contrast, GAIP expression is not detectable in brain but is highest in liver, muscle, and lung (40). Thus, brain SEMCAP-1(GIPC) is
not a potential partner for GAIP. In the lung, all four proteins (GAIP,
M-SemF, SEMCAP-1(GIPC), and SEMCAP-2) are expressed and might interact.
The majority of GAIP in liver and muscle tissue does not have access to
SEMCAP-1(GIPC) or SEMCAP-2.
SEMCAPs Interact with the Carboxyl Terminus of M-SemF--
An
in vitro binding assay was performed to determine whether
SEMCAP-1(GIPC) interacts directly with the cytoplasmic tail of M-SemF.
GST-M-SemF fusion proteins were expressed in E. coli and immobilized on glutathione-agarose beads. Cell lysates from HEK293 cells transiently expressing MycSEMCAP-1(GIPC) were incubated with
immobilized GST-M-SemF fusion proteins. SEMCAP-1(GIPC) interaction with
GST-M-SemF fusion proteins was monitored by immunoblot analysis of
proteins bound to the agarose beads. No interaction is observed with
GST leader sequence, but MycSEMCAP-1(GIPC) binds strongly to wild type
GST-SEMF (Fig. 4A). Removal of
3 amino acid residues from the carboxyl terminus (GST-SEMF-831*)
abolishes the interaction with MycSEMCAP-1(GIPC). Substitution of the
serine at 832 with either alanine or aspartate (GST-SEMF-S832A and
GST-SEMF-S832D) results in the complete loss of association with
MycSEMCAP-1(GIPC). In contrast, the mutation of valine to alanine at
834 (GST-SEMF-V834A) shows little change of binding with
MycSEMCAP-1(GIPC). Thus, the in vitro binding experiments
confirm the direct interaction between the cytoplasmic tail of M-SemF
and SEMCAP-1(GIPC). As expected for a class I PDZ protein,
SEMCAP-1(GIPC) binding depends on the extreme carboxyl terminus of
M-SemF.
To examine whether endogenous SEMCAP-1(GIPC) protein could bind to the
cytoplasmic tail of M-SemF, brain cytosol was incubated with
immobilized GST-SEMF and GST-SEMF-831* fusion proteins. Association of
brain SEMCAP-1(GIPC) with M-SemF is observed by anti-SEMCAP immunoblot
of retained proteins (Fig.
5A). The truncated form of
SemF exhibits no brain SEMCAP-1(GIPC) binding. Thus, endogenous brain
SEMCAP-1(GIPC) binds selectively to the cytoplasmic tail of M-SemF and
requires the presence of the terminal 3 amino acid residues.
Specificity of SEMCAP Interaction with M-SemF--
The presence of
the PDZ binding motif at the cytoplasmic tail of M-SemF prompted us to
examine the intracellular domains of other transmembrane semaphorins
for similar sequences. Of published semaphorin sequences, only SemC
contains a PDZ binding motif, Asp-Ser-Val-Val, at its carboxyl terminus
(41). Both SemC and M-SemF belong to semaphorin group IV. To determine
if SemC would interact with SEMCAPs, the cytoplasmic domain of SemC was
analyzed with SEMCAP-1(GIPC) or SEMCAP-2 in the yeast two-hybrid
system. We also examined the cytoplasmic tail from SemB, a type IV
semaphorin with no predicted PDZ binding motif at the carboxyl end, as
a control. The SemB tail does not interact with either SEMCAP-1(GIPC) or SEMCAP-2. In contrast, both SEMCAP-1(GIPC) and SEMCAP-2 associate with the SemC tail (Fig. 4B). In the yeast two-hybrid
analysis, SemC does not bind as avidly to SEMCAP-1(GIPC) or SEMCAP-2 as does M-SemF.
The similarity among proteins carrying class I PDZ binding motifs
raises the possibility that SEMCAP-1(GIPC) and SEMCAP-2 will bind to
other proteins with PDZ binding sequences although the yeast two-hybrid
screen did not yield any known PDZ proteins. To examine this
possibility, SEMCAPs were coexpressed with the cytoplasmic tail from
NMDA glutamate receptor type 2C (NR2C, carboxyl terminus
Glu-Ser-Glu-Val). Despite the similarity in the carboxyl termini of
NR2C and M-SemF, neither SEMCAP-1(GIPC) nor SEMCAP-2 interacts with
NR2C (Fig. 4B). The PDZ domain of PSD-95 is known to
associate with NR2C in vivo (42). While PSD-95 binds
strongly to NR2C in the yeast two-hybrid system, there is no
interaction of PSD-95 with SemC or M-SemF.
As reported by De Vries et al. (27), SEMCAP-1(GIPC)
interacts with GAIP in the yeast two-hybrid system (Fig. 4C). SEMCAP-2 also exhibits similar affinity for GAIP as that of SEMCAP-1(GIPC) in
this assay (Fig. 4C). However, the interaction of both
SEMCAPs with GAIP is approximately one-tenth as strong as with M-SemF in this yeast assay. These results suggest that SEMCAP-1(GIPC) and
SEMCAP-2 are selective partners for M-SemF, SemC, and GAIP.
Major SEMCAP-1(GIPC)-binding Proteins in Brain--
We considered
whether the SEMCAP-1(GIPC) proteins utilized in the yeast two-hybrid
analysis are the major SEMCAP-1(GIPC)-binding proteins in brain. Brain
extracts were separated by SDS-PAGE and then overlaid with
SEMCAP-1(GIPC) (Fig. 6). M-SemF expressed
in HEK293T cells supports SEMCAP-1 binding in this overlay assay. The
two most prominent SEMCAP-1(GIPC)-binding proteins in brain membranes
exhibit apparent molecular masses of 100 and 33 kDa. The higher
molecular mass binding protein precisely co-migrates with M-SemF
immunoreactivity from brain. The identity of the 33-kDa band is not
clear, but it is notable that the mass is nearly identical to that
reported for GAIP in nonneuronal tissues (43). Thus, these assays are
consistent with M-SemF serving as one of the major binding sites for
brain SEMCAP-1(GIPC).
SEMCAP-1(GIPC) Clusters M-SemF in Transfected Cells--
To
examine the interaction between M-SemF and SEMCAP-1(GIPC) in a cellular
environment, full-length M-SemF and MycSEMCAP-1(GIPC) cDNAs were
coexpressed in HEK293T cells. Cotransfected cells were analyzed by
either immunoprecipitation or immunohistology. Proteins precipitated by
anti-M-SemF or anti-SEMCAP antisera were analyzed by immunoblotting
with anti-M-SemF antisera or with anti-Myc antibody (Fig.
5B). As expected, anti-M-SemF antisera immunoprecipitate the
100-kDa M-SemF protein from transfected cells, and anti-SEMCAP antisera
precipitate the MycSEMCAP-1(GIPC) protein. SEMCAP-1(GIPC) protein
co-immunoprecipitates with M-SemF protein, and anti-SEMCAP precipitates
complexes containing M-SemF protein. These data indicate that
SEMCAP-1(GIPC) and M-SemF form a complex when coexpressed in HEK293T cells.
Some PDZ proteins are capable of forming microscopically visible
clusters in heterologous cells and shifting their binding partners into
these clusters (44). We examined the distribution of M-SemF and
SEMCAP-1(GIPC) in HEK293 cells. When expressed alone, M-SemF is
distributed in a uniform fashion on the cell surface (Fig.
7A). In contrast,
overexpression of MycSEMCAP-1(GIPC) results in large aggregates of
Myc-immunoreactive protein. When the two proteins are co-expressed,
M-SemF localization is dramatically altered to resemble the
SEMCAP-1(GIPC) pattern. The clusters of M-SemF antigen in double
transfected cells are precisely coincident with SEMCAP-1(GIPC)
clusters. It is noteworthy that co-expression of GAIP and
SEMCAP-1(GIPC) does not lead to co-clustering (27), indicating that
GAIP may have other binding partners in these cells or that the lower
affinity of the GAIP/SEMCAP-1(GIPC) interaction as compared with the
M-SemF/SEMCAP-1(GIPC) interaction does not support clustering.
The size of the SEMCAP-1(GIPC) clusters in 293 cells suggested the
creation of a unique subcellular domain by SEMCAP-1(GIPC). Since
clustering by the PDZ protein PSD-95 is thought to responsible in part
for the existence of the detergent-insoluble nature of the postsynaptic
density, we considered whether SEMCAP-1(GIPC) and co-expressed M-SemF
exist in a state not dissociable by nonionic detergents. Immunoblot
analysis of membrane and detergent-extracted insoluble fraction from
293 cells indicates that detergent insolubility is a correlate of the
SEMCAP-1(GIPC) clusters (Fig. 7B). In the absence of
SEMCAP-1(GIPC), M-SemF is solubilized by the addition of Triton X-100,
but coexpression of SEMCAP-1(GIPC) results in the redistribution of a
majority of M-SemF to a detergent-insoluble fraction.
The detergent-insoluble nature of M-SemF/SEMCAP-1(GIPC) clusters in
HEK293T cells might correspond to a physiologically relevant state of
these proteins or be a condition created by high level expression in a
nonneuronal cell. Brain membranes were fractionated by Triton X-100
extraction and analyzed for the presence of immunoreactivity. While a
significant portion (about 60%) of SEMCAP-1(GIPC) is cytosolic, particulate brain SEMCAP-1(GIPC) is not extracted by Triton X-100 (Fig.
7B). Similarly, a proportion of M-SemF immunoreactivity in
the brain cannot be solubilized with Triton X-100. These data raise the
possibility that M-SemF and SEMCAP-1(GIPC) comprise part of the
postsynaptic density. However, this is highly unlikely, since PSD
fractions highly enriched for known PSD proteins are not enriched in
either M-SemF or SEMCAP-1(GIPC) (data not shown). Instead, these
proteins appear to associate with a detergent-resistant structure with
properties similar to that observed in co-transfected HEK293T cells.
To further define the endogenous distribution of these proteins, we
performed an immunohistochemical study of embryonic cortical neurons.
SEMCAP-1(GIPC) immunoreactivity is detected in neurons, and within cell
soma discrete intensely stained puncta are visualized (Fig.
8). Although not quite as large, these
aggregates appear similar to those observed in transfected HEK293T
cells (Fig. 7A). Similar immunohistochemical analysis of
M-SemF localization in cortical neurons was not technically feasible
with the available antisera. Together, these HEK293T and neuronal
studies demonstrate that SEMCAP-1(GIPC) can physically associate in
clusters with M-SemF in cells and alter M-SemF microscopic distribution
and biochemical properties.
SEMCAP-1(GIPC) Binds to the Cytoplasmic Tail of Brain
M-SemF--
In this study, we have identified a family of SEMCAP
proteins that interact with the cytoplasmic domain of M-SemF. The
interaction of these proteins can be detected by yeast two-hybrid
analysis, affinity chromatography, immunoprecipitation, double
immunofluorescence, and subcellular fractionation. Thus, it appears
likely that at least a fraction of brain M-SemF exists in a complex
with SEMCAP-1(GIPC). Several factors indicate that this interaction is
relatively specific: 1) the glutamate receptor subunit NR2C does not
interact with SEMCAP-1(GIPC); 2) PSD-95 does not interact with M-SemF;
and 3) none of the many known PDZ proteins were isolated in the yeast two-hybrid screen with M-SemF as bait. However, there is some promiscuity in this SEMCAP/semaphorin interaction in that SEMCAP-2 has
a similar binding profile to SEMCAP-1(GIPC) and that SemC binds nearly
as well as M-SemF to SEMCAP-1(GIPC). The SEMCAP family of proteins is
highly conserved through evolution from worm to mouse. There is no
information now available on the distribution or binding partners of
the two C. elegans members of the SEMCAP family.
Since SEMCAP-2 is expressed in the kidney and lung, but not the brain,
it cannot be a major binding partner for neuronal M-SemF. The
expression of both M-SemF and SEMCAP-2 in lung suggests that they may
form a lung-specific complex in vivo.
Additional Binding Partners for SEMCAP-1(GIPC) and
SEMCAP-2--
It remains possible that other binding partners for
SEMCAPs exist in particular cell types. It is not unusual for a PDZ
domain to interact with multiple binding partners, and vice
versa. For example, the PDZ domain in neuronal nitric-oxide
synthase associates with PSD-95 and CAPON (45, 46). The third PDZ
domain of PSD-95 interacts with CRIPT and neuroligin (47, 48).
Additional SEMCAP-1(GIPC) or SEMCAP-2 interactions may occur in
discrete cell types and may contribute to the assembly of physiological
relevant multiprotein complexes. In the brain, SEMCAP-1(GIPC) is
capable of interacting with an unidentified 33-kDa protein. The
in vitro binding studies of Farquhar and colleagues (27)
identified SEMCAP-1(GIPC) as a GAIP-interacting protein. We show here
that both SEMCAP-1 and SEMCAP-2 can interact with GAIP. The tissue
distribution studies raise the possibility that all four proteins
(M-SemF, GAIP, SEMCAP-1(GIPC), and SEMCAP-2) could interact in lung. In
the brain, only SEMCAP-1(GIPC) and M-SemF are expressed, not SEMCAP-2
or GAIP. It is an intriguing possibility that the 33-kDa
SEMCAP-1(GIPC)-binding protein in brain extracts is a GAIP-related
protein. The major sites of GAIP expression, liver and muscle (40),
exhibit neither SEMCAP-1(GIPC) nor SEMCAP-2 expression, indicating that
the majority of GAIP cannot be complexed with the two PDZ domain
proteins studied here. The PDZ domain of human SEMCAP-1(GIPC) (TIP-2)
can also bind the carboxyl terminus of the transactivator protein (Tax)
of human T cell leukemia virus type 1 in vitro (39). This
pathologic interaction may or may not be relevant in vivo.
We have not specifically measured the level of SEMCAP-1(GIPC)
expression in lymphocytes.
Binding Is Mediated by a PDZ Domain Type Interaction--
The
interactions between the semaphorin cytoplasmic tails and SEMCAPs are
likely to be mediated by the PDZ domain for several reasons. Both
M-SemF and SemC carry the class I PDZ binding motif in the carboxyl
termini. The sequence requirements in the M-SemF carboxyl terminus for
interacting with SEMCAPs are very similar to those characterized in
other PDZ domain associations. The PDZ domain is a polypeptide module
of about 100 amino acids that mediates protein/protein interactions.
Based upon the consensus binding sequences at the carboxyl termini, PDZ
domains are divided into two classes. Class I PDZ domains selectively
bind to the sequence motif Ser/Thr-X-Val, and class II PDZ
domains favor Phe/Tyr-X-Ala/Phe/Val (36). The crystal
structures of PDZ domains from two class I families and one class II
family have been determined (37, 38). Although the primary sequences
show only limited similarity, the secondary structures of different PDZ
domains are highly conserved, containing a core of six Clusters of M-SemF/SEMCAP-1(GIPC)--
The binding of M-SemF to
SEMCAP-1(GIPC) appears to create an aggregated complex that is
resistant to nonionic detergent extraction. Since PDZ proteins are
frequently found at specialized cell contacts, the biochemical
properties of the M-SemF-SEMCAP-1(GIPC) complex raise the possibility
that they are part of a specialized cellular domain in the brain. We
have excluded the possibility that M-SemF and SEMCAP-1(GIPC) are
selectively localized to PSDs, but we have not determined the nature of
M-SemF/SEMCAP-1(GIPC) clusters in the brain. It is conceivable that
they define a heretofore unrecognized structure in the developing and
adult nervous system. Immunohistologic analysis at the ultrastructural
level may be required to explore this issue.
PDZ domain proteins are best known for their localization to cell-cell
junctions in epithelial cells and neurons, where they bind to
cytoplasmic domains of channels or receptors and coordinate the
assembly of multiprotein complexes. Such PDZ proteins generally have
more than one PDZ domain as well as other recognized functional domains, such as Src homology 3, guanylate kinase, CaM kinase, or
tyrosine phosphatase domains. These multivalent, multiprotein complexes
provide both a structural basis and a signaling function for the
regulation of specific physiological activities. In contrast, SEMCAP-1(GIPC) and SEMCAP-2 have only a single PDZ domain, and no other
protein interacting domain is identified. However, since SEMCAP-1(GIPC)
is capable of forming aggregates in the absence of M-SemF, it is
possible that the single PDZ domain of SEMCAP-1(GIPC) in a multivalent
aggregate might assemble multiple associated proteins. The mechanism of
SEMCAP-1(GIPC) aggregation is unclear. Neither SEMCAP-1(GIPC) nor
SEMCAP-2 protein interacts with itself or one another other in the
yeast two-hybrid system (data not shown).
Biologic Functions of M-SemF--
The function of M-SemF remains
ill defined, although other semaphorin family members are known to
function as axonal guidance signals in the developing nervous system.
The identification of SEMCAP-1(GIPC) as an intracellular M-SemF binding
partner is consistent with a role for M-SemF as a ligand functioning in
cell surface communication. We have found no proteins with known signal
transduction activity associated with the cytoplasmic tail of M-SemF.
However, if SEMCAP-1(GIPC) or SEMCAP-2 were to link M-SemF to the RGS
protein GAIP in lung, it is conceivable that this could underlie
receptor-type function.
It is tempting to speculate that M-SemF clustering by SEMCAP-1(GIPC) is
required for biologic activity as a ligand for receptors on adjacent
cells directing axonal growth in the nervous system. The function of
another axonal guidance molecule, fasciclin II, depends upon
co-expression with the PDZ protein, Dlg (49, 50). Fasciclin II is a
member of the extensively studied immunoglobulin superfamily of
homophilic cell adhesion molecules. Unless Dlg is co-expressed with
fasciclin II, the latter protein is not found at developing
neuromuscular junctions, and these junctions are functionally
defective. Our results indicate that at least some members of the
semaphorin family of axonal guidance signals will require similar
PDZ-dependent localization and clustering for activity.
Model studies of clustered and unclustered signal transduction cascades
in prokaryotes have emphasized the dramatic effects that oligomerization can have for ligand receptor signaling complexes (51).
It is also of interest that both M-SemF and SEMCAP-1(GIPC) are
expressed at high levels in the mature nervous system. It is conceivable that this complex plays a role in synaptic plasticity of
the adult brain. Further studies defining the subcellular localization and perturbing the expression of M-SemF and SEMCAP-1(GIPC) will illuminate these issues.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, Trp
,
His
) conditions were assayed for
-galactosidase
activity. Library-derived plasmids from LacZ-positive yeast cells were
recovered and purified for further analyses. Liquid
-galactosidase
activities were assayed with doubly transformed SFY526 yeast reporter
cells using o-nitrophenyl
-D-galactopyranoside as the substrate. The DNA sequence
analysis of isolated clones was carried out utilizing the fluorescently conjugated dideoxynucleotide termination method with a thermal cycling
protocol on automatic DNA sequencers (Applied Biosystems model 377) at
the W. M. Keck Biotechnology Laboratory of Yale University.
-32P]dCTP.
Hybridization was performed as described previously (31). Washed nylon
membranes were exposed to Kodak BioMax film for 12-36 h with a Kodak
BioMax MS intensifying screen at
80 °C.
80 °C. For transfected cell membranes, HEK293T cell membranes were
treated with DPBS containing 1% TX-100 for 1 h on ice.
Particulate fractions were collected by centrifuging at 13,000 rpm
(30,000 × g) for 15 min at 4 °C. The resulting
membrane pellet was sonicated in DPBS containing 1% TX-100 and
incubated for 1 h on ice. Insoluble membrane fractions were
collected by centrifugation as described above.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Tissue distribution of mouse M-SemF.
A, Northern blot analysis of M-SemF expression. Total RNA
(20 µg) from the indicated embryonic and adult mouse tissues was
separated on an agarose-formaldehyde gel, transferred onto nylon
membrane, and hybridized to 32P-labeled M-SemF cDNA
probe. The probe was a 2.3-kb HindIII fragment containing
the majority of the M-SemF coding region. The probe detects a single
4.5-kb M-SemF mRNA whose expression is developmentally regulated
and is enriched in the adult nervous system. The positions of 28 and 18 S ribosomal RNA are shown by the bars at the
left. B, immunoblot analysis of M-SemF
immunoreactivity in transfected cells and mouse tissues. Cell membranes
(1% of a 100-mm dish) from HEK293T cells not transfected
(lane 1), transfected with a control plasmid
(lane 2), and transfected with pcDNA-M-SemF
(lane 3) were separated on SDS-PAGE, transferred
to a nitrocellulose membrane, and probed with a rabbit polyclonal
anti-M-SemF antiserum (1:2500 dilution). Total tissue homogenates (100 µg) from the indicated P1 and adult mouse tissues were processed for
immunoblotting using anti-M-SemF antiserum. The abbreviations used for
postnatal tissues are as follows. B, forebrain;
C, cerebellum; H, heart; K, kidney;
V, liver; L, lung; M, muscle;
S, spinal cord. The abbreviations used for embryonic tissues
are as follows. E8, 8-day embryo; E12, 12-day
embryo; E15, 15-day embryonic head; E20, 20-day
embryonic brain; P1, neonatal brain.
-galactosidase reporter gene. Two clones were false positives that
encoded transcription factors interacting with the GAL4 DNA-binding domain itself. Fifteen of the 22 clones derived from the same cDNA
and interacted specifically with the M-SemF cytoplasmic domain (Fig.
2A). The other five
specifically interacting clones were derived from a single cDNA
encoding a novel muscle-specific PDZ protein.2 The four most
variable inserts from the 15 clones were sequenced, the longest insert
being 1.7 kb. All four of the sequences contain a polyadenylate tail
and have 5'-ends within 150 bases of one another. A single extended
open reading frame was identified and was in frame with the GAL4
transactivation domain. The interacting protein is designated SEMCAP-1.
The SEMCAP-1 cDNA encodes an open reading frame of 333 amino acids.
The longest clone has 27 base pairs of 5'-untranslated region, and the
shortest clone is missing the first 40 amino acids from the conceptual
sequence.
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Fig. 2.
Identification of SEMCAP-1(GIPC) and
SEMCAP-2. A, interactions of SEMCAP-1(GIPC) and
SEMCAP-2 with the cytoplasmic domain of M-SemF in the yeast two-hybrid
system. SFY526 yeast cells were cotransformed with the DNA-binding
domain (pGBT) and activation domain (pGAD) fusion protein expression
vectors as indicated. Colonies were selected on synthetic plates
lacking leucine and tryptophan and cultured in selective liquid medium.
-Galactosidase activity was measured using o-nitrophenyl
-D-galactopyranoside as the substrate. Data are the
means ± S.E. from four independent colonies. Error
bars are hidden by the symbol in some cases.
B, amino acid sequence alignment of SEMCAP-1(GIPC),
SEMCAP-2, C35D10.2, and F44D12.4. Deduced amino acid sequences from the
indicated gene products were aligned using the CLUSTALW program at the
GenomeNet server (Kyoto University, Japan). C, alignment of
the PDZ domains from SEMCAP-1(GIPC), SEMCAP-2, C35D10.2, and F44D12.4
with the third PDZ domain of rat PSD-95 (class I) and the PDZ domain of
rat CASK (class II) using the CLUSTALW program. The secondary struc tures of the PDZ domains determined by the crystallographic
studies are indicated above the aligned sequences. Several
residues that are critical for the formation of the binding pocket are
shown in boldface type. The positions of the
starting and ending amino acid residue are indicated in
parentheses. The GenbankTM accession numbers of
the cited proteins are as follows: SEMCAP-1(GIPC), AF061263; SEMCAP-2,
AF061262; C35D10.2, U21324; F44D12.4, Z68298; rat PSD-95, M96853; rat
CASK, U47710.
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Fig. 3.
Expression of SEMCAP-1(GIPC) and
SEMCAP-2. A, Northern blot analysis of SEMCAP-1(GIPC)
and SEMCAP-2 expression. Total RNA (20 µg) from the indicated adult
mouse tissues was prepared for RNA blotting as described in the legend
to Fig. 1. The probes were 1.7-kb cDNA for SEMCAP-1(GIPC) and
1.5-kb cDNA for SEMCAP-2. The positions of 28 and 18 S ribosomal
RNA are shown by the bars at the left. The
SEMCAP-1(GIPC) probe detects a single 1.7-kb mRNA enriched in
brain. The SEMCAP-2 probe detects a single 1.5-kb mRNA expressed
exclusively in kidney and lung. B, Northern blot analysis of
SEMCAP-1(GIPC) expression during development. Total RNA from the
indicated embryonic and postnatal tissues were prepared for RNA blot.
Note that SEMCAP-1(GIPC) is expressed early in development
(E8) and persists at a nearly constant level in the brain
through postnatal periods. C, immunoblot analysis of
SEMCAP-1(GIPC) and SEMCAP-2 immunoreactivity in transfected cells and
mouse tissues. Total cell lysates (2% of a 100-mm dish) from
untransfected (lane 1), pcDNA-M-
SemF-transfected (lane 2),
pcDNA-SEMCAP-1(GIPC)transfected (lane 3),
or pcDNA-SEMCAP-2-transfected (lane 4)
HEK293T cells were prepared for immunoblot as described in the legend
to Fig. 1. Rabbit anti-SEMCAP-1(GIPC) antiserum (1:2000 dilution)
detected a 39-kDa immunoreactive protein in SEMCAP-1(GIPC)-expressing
cells and a 37-kDa cross-reactive protein in SEMCAP-2-expressing cells.
Total tissue homogenates (100 µg) from the indicated P1 and adult
mouse tissue were prepared for immunoblot with the same
anti-SEMCAP-1(GIPC) antibody. The abbreviations are as described in the
legend to Fig. 1.
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Fig. 4.
Specificity of SEMCAP and M-SemF
interaction. A, interactions of SEMCAP-1(GIPC) with
GST-M-SemF fusion proteins in vitro. GST-M-SemF fusion
proteins were expressed in E. coli and immobilized on
glutathione-agarose beads. Wild type (GST-SEMF) and mutated M-SemF
constructs (GST-SEMF-831*, -SEMF-S832A, -SEMF-S832D, and -SEMF-V834A)
are described under "Experimental Procedures." Lysates from
transfected 293T cells transiently expressing MycSEMCAP-1(GIPC) were
incubated with immobilized GST-M-SemF fusion proteins (approximately
100 µg of protein/20 µl of beads) at 4 °C. After thorough
washing, bound SEMCAP-1(GIPC) proteins were analyzed by SDS-PAGE and
immunoblotting with the 9E10 (anti-Myc) monoclonal antibody.
B, selective interactions between SEMCAP-1(GIPC) and
SEMCAP-2 with M-SemF in the yeast two-hybrid system. SFY526 yeast cells
were cotransformed with the indicated pGBT and pGAD vectors expressing
the cytoplasmic domains of M-SemF, SemB, SemC, or NR2C along with the candidate binding partners,
full-length SEMCAP-1(GIPC), SEMCAP-2, or the PDZ domains of PSD-95.
Colonies growing on double selection plates were isolated and cultured
in selective liquid medium. -Galactosidase activity was determined
as described in the legend to Fig. 2. C, SEMCAP-1(GIPC) and
SEMCAP-2 are both capable of interacting with GAIP. SFY526 yeast cells
were cotransformed with pGBT and pGAD vectors expressing full-length
GAIP, the cytoplasmic domain of M-SemF, SEMCAP-1(GIPC), and SEMCAP-2 as
indicated. Colonies growing on double selection plates were isolated,
and
-galactosidase activity was determined using a liquid assay as
described above.
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Fig. 5.
Physical interaction between SEMCAP-1(GIPC)
and M-SemF. A, endogenous SEMCAP-1(GIPC) binds to
GST-M-SemF affinity matrix. Immobilized GST-SEMF and GST-SEMF-831*
fusion proteins were incubated with either mouse brain cytosol or
lysates prepared from transfected cells expressing full-length
SEMCAP-1(GIPC) or SEMCAP-2. Bound proteins were analyzed by immunoblot
with a polyclonal anti-SEMCAP-1(GIPC) antiserum. B,
coimmunoprecipitation of SEMCAP-1(GIPC) with M-SemF. Lysates from
transfected HEK293T cells expressing full-length M-SemF and
MycSEMCAP-1(GIPC) were subject to immunoprecipitation with anti-M-SemF
antiserum, anti-SEMCAP-1(GIPC) antiserum, and corresponding preimmune
sera. Protein G-associated immunocomplexes were prepared for
immunoblotting. Proteins with an apparent molecular mass of 55 kDa were
probed with anti-M-SemF antiserum, and proteins below 55 kDa were
probed with the 9E10 monoclonal antibody. The coprecipitation of
SEMCAP-1(GIPC) with anti-M-SemF antiserum and vice versa
shows that M-SemF and SEMCAP-1(GIPC) form complexes in transfected
cells.
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Fig. 6.
Major SEMCAP-1(GIPC)-binding proteins in
brain. Detection of SEMCAP-1-binding proteins in brain membrane
fraction using an overlay assay is shown. Fifty micrograms of
100,000 × g brain membrane pellet from 2-day-old rats
(lanes 1-3) and cell lysates from full-length
M-SemF-expressing (lane 4) or mock-transfected
(lane 5) HEK293 cells were fractionated by
SDS-PAGE and transferred to nitrocellulose membrane. Membrane strips
were probed separately with anti-SEMCAP antiserum (lane
1) or anti-M-SemF antiserum (lane 2)
or overlaid with purified MycSEMCAP-1 fusion proteins prebound with
rabbit polyclonal anti-SEMCAP antibodies (lanes
3-5). The reactive proteins were detected with
peroxidase-conjugated goat anti-rabbit IgG and followed by TMB
substrate reagent. MycSEMCAP-1 fusion proteins detects two major brain
membrane-associated proteins at 33 and 100 kDa. The 100-kDa protein
comigrates with endogenous and recombinant M-SemF.
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Fig. 7.
Distribution of M-SemF-SEMCAP-1(GIPC)
complexes. A, colocalization of M-SemF and
SEMCAP-1(GIPC) in transfected cells by immunofluorescence analysis.
HEK293T cells were transfected with expression plasmid for M-SemF or
MycSEMCAP-1(GIPC) or both, as indicated in white at the
lower left of each image. cells were
processed for indirect immunofluorescence localization of M-SemF using
a polyclonal anti-M-SemF antiserum (green) and for
SEMCAP-1(GIPC) using the 9E10 monoclonal antibody recognizing the Myc
tag (red). The three right hand
images display the distribution of one or both antigens in
the same microscopic field. Coexpression of SEMCAP-1(GIPC) causes the
redistribution of surface M-SemF with SEMCAP-1(GIPC) clusters.
Scale bar, 24 µm. B,
detergent-insoluble M-SemF and SEMCAP-1(GIPC) in brain and transfected
cell membranes. Membranes from transfected HEK293 cells expressing
M-SemF and/or MycSEMCAP-1(GIPC) were treated with Triton X-100 (1%
TX-100) as described under "Experimental Procedures." Crude
membrane fraction (2% of the total preparation) and extracted membrane
fraction (5%) were prepared for immunoblot analysis. Brain membrane
fractions from P2 rats were processed in a similar fashion. Fifty
micrograms of proteins from the crude membrane or the TX-100-insoluble
membrane were analyzed by immunoblotting. The presence of M-SemF was
detected by anti-M-SemF antiserum. Recombinant SEMCAP-1(GIPC) was
detected using the 9E10 antibody, and brain SEMCAP-1(GIPC) was detected
by anti-SEMCAP-1(GIPC) antiserum. The majority of membrane- associated
SEMCAP-1(GIPC) resists TX-100 extraction. The proportion of M-SemF in
the detergent-insoluble fraction is greatly increased by SEMCAP-1(GIPC)
coexpression in HEK293 cells.
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Fig. 8.
SEMCAP-1(GIPC) clusters in embryonic cortical
neurons. Indirect immunofluorescence analysis of SEMCAP-1(GIPC)
distribution in cultured embryonic cortical neurons. Dissociated E15
cortical cells were cultured for 2 days, fixed, and stained with
anti-SEMCAP-1(GIPC) antiserum or preimmune serum. Clusters of
SEMCAP-1(GIPC) immunoreactivity are detected in the cell soma
(arrow). Scale bar, 30 µm.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheets
(
A-
F) and two
-helices (
A and
B). When the amino acid
sequences encoding PDZ domains from SEMCAP-1(GIPC) and SEMCAP-2 are
aligned with those from PSD-95 and CASK, it is found that the amino
acid residues forming the binding pockets are well conserved. Both
SEMCAP-1(GIPC) and SEMCAP-2 encode class 1A PDZ domains that have a His
residue at the initiation of
-helix B. The specificity of SEMCAP
binding to variable amino acids at the carboxyl end could also be
rationalized from the results of the structural analysis. A Leu residue
at position
B1 of the carboxylate binding pocket in SEMCAP-1(GIPC)
and SEMCAP-2 instead of the residue Phe in PSD-95 may render the
flexibility to accommodate different amino acids at the terminal
position of the PDZ binding consensus.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. S. Inagaki (Osaka University), Dr. A. W. Püschel (Max-Planck-Institut), Dr. M. B. Kennedy (California Institute of Technology), and Dr. J. Boulter (Salk Institute) for providing mouse M-SemF cDNA, mouse SemB cDNA, rat PSD-95 cDNA, and rat NR2C cDNA, respectively.
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FOOTNOTES |
---|
* This work was supported by Grants NS33020 and NS29837 from the National Institutes of Health (to S. M. S. and R. G. K.) and from the American Paralysis Association (to S. M. S.).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.
A Coxe-Brown Fellow of Yale University.
§ A John Merck Scholar in the Biology of Developmental Disorders in Children and an Investigator of the Patrick and Catherine Weldon Donaghue Medical Research Foundation. To whom all correspondence should be addressed: Dept. of Neurology, Yale University School of Medicine, P. O. Box 208018, 333 Cedar St., New Haven, CT 06520. Tel.: 203-785-4878; Fax: 203-785-5098; E-mail: Stephen.Strittmatter{at}yale.edu.
2 L.-H. Wang, H.-Y. Tang, and S. M. Strittmatter, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: SEMCAP-1 and -2, M-SemF cytoplasmic domain-associated protein 1 and 2, respectively; PCR, polymerase chain reaction; GST, glutathione S-transferase; DPBS, Dulbecco's phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; kb, kilobase pair(s); PSD, postsynaptic density/densities; TIP, Tax interacting protein clone 2.
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