(Received for publication, May 15, 1995)
From the
CD45, a leukocyte-specific transmembrane protein tyrosine phosphatase, is required for critical signal transduction pathways in immune responses. To elucidate the molecular interactions of CD45 with other proteins involved in CD45-mediated signal transduction pathways, we have recently cloned a 30-kDa phosphorylated protein, CD45-AP, which specifically associates with CD45. Binding analysis employing several deleted or chimeric forms of CD45-AP and CD45 demonstrated that the potential transmembrane segment of CD45-AP bound to the transmembrane portion of CD45. CD45-AP was found in particulate fractions of lymphocytes along with CD45, indicating that it is likely to be a transmembrane protein. In addition, CD45-AP was resistant to proteolysis by tosylphenylalanyl chloromethyl ketone-treated trypsin applied to intact cells. This is consistent with the most likely membrane orientation of CD45-AP predicted from the amino acid sequence, that is, only a short amino-terminal segment of CD45-AP is extracellular. We propose that CD45-AP interacts with CD45 at the plasma membrane and that the bulk of CD45-AP located in the cytoplasm act as an adapter which directs the interaction between CD45 and other molecules involved in CD45-mediated signal transduction pathways.
CD45 plays a critical role in signal transduction pathways
essential for immune responses(1) . The intracellular domain of
CD45 exhibits protein tyrosine phosphatase activity (2) and
differential usage of extracellular NH-terminal exons
results in several isotypes of CD45 which are variably expressed in a
cell lineage- and developmental stage-specific
manner(1, 3) . It has been proposed that CD45
activates src family protein tyrosine kinases associated with
the T cell receptor signaling complex, p56
(4, 5) and
p59
(6, 7) , by
dephosphorylating their down-regulatory tyrosyl residues. Thus, CD45
appears to be required at the onset of the T cell receptor-mediated
signal transduction pathway. CD45 appears to play a similar role in B
lymphocytes in combination with their src protein tyrosine
kinases(8) . Furthermore, CD45 is required for signal
transduction pathways of monocytes(9) , natural killer
cells(10) , and mast cells (11) . Physical associations
between CD45 and various membrane proteins involved in T cell
activation, such as CD2(12) , CD4(13, 14) ,
CD8(13, 14) , Thy-1(15) , T cell
receptor(14) , CD26(16) , and LFA-1(14) , as
well as components of the B cell antigen receptor complex(17) ,
have been documented under certain circumstances. It is not clear how
specifically these proteins interact with CD45 and what their binding
sites are. A cytoskeletal protein, fodrin, has been reported to
interact with the cytoplasmic portion of CD45 and stimulate its protein
tyrosine phosphatase activity(18) . In addition, CD45 has been
reported to be one of surface glycoproteins to which CD22, the B
lineage-specific cell surface glycoprotein and a sialic acid-binding
lectin, binds(19) .
We have recently reported the molecular cloning of a novel 30-kDa phosphorylated protein, CD45-AP, which is specifically associated with CD45(20) . CD45-AP shares no homology with previously known sequences and appears to be expressed specifically in leukocytes. The association of CD45-AP with CD45 has been observed in various types of lymphocytes (21) , indicating that the association involves the isotypically invariant portion of CD45. No tyrosine phosphorylation of CD45-AP has been detected either in vivo or in vitro, and the predicted sequence contains no consensus tyrosine phosphorylation sites (20) , indicating that it is not a substrate for protein tyrosine kinases. In addition, the predicted sequence of CD45-AP does not contain conserved sequences of GTP-binding proteins. Given the importance and complexity of CD45-mediated signal transduction, it is essential to determine how this novel protein, CD45-AP, interacts with CD45. In the present study, we characterize the interaction between CD45 and CD45-AP and summarize our findings in a model.
Figure 1: Deleted forms of CD45-AP cDNA. Various deleted forms of CD45-AP were derived from the full-length (F) cDNA as described under ``Experimental Procedures.'' The potential transmembrane segment is indicated with filled bars. Consensus phosphorylation sites are indicated with arrows. Downward arrows indicate phosphorylation sites favored by more than one enzyme.
Figure 2: Binding of in vitro translated deleted forms of CD45-AP to CD45. Full-length (F) and deleted (N and C) forms of CD45-AP cDNA were tested for specific binding to CD45. The first lane of each panel is direct analysis of in vitro translated products by SDS-PAGE. The next three lanes are material bound to immunocomplexes of CD45, LFA-1, and transferrin receptor, respectively. The F and C forms were analyzed in an 8-15% acrylamide gradient and the N form in a 12-19.2% acrylamide gradient, under reducing conditions. The positions of molecular mass markers (expressed in kilodaltons) are shown.
Figure 3:
Binding of recombinant deleted forms of
CD45-AP to CD45. Control glutathione-Sepharose 4B beads with GST
alone(-) or beads bound to recombinant GST-fusion proteins of
CD45-AP were incubated with lysates of YAC-1 cells metabolically
labeled by C-labeled amino acid incorporation. F, N, C, pN1, and pN2 correspond to
the full-length and deleted forms of CD45-AP depicted in Fig. 1.
Material bound to equal amounts of beads, along with material
immunoprecipitated with anti-CD45 mAb from the same lysate, were
analyzed by SDS-PAGE in an 8-15% acrylamide gradient under
reducing conditions. The positions of molecular mass markers (expressed
in kilodaltons) are shown.
Figure 4: Binding of recombinant cytoplasmic CD45 to CD45-AP. Panel A, GST fusion proteins of CD45-AP bound to glutathione-Sepharose 4B beads and control beads with GST alone(-) were incubated with CD45 isolated from YAC-1 cells. F, N, and C refer to the full-length and deleted forms shown in Fig. 1. CD45 isolated from YAC-1 cells (YAC-1 CD45) and material bound to equal amounts of beads were subjected to SDS-PAGE in an 8-15% acrylamide gradient under reducing conditions and were analyzed by Western blotting with antiserum against the cytoplasmic portion of CD45. Panel B, GST fusion proteins of CD45-AP bound to glutathione-Sepharose 4B beads and control beads with GST alone(-) were incubated with purified recombinant cytoplasmic CD45. Recombinant cytoplasmic CD45 (Cyto-CD45) and material bound to equal amounts of beads were subjected to SDS-PAGE in an 8-15% acrylamide gradient under reducing conditions and were analyzed by Western blotting with antiserum against the cytoplasmic portion of CD45. The positions of molecular mass markers (expressed in kilodaltons) are shown.
Figure 5: Binding of recombinant noncytoplasmic CD45 to CD45-AP. Panel A, immunoprecipitates were obtained from untransfected L cells (L40B7), L cells transfected with full-length CD45RABC (L106A6), L cells transfected with the noncytoplasmic portion of CD45RABC (L12B5), or YAC-1 cells using anti-CD45 mAb and were subjected to SDS-PAGE in an 8-15% acrylamide gradient under reducing conditions. They were then analyzed by Western blotting with antiserum against the extracellular domain of CD45. Panel B, full-length CD45-AP GST fusion protein (F) or its deleted forms (N and C, shown in Fig. 1) bound to glutathione-Sepharose 4B beads were incubated with lysate of L40B7, L106A6, L12B5, or YAC-1 cells. Material bound to equal amounts of beads was subjected to SDS-PAGE in an 8-15% acrylamide gradient under reducing conditions and was analyzed by Western blotting with antiserum against the extracellular portion of CD45. The positions of molecular mass markers (expressed in kilodaltons) are shown.
For binding analysis, GST fusion proteins of the full-length (F) or the deleted forms (N or C) of CD45-AP bound to glutathione-Sepharose 4B beads were incubated with a cell lysate of untransfected control L cells (L40B7), L106A6, L12B5, or YAC-1 cells. Material bound to the recombinant CD45-AP beads was subjected to SDS-PAGE and was analyzed by Western blotting with antiserum against the extracellular portion of CD45 (Fig. 5B). The full-length as well as the noncytoplasmic form of CD45RABC bound to the full-length and N forms of CD45-AP. This indicates that CD45-AP interacts with the noncytoplasmic portion of CD45.
Figure 6: Binding of the CD45 transmembrane domain to CD45-AP. Panel A, immunoprecipitates were obtained from untransfected L cells (L40B7) or L cells transfected with CD44.TM2 (CD44.TM2) using anti-CD44 mAb and were subjected to SDS-PAGE in an 8-15% acrylamide gradient under nonreducing conditions. They were then analyzed by Western blotting with the same mAb. Panel B, full-length CD45-AP GST fusion protein (F) or its deleted forms (N and C, shown in Fig. 1) bound to glutathione-Sepharose 4B beads were incubated with lysate of L40B7 or the CD44.TM2 transfectant. Material bound to equal amounts of beads was subjected to SDS-PAGE in an 8-15% acrylamide gradient under nonreducing conditions and was analyzed by Western blotting with anti-CD44 mAb. The positions of molecular mass markers (expressed in kilodaltons) are shown.
For binding analysis, GST-fusion proteins of the full-length (F) or the deleted forms (N or C) of CD45-AP bound to glutathione-Sepharose 4B beads were incubated with a cell lysate of the control L cells or the L cells transfected with CD44.TM2. Material bound to the recombinant CD45-AP beads was subjected to SDS-PAGE and was analyzed by Western blotting with the anti-CD44 mAb (36) (Fig. 6B). In the control L cells, endogenous CD44 did not bind to any form of CD45-AP. In the L cells transfected with CD44.TM2, on the other hand, CD44 bound to the full-length and N forms of CD45-AP but not to the C form. This indicates that the transmembrane portion of CD45 is sufficient for specific interaction with CD45-AP.
Figure 7: Subcellular localization of CD45-AP. Panel A, subcellular fractions derived from equal numbers of YAC-1 cells were subjected to SDS-PAGE in an 8-15% acrylamide gradient under reducing conditions and were analyzed by Western blotting with antiserum against CD45-AP. Panel B, a portion of the same fractions used in A was immunoprecipitated by anti-CD45 mAb and the immunoprecipitates were subjected to SDS-PAGE in an 8-15% acrylamide gradient under reducing conditions. The SDS-PAGE gel was then stained in Coomassie Blue. The positions of molecular mass markers (expressed in kilodaltons) are shown.
Figure 8:
TPCK-trypsin treatment of lysates and
cells. YAC-1 cell lysates or intact YAC-1 cells were treated without or
with TPCK-treated trypsin, and a portion of each sample derived from
equal numbers of cells was subjected to SDS-PAGE in an 8-15%
acrylamide gradient under reducing conditions. They were then analyzed
by Western blotting with antiserum against either CD45-AP (panel
A) or the extracellular domain of CD45 (panel B). Panel C, YAC-1 cells were radiolabeled by incorporation of C-labeled amino acid mixture and were treated without or
with TPCK-treated trypsin. Immunoprecipitates were obtained from these
cells with anti-LFA 1 or anti-transferrin receptor mAb and were
analyzed by SDS-PAGE in an 8-15% acrylamide gradient under
reducing conditions. The positions of molecular mass markers (expressed
in kilodaltons) are shown.
On the other hand,
TPCK-trypsin treatment of intact cells did not alter the amount of
CD45-AP detected at all, indicating that no arginine or lysine residue
of CD45-AP is located extracellularly or is accessible to TPCK-treated
trypsin. The 28-kDa band seen both in the presence and absence of
TPCK-treated trypsin (Fig. 8A) was detected in control
samples lacking cells as well and represented a component
present in the fetal calf serum used for washing cells at the end of
the incubation. In contrast to CD45-AP, the amount of CD45 detected was
significantly reduced after TPCK-trypsin treatment of intact cells due
to degradation of the extracellular domain. The 120-kDa fragment is not
seen with TPCK-trypsin treatment of intact cells indicating that it
probably results from digestion of the cytoplasmic domain of CD45.
In order to confirm that the proteolytic conditions employed above
for intact cells were appropriate for degrading other transmembrane
proteins as well, a similar experiment was carried out using cells that
were radiolabeled by incorporation of C-labeled amino acid
mixture. The labeled intact cells were treated without or with
TPCK-treated trypsin as described above, and immunoprecipitates of
LFA-1 (27) or the transferrin receptor (28) were
analyzed by SDS-PAGE (Fig. 8C). The TPCK-trypsin
treatment dramatically reduced the amounts of LFA-1 (the 180-kDa
chain and the 95-kDa
chain) and the mature form of the
transferrin receptors (95 kDa) but not the nascent intracellular form
(90 kDa) demonstrating that extracellular proteins were degraded under
the proteolytic conditions employed.
Binding analysis employing several different deleted forms of CD45-AP demonstrated that the potential transmembrane segment of CD45-AP is required for binding to CD45 and that the rest of CD45-AP, including most of the consensus phosphorylation sites, is not directly involved in the interaction ( Fig. 2and Fig. 3). Interestingly, binding analysis employing recombinant cytoplasmic or noncytoplasmic forms of CD45, and various recombinant forms of CD45-AP showed that the noncytoplasmic portion of CD45 bound to CD45-AP ( Fig. 4and Fig. 5). Furthermore, binding analysis employing a recombinant chimeric transmembrane protein which consists of the extracellular and intracellular domains of CD44 and the transmembrane segment of CD45 demonstrated that the transmembrane segment of CD45 is sufficient for specific interaction with CD45-AP (Fig. 6). These data indicate that CD45 and CD45-AP interact at the plasma membrane through their respective transmembrane domains.
Consistent with this notion are the following observations: (i)
CD45-AP can associate with various isotypes of CD45(21) ,
indicating that the interaction involves the isotypically invariant
portion of CD45, (ii) the association between CD45-AP and CD45 is
disrupted by some nonionic detergents(20) , suggesting that the
interaction between the two proteins involves hydrophobic segments, and
(iii) CD45-AP is found in particulate fractions along with CD45 by
subcellular localization studies (Fig. 7), indicating that
CD45-AP is likely to be a membrane protein. The recombinant
noncytoplasmic and transmembrane forms of CD45 used for binding studies
were produced in L cell fibroblasts and the recombinant forms of
CD45-AP were purified from E. coli. Therefore, these binding
studies establish that the physical association between CD45 and
CD45-AP does not require the presence of other leukocyte-specific
proteins, such as p56 and
p59
.
When the CD45-AP cDNA was in
vitro transcribed and translated with the rabbit reticulocyte
lysate system in the absence of microsomal fraction, the product
migrated as 30 kDa in SDS-PAGE (Fig. 2). Moreover, the in
vitro translated product comigrated with the mature form of
CD45-AP obtained from YAC-1 cells(20) . In the absence of
microsomal fraction, NH-terminal signal peptides of nascent
proteins cannot be cleaved(39) . Therefore, it seems unlikely
that the predicted sequence of the CD45-AP cDNA contains an
NH
-terminal signal peptide for protein translocation
because, in that case, the mature form of CD45-AP would be smaller than
the in vitro translated product. Instead, CD45-AP is likely to
be a single-pass membrane protein with an internal signal peptide that
remains as a membrane spanning segment. All three charged amino acid
residues in the NH
-terminal side of the potential
transmembrane segment of CD45-AP are acidic amino acids. In contrast,
the 42-residue peptide segment immediately on the carboxyl side of the
potential transmembrane segment contains nine charged residues which
are all basic. This charge distribution of the amino acids in the
vicinity of the potential signal/transmembrane segment of CD45-AP would
most likely cause translocation of the amino-terminal end into the
endoplasmic reticulum lumen(40, 41) , as seen with the
-adrenergic receptor (42) and glycophorin C(43) .
As a result, only a short segment at the NH
terminus of
CD45-AP would be located extracellularly, and the bulk of the protein
would be intracellular. This is consistent with the results obtained by
treating cell lysates and intact cells with TPCK-treated trypsin (Fig. 8A). CD45-AP was completely resistant to
proteolysis by TPCK-treated trypsin applied to intact cells but was
susceptible when the enzyme was applied to cell lysates. CD45-AP
contains 10 arginine and one lysine residues, the TPCK-treated trypsin
target sites, and all of them are located in the carboxyl-terminal side
of the potential transmembrane segment(20) . A proposed model
of the physical orientation of CD45-AP is depicted in Fig. 9.
Figure 9: A model for the association of CD45 with CD45-AP. PTP, protein tyrosine phosphatase.
Recently, a cDNA clone of a CD45-AP human homologue has been
reported(44) . The predicted amino acid sequence of the human
homologue has a high percentage of sequence identity with murine
CD45-AP. Interestingly, the human homologue differs from murine CD45-AP
in two important aspects. (i) The human homologue appears to have an
NH-terminal signal peptide that is cleaved to produce the
mature form, and (ii) the human homologue has a potential tyrosine
phosphorylation site. It is of interest to know (i) whether the two
highly conserved proteins have different mechanisms of protein
translocation, and (ii) why is the tyrosine phosphorylation lacking in
murine CD45-AP if the human homologue plays a role as a physiological
substrate for CD45 as proposed(44) . It is possible that a
family of CD45-AP proteins of different structures exist. In fact, we
have obtained and analyzed a human genomic clone of CD45-AP that also
predicts a protein of very high homology to the murine CD45-AP, but
differs in part from the reported sequence of the human homologue. (
)The homology between the human CD45-AP characterized in
our laboratory and the murine CD45-AP is higher than that between the
recently reported human homologue (44) and the murine CD45-AP.
The present study predicts that the major portion of CD45-AP including most of the potential phosphorylation sites is located intracellularly (Fig. 9). The cytoplasmic portion of CD45-AP may act as an adapter which directs the interaction between CD45 and other molecules involved in CD45-mediated signal transduction pathways. Proteins that interact with the intracellular portion of CD45-AP may be a substrate or a regulator of CD45 protein tyrosine phosphatase, and such proteins are currently under investigation. Distinct differences in the specific protein tyrosine phosphatase activity of CD45 have been detected among various populations of CD45 separated by sucrose gradient ultracentrifugation with a nondisruptive detergent(21) . It is possible that CD45-AP affects the protein tyrosine phosphatase activity of CD45 directly or indirectly since a population of CD45 without associated CD45-AP appeared to have a higher specific activity. This possibility can now be more closely examined by using various forms of recombinant CD45-AP.