Integrin-associated Protein Is a Ligand for the P84 Neural Adhesion Molecule

P84 (also known as SHPS-1, BIT, and SIRP) is a heterophilic adhesive membrane protein involved in receptor tyrosine kinase signaling that is found at synapses in the mammalian central nervous system and in non-neural tissues. We have identified a binding partner for P84 using an expression cloning strategy. Here we report that integrin-associated protein (IAP/CD47) is a predominant binding partner of P84. Immunohistochemistry reveals a virtually identical distribution of P84 and IAP in a variety of adult brain regions. Because IAP has been implicated in cell signaling in cells of the immune system, P84 and IAP represent a heterophilic binding pair that is likely to be involved in bi-directional signaling at the synapse and in other tissues. P84 (also known as SHPS-1, BIT, and SIRP) is a heterophilic adhesive membrane protein involved in receptor tyrosine kinase signaling that is found at synapses in the mammalian central nervous system and in non-neural tissues. We have identified a binding partner for P84 using an expression cloning strategy. Here we report that integrin-associated protein (IAP/CD47) is a predominant binding partner of P84. Immunohistochemistry reveals a virtually identical distribution of P84 and IAP in a variety of adult brain regions. Because IAP has been implicated in cell signaling in cells of the immune system, P84 and IAP represent a heterophilic binding pair that is likely to be involved in bi-directional signaling at the synapse and in other tissues. A number of adhesive molecules have been shown to be important for formation and maintenance of synaptic connections in the CNS. 1The abbreviations used are: CNS, central nervous system; IAP, integrin-associated protein (CD47); BCIP, 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt; NBT, nitro blue tetrazolium chloride; AP, alkaline phosphatase; RAP, receptor alkaline phosphatase; PBS, phosphate-buffered saline; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; N-CAM, neural cell adhesion molecule. 1The abbreviations used are: CNS, central nervous system; IAP, integrin-associated protein (CD47); BCIP, 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt; NBT, nitro blue tetrazolium chloride; AP, alkaline phosphatase; RAP, receptor alkaline phosphatase; PBS, phosphate-buffered saline; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; N-CAM, neural cell adhesion molecule. Immunoglobulins, cadherins, integrins, as well as proteoglycans and their receptors appear to contribute to synapse formation (1Mayford M. Barzilai A. Keller F. Schacher S. Kandel E.R. Science. 1992; 256: 638-644Crossref PubMed Scopus (347) Google Scholar, 2Fannon A.M. Colman D.R. Neuron. 1996; 17: 423-434Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 3Einheber S. Schnapp L.M. Salzer J.L. Cappiello Z.B. Milner T.A. J. Comp. Neurol. 1996; 370: 105-134Crossref PubMed Scopus (111) Google Scholar, 4Hsueh Y.P. Yang F.C. Kharazia V. Naisbitt S. Cohen A.R. Weinberg R.J. Sheng M. J. Cell Biol. 1998; 142: 139-151Crossref PubMed Scopus (284) Google Scholar). We originally identified the immunoglobulin family member P84 by virtue of its adhesive properties when tested with cerebellar and neocortical neurons (5Chuang W. Lagenaur C.F. Dev. Biol. 1990; 137: 219-232Crossref PubMed Scopus (86) Google Scholar). The ability of P84 to promote neurite outgrowth and stimulate robust filopodial extension in growth cones suggested that its ligand was also capable of signal transduction (5Chuang W. Lagenaur C.F. Dev. Biol. 1990; 137: 219-232Crossref PubMed Scopus (86) Google Scholar, 6Abosch A. Lagenaur C. J. Neurobiol. 1993; 24: 344-355Crossref PubMed Scopus (33) Google Scholar). The association of P84 with synaptic regions in the CNS suggested that it might be another candidate molecule for mediation of synaptic adhesion. We recently cloned the P84 gene and discovered that it was identical to SHPS-1, BIT, and SIRP-α molecules that had been identified by their binding to the cytoplasmic tyrosine phosphatase, SHP-2 (7Comu S. Weng W. Olinsky S. Ishwad P. Mi Z. Hempel J. Watkins S. Lagenaur C.F. Narayanan V. J. Neuroscience. 1997; 17: 8702-8710Crossref PubMed Google Scholar, 8Fujioka Y. Matozaki T. Noguchi T. Iwamatsu A. Yamao T. Takahashi N. Tsuda M. Takada T. Kasuga M. Mol. Cell. Biol. 1996; 16: 6887-6899Crossref PubMed Scopus (383) Google Scholar, 9Sano S. Ohnishi H. Omori A. Hasegawa J. Kubota M. FEBS Lett. 1997; 411: 327-334Crossref PubMed Scopus (75) Google Scholar, 10Kharitonenkov A. Chen Z. Sures I. Wang H. Schilling J. Ullrich A. Nature. 1997; 386: 181-186Crossref PubMed Scopus (541) Google Scholar). P84 contains a single transmembrane domain, three extracellular Ig-like domains, and a cytoplasmic segment that includes tyrosine phosphorylation sites. These phosphotyrosines correspond to sites recognized by SH2-containing segments of the SHP-2 phosphatase. There remain many intriguing questions regarding P84 that are to be answered, including how and why this molecule is localized at the synapse, what the downstream effects of P84 activation are, and what extracellular molecules are involved in initiating signaling via P84. To address this last question, we undertook an expression-cloning approach to identify ligands that bound to the extracellular segment of P84. We have discovered that a predominant ligand for P84 is the integrin-associated protein (IAP/CD47). In addition to its expression in lymphocytes and other extraneural tissues, IAP was known to be expressed in the brain, and its expression has recently been associated with memory formation (11Huang A-M. Wang H.L. Tang Y.P. Lee E.H.Y. J. Neurosci. 1998; 18: 4305-4313Crossref PubMed Google Scholar). Antibodies against IAP blocked the adhesion of cerebellar neurons, erythrocytes, and thymocytes to P84-coated substrates. We also showed that the distribution of P84 and IAP in the cerebellum and retina were very similar and consistent with their participation in a heterophilic synaptic adhesion complex. Alkaline phosphatase (AP)-tag2 and AP-tag4 plasmids were a generous gift of J. Flanagan (Harvard University, School of Medicine). The sequences corresponding to the signal peptide and extracellular segment of murine P84 were amplified by polymerase chain reaction, and cloned into theBglII site of the AP-tag 2 vector. This plasmid was transfected with LipofectAMINE (Life Technologies Inc.) into 293TWT cells (Edge Biosystems), and the supernatant was collected between days 4 and 8. The supernatant was monitored for phosphatase activity using a synthetic substrate,p-nitrophenyl phosphate (Sigma 104 phosphatase substrate) (12Flanagan J.G. Leder P. Cell. 1990; 63: 185-194Abstract Full Text PDF PubMed Scopus (630) Google Scholar). The fusion protein was affinity purified and analyzed by SDS-polyacrylamide gel electrophoresis. As a control, 293T cells were transfected with the AP-tag4 plasmid, which directed the secretion of AP alone. For P84 ligand staining in situ, the P84-AP fusion protein (culture supernatant) was incubated with tissue sections or cells (receptor alkaline phosphatase (RAP) in situ) following the method of Cheng and Flanagan (13Cheng H-J. Flanagan J.G. Cell. 1994; 79: 157-168Abstract Full Text PDF PubMed Scopus (326) Google Scholar). The extracellular domain of IAP was cloned into the AP-tag2 vector, and an IAP-AP fusion protein was produced as described above for P84-AP. Immunopurified P84, L1, and neural cell adhesion molecule (N-CAM) were spotted on nitrocellulose, blocked, and incubated with IAP-AP for 30 min. After washing, AP activity was detected with NBT/BCIP. An adult mouse brain cDNA library was obtained from Edge Biosystems Inc. (Gaithersburg, MD). This library was constructed in the pEAK8 plasmid vector. Forty pools of about 2500 colonies each were plated. These were harvested into liquid medium, and an aliquot of each was saved. DNA was extracted from the remainder of each sample (Perfect Prep Kit, 5 Prime–3 Prime, Inc.). DNA from each pool (0.3–0.5 μg) was transiently transfected into COS-7 cells in 6-well plates using LipofectAMINE. After 48 h, cells were incubated with P84-AP fusion protein and stained. From a single positive pool, a single cDNA clone was purified by sib selection. Briefly, the culture corresponding to the positive pool was replica plated onto nylon membranes, and one of these membranes divided into ten segments. DNA was extracted from each of the subpools and tested for staining with the P84-AP fusion protein. This process was iterated until a single positive cDNA clone was obtained. As a control, transfected and untransfected cells were stained with P84-AP and AP alone. The positive clones were sequenced using vector-specific primers (pEAK8.for -ggatcttggttcattctcaa; and pEAK8.rev -ctggatgcaggctactctag) and with gene-specific primers. Animals were perfused with 4% paraformaldehyde in PBS, and dissected tissues were cryoprotected in 30% sucrose. Cryostat sections were collected on pretreated slides. The monoclonal P84 and IAP (miap301, PharMingen) antibodies were previously described (5Chuang W. Lagenaur C.F. Dev. Biol. 1990; 137: 219-232Crossref PubMed Scopus (86) Google Scholar, 14Lindberg F.P. Bullard D.C. Caver T.E. Gresham H.D. Beaudet A.L. Brown E.J. Science. 1996; 274: 795-798Crossref PubMed Scopus (297) Google Scholar). Sections were stained with primary antibodies for 1 h and incubated with FITC-conjugated goat anti-rat secondary antibody (Cappel) for 30 min. The staining was examined with fluorescence microscopy. Coverslips were first coated with nitrocellulose (Schleicher and Schuell, Inc.) as described previously (6Abosch A. Lagenaur C. J. Neurobiol. 1993; 24: 344-355Crossref PubMed Scopus (33) Google Scholar). Purified proteins (P84, P84-AP, laminin, or miap301 antibody) were spotted at the center of the coated coverslips in 5-mm spots. The substrate solution was aspirated after 5 min. Coverslips were blocked with 1% BSA in PBS followed by medium containing 10% horse serum. Laminin was a generous gift of Dr. J. Hassel (University of Pittsburgh). Cerebellar cells were prepared as described previously (15Schnitzer J. Schachner M. J. Neuroimmunol. 1981; 1: 429-456Abstract Full Text PDF PubMed Scopus (139) Google Scholar). Blood was collected from adult mice in heparinized tubes, washed twice, and suspended in 1% BSA containing PBS at a titer of 5 × 106 cell/ml. Thymus was removed from young mice, washed several times with PBS, and cut into several small pieces to release free thymocytes. After several washes, thymocytes were resuspended in PBS containing 1% BSA at a titer of 5 × 106 cell/ml. Purified miap301 (0.5 mg/ml) was added to suspended cells at 1:100 ratio and then allowed to bind for 20 min at 0 °C. After plating on P84-coated coverslips and incubation for 2 h, cells were washed three times with PBS, fixed with 4% paraformaldehyde for 10 min, and counted. To facilitate the cloning of P84-binding proteins, a cDNA encoding the P84 ectodomain was inserted into the AP-tag2 vector (13Cheng H-J. Flanagan J.G. Cell. 1994; 79: 157-168Abstract Full Text PDF PubMed Scopus (326) Google Scholar). This plasmid directs expression of a soluble fusion protein with human alkaline phosphatase at the carboxyl-terminal end (Fig.1 A). 293T cells transiently transfected with this construct secreted the recombinant P84-AP, and this fusion protein was purified with an anti-P84 affinity column (Fig.1 B). Because native P84 was known to be a good substrate for cerebellar cell attachment and neurite outgrowth, we tested the purified P84-AP fusion protein in a cell adhesion assay. The purified P84-AP was immobilized on Petri dishes, and mouse cerebellar cells were allowed to attach and grow for 24 h. There was no obvious difference in the attachment of neurons or pattern of neurite growth on native P84 or P84-AP (Fig. 1 C, and see Ref. 5Chuang W. Lagenaur C.F. Dev. Biol. 1990; 137: 219-232Crossref PubMed Scopus (86) Google Scholar). RAP in situ staining (13Cheng H-J. Flanagan J.G. Cell. 1994; 79: 157-168Abstract Full Text PDF PubMed Scopus (326) Google Scholar) was done to examine P84 binding activity in the brain regions which were known to contain P84. In the cerebellum, the molecular layer was heavily stained, and a pattern consistent with synaptic glomeruli in the granule cell layer was observed (Fig.1 D). This P84-AP cerebellar staining was very similar to that seen with P84 antibody staining (7Comu S. Weng W. Olinsky S. Ishwad P. Mi Z. Hempel J. Watkins S. Lagenaur C.F. Narayanan V. J. Neuroscience. 1997; 17: 8702-8710Crossref PubMed Google Scholar), which suggested that the P84-AP probe detected an endogenous extracellular binding partner for P84. No staining was observed with AP alone (Fig. 1 E). To search for P84-binding proteins, we screened a mouse brain cDNA library with the P84-AP fusion protein. Transfected cells were screened for P84-AP binding using the procedure described by Cheng and Flanagan (13Cheng H-J. Flanagan J.G. Cell. 1994; 79: 157-168Abstract Full Text PDF PubMed Scopus (326) Google Scholar). AP without the P84 ectodomain was included as a negative control. From the 40 pools screened (about 2,500 colonies in each pool), two positive pools were identified. From one of these, a single positive cDNA clone was purified (Fig. 2). Sequencing of the cDNA derived from this clone revealed that it was the brain-specific form of mouse IAP (form 4) (16Reinhold M.I. Lindberg F.P. Plas D. Reynolds S. Peters M.G. Brown E.J. J. Cell Sci. 1995; 108: 3419-3425Crossref PubMed Google Scholar). This form of IAP has the longest cytoplasmic domain of all the forms of IAP. We also noticed that a region of 63 nucleotides that encode 21 amino acids in the extracellular domain near the first transmembrane region (but not in IgV-like domain) is lacking in this particular clone. This 21 amino acid region is also lacking in some mouse, rat, and human IAP forms (Fig. 3 A) (17Furusawa T. Yanai N. Hara T. Miyajima A. Obinata M. J. Biochem. 1998; 123: 101-106Crossref PubMed Scopus (23) Google Scholar). An IAP-AP fusion protein was tested for binding on purified P84, L1, and N-CAM. Binding was observed on P84 but not on the other molecules (Fig.3 B).Figure 3Comparison of P84 ligand to IAP. A, diagram showing the structure of mouse IAP and the P84 ligand. B, IAP-AP fusion protein binds directly to purified brain P84 but not L1 or N-CAM. No binding is seen with AP alone.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Freshly dissociated cerebellar cells can bind to native P84 within 10 min. We tested whether IAP was present on trypsin-dissociated cerebellar cells. Anti-IAP (miap301) was bound to nitrocellulose-coated coverslips, and cerebellar cells were tested for their ability to bind to these coverslips. Cell binding to this antibody occurred within 10 min; with overnight incubation, the cells remained attached to the antibody substrate and extended neurites (data not shown). To determine whether IAP represented a major binding partner for P84, we attempted to block neuronal IAP with the miap301 antibody which is directed against the IgV domain of IAP. We then tested antibody-treated neurons for their ability to bind to purified brain P84. Freshly dissociated cerebellar cells were incubated with anti-IAP antibody or in antibody-free media for 30 min at 0 °C and then plated on P84-coated coverslips. Cells were allowed to attach for 2 h at 37 °C, washed with PBS, and photographed. The antibody-blocking effect was dramatic; virtually no antibody-treated neurons attached, in contrast to the large number of cells that attached in untreated controls (Fig.4, A and B). To be sure that the blocking effect was specific, we tested the ability of anti-IAP to interfere with neuronal binding to laminin. As shown in Fig. 4, D and E, anti-IAP had no effect on neuronal binding to laminin. Both P84 and IAP are found on a number of non-neuronal tissues, with IAP found on lymphocytes and erythrocytes (as well other tissues) and P84 found on dendritic cells and macrophages (18Adams S. van der Laan L.J.W. Vernon-Wilson E. de Lavalette C.R. Döpp E.A. Dijkstra C.D. Simmons D.L. van den Berg T.K. J. Immunol. 1998; 161: Google Scholar, H. Chen H. S. Mol. Cell. Biol. 1998; 18: PubMed Scopus Google Scholar). To determine whether some of the other IAP cells bind to P84, we tested thymocytes and erythrocytes for their ability to bind to brain P84. Both erythrocytes and thymocytes attached to P84-coated coverslips, and this binding be blocked by anti-IAP antibody (Fig. These the that IAP might be a receptor for P84. To be a binding pair in the brain, P84 and IAP be expressed on membranes of cells. with a P84 antibody an distribution of P84 in cerebellum and We stained sections from these two with anti-IAP and anti-P84 antibodies for The distribution of P84 and IAP was In the cerebellum, the molecular layer the P84 and IAP and in the granule cell staining was observed that to be associated with synaptic glomeruli A and see also Ref. 6Abosch A. Lagenaur C. J. Neurobiol. 1993; 24: 344-355Crossref PubMed Scopus (33) Google Scholar). In the P84 and IAP were found in the and with or no staining of these synaptic (Fig. This of P84 and IAP is consistent with an adhesive association between these two molecules in P84 is a cell adhesion molecule that is a member of the immunoglobulin (5Chuang W. Lagenaur C.F. Dev. Biol. 1990; 137: 219-232Crossref PubMed Scopus (86) Google Scholar, S. Weng W. Olinsky S. Ishwad P. Mi Z. Hempel J. Watkins S. Lagenaur C.F. Narayanan V. J. Neuroscience. 1997; 17: 8702-8710Crossref PubMed Google Scholar). P84 is identical to SHPS-1, BIT, and molecules that are known to bind the tyrosine phosphatase SHP-2 and cell signaling Y. Matozaki T. Noguchi T. Iwamatsu A. Yamao T. Takahashi N. Tsuda M. Takada T. Kasuga M. Mol. Cell. Biol. 1996; 16: 6887-6899Crossref PubMed Scopus (383) Google Scholar, 9Sano S. Ohnishi H. Omori A. Hasegawa J. Kubota M. FEBS Lett. 1997; 411: 327-334Crossref PubMed Scopus (75) Google Scholar, 10Kharitonenkov A. Chen Z. Sures I. Wang H. Schilling J. Ullrich A. Nature. 1997; 386: 181-186Crossref PubMed Scopus (541) Google Scholar). The human a large family of signaling by with from these by A. Chen Z. Sures I. Wang H. Schilling J. Ullrich A. Nature. 1997; 386: 181-186Crossref PubMed Scopus (541) Google Scholar). Because P84 has been associated with regions in the CNS, and P84 was known to bind to a heterophilic the intriguing that P84 and its receptor were involved in or of synaptic of a membrane ligand that bound to the extracellular domain of P84 was for the of by which P84 and this ligand to the formation of We have cloned a ligand for the P84 molecule and identified it as IAP This is by the following The distribution of P84 binding activity situ staining with P84-AP fusion and IAP by are The distribution of P84 and its IAP, within the brain and retina are also with expressed in synaptic regions. A monoclonal antibody against IAP blocked the attachment of cerebellar neurons, erythrocytes, and thymocytes to P84-coated substrates. These that IAP was a ligand for the P84 adhesion molecule. IAP was described as a cell protein that was involved in the of and by extracellular molecules Gresham H.D. Brown E.J. J. 1992; PubMed Scopus Google Scholar, E. T. Gresham H. J. Cell Biol. 1990; PubMed Scopus Google Scholar). IAP not directly with ligands but with and E. T. Gresham H. J. Cell Biol. 1990; PubMed Scopus Google Scholar). IAP to be a of several including and of D. Lindberg F.P. Brown E.J. Sci. S. A. 1995; PubMed Scopus Google Scholar, F.P. Gresham H.D. M.I. Brown E.J. J. Cell Biol. 1996; PubMed Scopus Google Scholar, A. J.L. J. Cell Biol. 1996; PubMed Scopus Google activation of F.P. Bullard D.C. Caver T.E. Gresham H.D. Beaudet A.L. Brown E.J. Science. 1996; 274: 795-798Crossref PubMed Scopus (297) Google Scholar, E. T. Gresham H. J. Cell Biol. 1990; PubMed Scopus Google of activation M. T. M. T. W. J. Immunol. 1997; Google Scholar, M. M. F. Brown E.J. A. J. 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Cell Sci. 1995; 108: 3419-3425Crossref PubMed Google Scholar). The human gene family contains at that in the amino acid of the extracellular A. Chen Z. Sures I. Wang H. Schilling J. Ullrich A. Nature. 1997; 386: 181-186Crossref PubMed Scopus (541) Google Scholar). these forms of all bind to IAP or to other to be an has been between of IAP in the and memory in (11Huang A-M. Wang H.L. Tang Y.P. Lee E.H.Y. J. Neurosci. 1998; 18: 4305-4313Crossref PubMed Google Scholar). These that IAP have a in synaptic and memory an consistent with that IAP is a ligand for the synaptic neural adhesion P84. P84 and are known to be involved in cell signaling via tyrosine phosphorylation in a variety of cell As IAP is also known to in cell signaling in cells of the immune identify P84 and IAP as a bi-directional signaling pair that be a variety of tissues, including the nervous system and immune the of the of signaling of each of these molecules their that the are likely to be in the cells that P84 and that IAP. on their synaptic and signaling we that P84 and IAP also be involved in of synaptic and We Dr. J. Flanagan for the plasmids and for a We Olinsky and for and Dr. for at