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First published online April 3, 2008
doi: 10.1242/10.1242/jcs.025163

Journal of Cell Science 121, 1235-1242 (2008)
Published by The Company of Biologists 2008
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An alternatively spliced isoform of PECAM-1 is expressed at high levels in human and murine tissues, and suggests a novel role for the C-terminus of PECAM-1 in cytoprotective signaling

Carmen Bergom1,2,*, Cathy Paddock1,*, Cunji Gao1, Trudy Holyst1, Debra K. Newman1,3,4 and Peter J. Newman1,2,4,5,{ddagger}

1 Blood Research Institute, BloodCenter of Wisconsin, 8727 Watertown Plank Road, Milwaukee, WI 53201, USA
2 Department of Cell Biology, Medical College of Wisconsin, Milwaukee, WI, USA
3 Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI, USA
4 The Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, WI, USA
5 Department of Pharmacology, Medical College of Wisconsin, Milwaukee, WI, USA


Figure 1
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  		Fig. 1. Cytoplasmic domain amino acid sequences for full-length and {Delta}15-containing isoforms of PECAM-1. (A) Illustration of the cDNA and predicted amino acid sequences for the terminal cytoplasmic tail regions of the PECAM-1 splice variants described in this study. The new C-terminus created by deletion of exon 15 is boxed, and dotted lines indicate splice junctions. Stop codons are also indicated. A schematic diagram of the full-length (filled circles) and alternatively spliced {Delta}15-containing PECAM-1 cytoplasmic domains (unfilled circles) is shown in (B). Phylogenetically conserved tyrosine residues are colored yellow, whereas conserved serine residues are colored orange. Note that the {Delta}15 form is missing two serine residues and one tyrosine residue and has a novel six-amino-acid C-terminal sequence. (C) A rabbit polyclonal antibody produced against the novel {Delta}15 PECAM-1 C-terminal region reacts with human {Delta}15 PECAM-1, but not full-length WT PECAM-1, in HEK293 cells transfected with specific cDNAs encoding these isoforms. Fig. 1A,B are adapted, with permission, from Newman and Newman (Newman and Newman, 2003Go).

 

Figure 2
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  		Fig. 2. PECAM-1 isoforms lacking exon 15 are expressed in variable amounts in human and murine tissues. (A) Normal human tissue lysates were immunoblotted for {Delta}15 and total PECAM-1 protein. To determine {Delta}15 PECAM-1 expression in platelets, HUVECs and transformed human hematopoietic cells, antibody against PECAM-1 was incubated with whole cells, after which the cells were washed, lysed and cell-bound PECAM-1 was immunoprecipitated. PECAM-1 immunoprecipitates were probed for {Delta}15 and total PECAM-1 antigen. Note that varying relative amounts of {Delta}15 PECAM-1 are present in human ovary, brain and testes and that {Delta}15 PECAM-1 is on the surface of HUVECs, platelets and cancer cells. PECAM-1 antigen migrates with different electrophoretic mobilities in different tissues, probably owing to differences in glycosylation. (B) Murine PECAM-1 immunoprecipitates were probed for expression of total PECAM-1 and {Delta}15 PECAM-1. Note that a large proportion of the total PECAM-1 expressed in murine platelets and lung tissue is lacking exon 15. All results are representative of at least two independent experiments.

 

Figure 3
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  		Fig. 3. The {Delta}15 PECAM-1 isoform becomes tyrosine phosphorylated on its ITIMs and binds to the phosphatase SHP-2. REN mesothelioma cells expressing equivalent amounts of WT, ITIM-less or {Delta}15 PECAM-1 were stimulated with pervanadate and PECAM-1 was immunoprecipitated. Upon stimulation, WT PECAM-1 became tyrosine phosphorylated, as indicated by the phosphotyrosine immunoblot, and bound to SHP-2. By contrast, the Y663,686F ITIM-less form of PECAM-1 neither becomes tyrosine phosphorylated nor binds to SHP-2. Similar to WT PECAM-1, the {Delta}15 PECAM-1 isoform was able to become tyrosine phosphorylated and bind to SHP-2.

 

Figure 4
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  		Fig. 4. The {Delta}15 PECAM-1 isoform localizes to cell-cell borders, similar to full-length PECAM-1. REN mesothelioma cells expressing equivalent amounts of WT, ITIM-less, {Delta}15 and K89A PECAM-1 were grown to confluence, fixed, permeabilized and then subsequently stained with fluorescently labeled antibodies against PECAM-1 and analyzed using confocal microscopy. The panels on the left show the XY views, whereas panels on the right illustrate the xz cross-sections. As described previously, WT PECAM-1 is concentrated at cell-cell junctions (A), whereas a mutant form (K89A) unable to mediate homophilic binding is distributed along the apical surface of the cells, as illustrated in the xz panels (D). Both the ITIM-less (B) and {Delta}15 PECAM-1 (C) forms localized normally at cell-cell borders, similar to WT PECAM-1.

 

Figure 5
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  		Fig. 5. The PECAM-1 {Delta}15 isoform fails to mediate full protection against apoptosis induced by overexpression of Bax and by treatment with etoposide. (A) HEK293T cells were transiently transfected with plasmids encoding the indicated proteins. 24 hours after transfection, the cells were lysed and their caspase-3 activity was determined, as described in Materials and Methods. As reported previously, WT PECAM-1 protected against apoptosis induced by overexpression of Bax, but neither the ITIM-less nor the {Delta}15 form of PECAM-1 provided cytoprotection against overexpression of Bax. (B) REN mesothelioma cells expressing approximately equivalent amounts of WT PECAM-1 or {Delta}15 PECAM-1 were generated. After 48 hours, caspase-3 activity was determined. Cells expressing {Delta}15 PECAM-1 provided far less cytoprotection than that afforded by WT PECAM-1 (C). The values shown are the mean±s.d. The results are representative of at least two independent experiments performed in duplicate or triplicate; *P<=0.0002, **P<=0.05, derived using unpaired Student's t test.

 

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© The Company of Biologists Ltd 2008