Src homology 2 domain-containing protein-tyrosine phosphatases, SHP-1 and SHP-2, are required for platelet endothelial cell adhesion molecule-1/CD31-mediated inhibitory signaling.
Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a newly assigned member of the Ig immunoreceptor tyrosine-based inhibitory motif superfamily, and its functional role is suggested to be an inhibitory receptor that modulates immunoreceptor tyrosine-based activation motif-dependent signaling cascades. To test whether PECAM-1 is capable of delivering inhibitory signals in B cells and the functional requirement of protein-tyrosine phosphatases (PTPs) for this inhibitory signaling, we generated chimeric Fc gamma RIIB1-PECAM-1 receptors containing the extracellular and transmembrane portions of murine Fc gamma RIIB1 and the cytoplasmic domain of human PECAM-1. These chimeric receptors were stably expressed in chicken DT40 B cells either as wild-type or mutant cells deficient in SHP-1(-/-), SHP-2(-/-), SHIP(-/-), or SHP-1/2(-/-) and then assessed for their ability to inhibit B cell Ag receptor (BCR) signaling. Coligation of wild-type Fc gamma RIIB1-PECAM-1 with BCR resulted in inhibition of intracellular calcium release, suggesting that the cytoplasmic domain of PECAM-1 is capable of delivering an inhibitory signal that blocks BCR-mediated activation. This PECAM-1-mediated inhibitory signaling correlated with tyrosine phosphorylation of the Fc gamma RIIB1-PECAM-1 chimera, recruitment of SHP-1 and SHP-2 PTPs by the phosphorylated chimera, and attenuation of calcium mobilization responses. Mutational analysis of the two tyrosine residues, 663 and 686, constituting the immunoreceptor tyrosine-based inhibitory motifs in PECAM-1 revealed that both tyrosine residues play a crucial role in the inhibitory signal. Functional analysis of various PTP-deficient DT40 B cell lines stably expressing wild-type chimeric Fc gamma RIIB1-PECAM-1 receptor indicated that cytoplasmic Src homology 2-domain-containing phosphatases, SHP-1 and SHP-2, were both necessary and sufficient to deliver inhibitory negative regulation upon coligation of BCR complex with inhibitory receptor.
Henshall
T L
TL
Division of Haematology, Hanson Centre for Cancer Research, IMVS, Adelaide, South Australia.
Jones
K L
KL
Wilkinson
R
R
Jackson
D E
DE
D020816:Amino Acid Motifs / Q000235:genetics / Q000276:immunology; D000818:Animals; D001402:B-Lymphocytes / Q000201:enzymology* / Q000276:immunology* / Q000378:metabolism; D002460:Cell Line; D002645:Chickens; D016384:Consensus Sequence / Q000235:genetics / Q000276:immunology; D003593:Cytoplasm / Q000276:immunology; D015536:Down-Regulation / Q000235:genetics / Q000276:immunology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008213:Lymphocyte Activation / Q000235:genetics; D051379:Mice; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000235:genetics / Q000502:physiology*; D017434:Protein Structure, Tertiary / Q000235:genetics; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D017452:Receptors, IgG / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000494:pharmacology; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology*; D014443:Tyrosine / Q000235:genetics / Q000502:physiology; D018909:src Homology Domains / Q000235:genetics / Q000276:immunology*
D047908:Intracellular Signaling Peptides and Proteins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; C516724:PTPN11 protein, human; C509249:PTPN6 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; C516725:Ptpn11 protein, mouse; C509248:Ptpn6 protein, mouse; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases