Cloning of two new splice variants of Siglec-10 and mapping of the interaction between Siglec-10 and SHP-1.
Using a three-hybrid strategy in yeast, we have cloned a new splice variant of Siglec-10, called Siglec-10 Sv3. This splice variant lacks part of exon 3, but keeps the reading frame, as well as the crucial regions for interaction with Sias and the motifs for intracellular signaling. The expression of Siglec-10 Sv3 in T- and B-cells was detected by RT-PCR. Moreover, cDNA of another new splicing form of Siglec-10, named Siglec-10 Sv4, was identified by RT-PCR. One common characteristic of all Siglec-10 splice forms (except for Siglec-10 Sv2) is their cytoplasmic tail with two ITIMs and one CD150-like sequence. We confirmed the recruitment of SHP-1 to the Siglec-10 cytoplasmic tail by Western blot analysis and demonstrated that this interaction depends on tyrosine phosphorylation. Mutational analyses showed that ITIM Y609 of Siglec-10 and the N-terminal SH2 domain of SHP-1 play a pivotal role in the interaction between Siglec-10 and SHP-1. Finally, we demonstrated that Siglec-10 was not able to bind SAP/SH2d1A, indicating that the so-called CD150-like motif in Siglec-10 might be a docking site for other signal transduction mediators.
Kitzig
Friederike
F
Molecular Immunopathology Unit, DCEXS, Universitat Pompeu Fabra, Barcelona, Spain.
Martinez-Barriocanal
Agueda
A
López-Botet
Miguel
M
Sayós
Joan
J
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D037102:Lectins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D054631:Receptor-Like Protein Tyrosine Phosphatases, Class 3; D011956:Receptors, Cell Surface; D020798:Two-Hybrid System Techniques; D014443:Tyrosine / Q000378:metabolism
D047908:Intracellular Signaling Peptides and Proteins; D037102:Lectins; D011956:Receptors, Cell Surface; C426228:SIGLEC10 protein, human; D014443:Tyrosine; C509249:PTPN6 protein, human; C431695:PTPRH protein, human; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; D054631:Receptor-Like Protein Tyrosine Phosphatases, Class 3