Besides an ITIM/SHP-1-dependent pathway, CD22 collaborates with Grb2 and plasma membrane calcium-ATPase in an ITIM/SHP-1-independent pathway of attenuation of Ca2+i signal in B cells.
CD22 is a surface immunoglobulin implicated in negative regulation of B cell receptor (BCR) signaling; particularly inhibiting intracellular Ca2+ (Ca2+i)signals. Its cytoplasmic tail contains six tyrosine residues (Y773/Y783/Y817/Y828/Y843/Y863, designated Y1~Y6 respectively), including three (Y2/5/6) lying within immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that serve to recruit the protein tyrosine phosphatase SHP-1 after BCR activation-induced phosphorylation. The mechanism of inhibiting Ca2+i by CD22 has been poorly understood. Previous study demonstrated that CD22 associated with plasma membrane calcium-ATPase (PMCA) and enhanced its activity (Chen, J. et al. Nat Immunol 2004;5:651-7). The association is dependent on BCR activation-induced cytoplasmic tyrosine phosphorylation, because CD22 with either all six tyrosines mutated to phenylalanines or cytoplasmic tail truncated loses its ability to associate with PMCA. However, which individual or a group of tyrosine residues determine the association and how CD22 and PMCA interacts, are still unclear. In this study, by using a series of CD22 tyrosine mutants, we found that ITIM Y2/5/6 accounts for 34.3~37.1% Ca2+i inhibition but is irrelevant for CD22/PMCA association. Non-ITIM Y4 and its YEND motif contribute to the remaining 69.4~71.7% Ca2+i inhibition and is the binding site for PMCA-associated Grb2. Grb2, independently of BCR cross-linking, is constitutively associated with and directly binds to PMCA in both chicken and human B cells. Knockout of Grb2 by CRISPR/Cas9 completely disrupted the CD22/PMCA association. Thus, our results demonstrate for the first time that in addition to previously-identified ITIM/SHP-1-dependent pathway, CD22 holds a major pathway of negative regulation of Ca2+i signal, which is ITIM/SHP-1-independent, but Y4/Grb2/PMCA-dependent.
Department of Cardiology, Affiliated Xinhua Hospital, Shanghai Jiaotong University (SJTU) School of Medicine, Shanghai, China.
Jie
J
Chen
Department of Cardiology, Affiliated Xinhua Hospital, Shanghai Jiaotong University (SJTU) School of Medicine, Shanghai, China.
Hong
H
Wang
Department of Cardiology, Affiliated Xinhua Hospital, Shanghai Jiaotong University (SJTU) School of Medicine, Shanghai, China.
Wei-Ping
WP
Xu
Department of Pediatrics, Affiliated Xinhua Hospital, Shanghai Jiaotong University (SJTU) School of Medicine, Shanghai, China.
Si-Si
SS
Wei
Department of Medicine, University of Massachusetts School of Medicine, Worcester, MA, USA.
Hui Joyce
HJ
Li
Department of Cardio-Thoracic Surgery, Shanghai Tongji Hospital, Tongji University School of Medicine, Shanghai, China.
Yun-Qing
YQ
Mei
Department of Cardiology, Affiliated Xinhua Hospital, Shanghai Jiaotong University (SJTU) School of Medicine, Shanghai, China.
Yi-Gang
YG
Li
Department of Cardiology, Affiliated Xinhua Hospital, Shanghai Jiaotong University (SJTU) School of Medicine, Shanghai, China.
Yue-Peng
YP
Wang
D000328:Adult; D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002645:Chickens; D005260:Female; D051380:GRB2 Adaptor Protein / Q000235:genetics / Q000378:metabolism*; D055786:Gene Knockout Techniques; D064368:Healthy Volunteers; D006801:Humans; D008297:Male; D008875:Middle Aged; D010766:Phosphorylation; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000378:metabolism*; D017354:Point Mutation; D054730:Protein Interaction Domains and Motifs; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6 / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism; D051918:Sialic Acid Binding Ig-like Lectin 2 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
C497867:CD22 protein, human; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D034741:RNA, Small Interfering; D011947:Receptors, Antigen, B-Cell; D051918:Sialic Acid Binding Ig-like Lectin 2; D014443:Tyrosine; C509249:PTPN6 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D053499:Plasma Membrane Calcium-Transporting ATPases; D002118:Calcium
B cells activation; calcium signaling; plasma membrane calcium-transporting ATPase 4b; protein-protein interaction