Membrane raft association of CD47 is necessary for actin polymerization and protein kinase C theta translocation in its synergistic activation of T cells.
CD47 is a ubiquitously expressed membrane protein with an extracellular Ig domain and a multiple membrane-spanning domain that can synergize with antigen to induce interleukin (IL)-2 secretion by T lymphocytes. Ligation of CD47 induced actin polymerization and increased protein kinase Ctheta (PKCtheta) association with the cytoskeleton independent of antigen receptor ligation, but ligation of mutant forms of the molecule missing either the Ig domain or the multiple membrane-spanning domain did not. Simultaneous ligation of CD47 and CD3 led to additive effects on F-actin and synergistic effects on PKCtheta cytoskeletal association. Disruption of membrane rafts by removal of cholesterol with cyclodextrin blocked CD47-induced actin polymerization, and mutant forms of CD47 that localized poorly to rafts failed to effect cytoskeletal rearrangement. However, raft association alone was not sufficient, because a raft-localized CD47 Ig domain bound to the membrane by a glycan phosphoinositol anchor was unable to induce actin polymerization. A mutant form of CD47 without its Ig domain that did not induce actin polymerization or localize to rafts still enhanced T cell receptor (TCR)-dependent tyrosine phosphorylation of PLCgamma and associated Ca(2+) signaling but did not augment IL-2 secretion. Thus, CD47 synergy with TCR to increase [Ca(2+)](i) is independent of actin and rafts but is insufficient to explain CD47 cooperation with TCR in IL-2 synthesis. Full synergy with TCR requires CD47 localization to membrane rafts where ligation leads to TCR-independent signals causing actin polymerization and PKCtheta translocation.
Rebres
R A
RA
Center for Host/Pathogen Interactions, University of California, San Francisco 94143, USA.
Green
J M
JM
Reinhold
M I
MI
Ticchioni
M
M
Brown
E J
EJ
D000199:Actins / Q000378:metabolism*; D015703:Antigens, CD / Q000737:chemistry / Q000378:metabolism*; D051928:CD47 Antigen; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D003505:Cyclodextrins / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D006801:Humans; D015151:Immunoblotting; D007376:Interleukin-2 / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D019169:Jurkat Cells; D008213:Lymphocyte Activation; D021962:Membrane Microdomains / Q000378:metabolism*; D051966:Phospholipase C gamma; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D000074121:Protein Kinase C-theta; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism; D047392:beta-Cyclodextrins
D000199:Actins; D015703:Antigens, CD; D051928:CD47 Antigen; C497894:CD47 protein, human; D002352:Carrier Proteins; D003505:Cyclodextrins; D007376:Interleukin-2; D007527:Isoenzymes; D020033:Protein Isoforms; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C076233:PRKCQ protein, human; D011493:Protein Kinase C; D000074121:Protein Kinase C-theta; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002118:Calcium