Analysis of the tyrosine phosphorylation and calcium fluxing of human CD6 isoforms with different cytoplasmatic domains.
CD6 is a cell surface glycoprotein that functions both as a co-stimulatory and adhesion receptor on T cells. Recently we have described CD6 isoforms (CD6a, b, c, d, e) that arise via alternative splicing of exons encoding the cytoplasmic region of the molecule. CD6 becomes phosphorylated on tyrosine (Tyr) residues following stimulation through the T cell receptor (TCR) complex. Since the phosphorylation of Tyr residues renders some cell surface receptors competent for interactions with proteins of intracellular signaling pathways, we wanted to determine which region(s) and residues in the cytoplasmic domain of CD6 were important for phosphorylation on Tyr residues. We engineered and stably expressed chimeric receptors that consisted of the extracellular region of mouse CD6 and the cytoplasmic regions of either naturally occurring isoforms of human CD6, truncated proteins, or point mutants. We were able to demonstrate that of the nine Tyr residues in the cytoplasmic domain of the largest isoform CD6a, the two C-terminal Tyr residues (Tyr 629/662) are critical for the phosphorylation of CD6 following TCR cross-linking. Isoform CD6e, which is missing a region that contains two proline-rich motifs, is not phosphorylated. We further analyzed the ability of the different CD6 isoforms and truncated receptors to mobilize intracellular calcium after CD6/TCR co-ligation. All CD6 isoforms, including CD6e, as well as the truncation mutant delta 555, which is missing approximately the C-terminal half of the cytoplasmic domain, are able to increase Ca2+ influx. Taken together, these results suggest that the region of CD6 which is critical for Ca2+ mobilization is located N-terminal from amino acid 555 and is therefore different from the region located at the C terminus of CD6, which is necessary for tyrosine phosphorylation.
Kobarg
J
J
Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, USA. Jorg-Kobarg@ccmail.bms.com
Whitney
G S
GS
Palmer
D
D
Aruffo
A
A
Bowen
M A
MA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000945:Antigens, Differentiation, T-Lymphocyte / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D018929:Cell Culture Techniques; D003593:Cytoplasm / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D006801:Humans; D007536:Isomerism; D019169:Jurkat Cells; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; C062279:CD6 antigen; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D002118:Calcium