Figure 1.

Recruitment of TRAF2 and TRAF3 by hCD40LMP1AEDL and hCD40ADD in B cells. A, Domain composition of hCD40LMP1, hCD40LMP1AEDL, hCD40, and hCD40ADD. The LMP1 chimeric receptors are composed of the extracellular and transmembrane domains of hCD40 and either the full length CY domain of LMP1 (aa 187–386 of LMP1) or a TBS mutant version of this LMP1 CY domain (hCD40LMP1AEDL) where the sequence of the TBS has been changed from PQQATDD to PQQETLD (the CD40 TBS sequence). hCD40 is WT hCD40 while hCD40ADD has had its TBS mutated from that of CD40 (PVQETLH) to that of LMP1 (PVQATDD). B, M12 and M12 B cell clones were expression matched into sets expressing similar levels of hCD40, hCD40LMP1, hCD40LMP1AEDL, and hCD40ADD as determined by immunofluorescence flow cytometry. Similar results were obtained for CH12.LX subclones (not shown). C, M12.4.1 B cells were stably transfected with hCD40LMP1, hCD40, hCD40ADD, or hCD40LMP1AEDL and stimulated with 10 μl of Dynabeads coated with anti-hCD40 Ab for 15 min. The post lysis (PL) control sample was lysed before addition of anti-hCD40 Ab coated Dynabeads as in Methods. Samples were blotted for TRAF2, TRAF3, and hCD40. D, Quantification of TRAF binding in B cells was performed by measuring the bands with a low-light imaging system. The desitometric quantification of the TRAF band was normalized to the desitometric quantification of the corresponding CD40 band. The normalized densitometric quantification of TRAFs co-immunoprecipitated with a receptor following cell lysis (“PL”) was subtracted from the normalized value of the stimulated (“Stim”) samples. The normalized values for hCD40 ADD and hCD40LMP1AEDL are represented graphically as percentages of the normalized values of hCD40 and hCD40LMP1. Data shown are representative of six or more independent experiments utilizing two or more clones of each transfectant.