Rj. Peach et al., COMPLEMENTARITY-DETERMINING REGION-1 (CDR1)-ANALOGOUS AND CDR3-ANALOGOUS REGIONS IN CTLA-4 AND CD28 DETERMINE THE BINDING TO B7-1, The Journal of experimental medicine, 180(6), 1994, pp. 2049-2058
T cell surface receptors CD28 and CTLA-4 are homologous members of the
immunoglobulin superfamily (IgSF), each comprising a single V-like ex
tracellular domain. CD28 and CTLA-4 bind to the B7-1 and B7-2 counter-
receptors on antigen presenting cells (APCs), thereby triggering a cos
timulatory pathway important for optimal T cell activation in vitro an
d in vivo. Soluble forms of CD28 and CTLA-4 in which the V-like extrac
ellular domains were fused to Ig constant domains (CD28Ig and CTLA4Ig)
, have been used to study their interactions with B7-1 and B7-2, with
CTLA4Ig binding B7-1 more strongly than CD28Ig (similar to 20-fold hig
her avidity). We have now, by site-specific and homologue mutagenesis,
identified regions in CTLA4Ig important for strong binding to B7-1. A
hexapeptide motif (MYPPPY) in the complementarity determining region
3 (CDR3)-like region is fully conserved in all CD28 and CTLA-4 family
members. Alanine scanning mutagenesis through the motif in CTLA4Ig and
at selected residues in CD28Ig reduced or abolished binding to B7-1.
Chimeric molecules HS4, HS4-A, and HS4-B were constructed in which CDR
3-like regions of CTLA-4, COOH-terminally extended to include nonconse
rved residues, were grafted onto CD28Ig. These homologue mutants showe
d stronger binding to B7-1 than did CD28Ig. Grafting of the CDR1-like
region of CTLA-4, which is not conserved in CD28 and is predicted to b
e spatially adjacent to CDR3, into HS4 and HS4-A, resulted in chimeric
molecules (HS7 and HS8) which bound B7-1 even better. Inclusion of th
e CDR2-like domain of CTLA-4 into HS7 and HS8 did not further increase
binding. Thus, the MYPPPY motifs of CTLA4Ig and CD28Ig are important
for their binding to B7-1, but the increased strength of this binding
by CTLA4Ig is mediated by nonconserved residues in the CDR1- and CDR3-
analogous regions.