Identification of distinct C3b and C4b recognition sites in the human C3b/C4b receptor (CR1, CD35) by deletion mutagenesis.
Complementary DNA clones encoding the NH2-terminal region of human CR1 have been isolated and sequenced. The deduced complete amino acid sequence of the F allotype of human CR1 contains 2,039 residues, including a 41-residue signal peptide, an extracellular domain of 1,930 residues, a 25-amino acid transmembrane domain, and a 43-amino acid cytoplasmic region. The extracellular domain is composed exclusively of 30 short consensus repeats (SCRs), characteristic of the family of C3/C4-binding proteins. The 28 NH2-terminal SCRs are organized as four long homologous repeats (LHRs) of seven SCRs each. The newly sequenced LHR, LHR-A, is 61% identical to LHR-B in the NH2-terminal two SCRs and greater than 99% identical in the COOH-terminal five SCRs. Eight cDNA clones were spliced to form a single construct, piABCD, that contained the entire CR1 coding sequence downstream of a cytomegalovirus promoter. COS cells transfected with piABCD transiently expressed recombinant CR1 that comigrated with the F allotype of erythrocyte CR1 on SDS-PAGE and that mediated rosette formation with sheep erythrocytes bearing C4b and C3b. Recombinant CR1 also had factor I-cofactor activity for cleavage of C3(ma). Analyses of six deletion mutants expressed in COS cells indicated that the NH2-terminal two SCRs of LHR-A contained a site determining C4 specificity and the NH2-terminal two SCRs of LHR-B and -C each had a site determining C3 specificity. The presence of these three distinct sites in CR1 may enable the receptor to interact multivalently with C4b/C3b and C3b/C3b complexes generated during activation of the classical and alternative pathways.
Klickstein
L B
LB
Program in Cell and Developmental Biology, Harvard Medical School, Boston, Massachusetts 02115.
Bartow
T J
TJ
Miletic
V
V
Rabson
L D
LD
Smith
J A
JA
Fearon
D T
DT
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003179:Complement C3b / Q000378:metabolism*; D003181:Complement C4 / Q000378:metabolism*; D015935:Complement C4b; D004252:DNA Mutational Analysis; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011951:Receptors, Complement / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D017463:Receptors, Complement 3b; D015183:Restriction Mapping; D014162:Transfection
D000911:Antibodies, Monoclonal; D003181:Complement C4; D011951:Receptors, Complement; D017463:Receptors, Complement 3b; D003179:Complement C3b; D015935:Complement C4b