Membrane cofactor protein (MCP; CD46) is a widely distributed C3b/C4b-binding cell surface glycoprotein which serves as an inhibitor of complement activation on host cells. The protein has been purified, multiple cDNAs cloned and sequenced, and the genomic organization determined. MCP belongs to a family known as the regulators of complement activation (RCA) gene cluster. The RCA members are related structurally [possess approximately 60 amino acid repeating motifs termed short consensus repeats (SCR)], functionally (bind C3b/C4b), and genetically (genes are tightly clustered on chromosome 1 at q3.2). Beginning at its amino-terminus, MCP is composed of four SCRs, a ser/thr/pro-enriched region, an area of undefined function, a transmembrane hydrophobic domain, a cytoplasmic anchor and cytoplasmic tail. On SDS-PAGE, MCP migrates as two broad forms with Mrs of 59,000-68,000 and 51,000-58,000. The quantity of each form expressed is inherited in an autosomal codominant fashion. This structural heterogeneity is partly explained by the expression of multiple cDNA/protein isoforms that arise by alternative splicing of ser/thr/pro-rich exons (sites of heavy O-glycosylation) and of cytoplasmic tails. This protein is of interest to immunologists and clinicians because of its role in regulation of the complement pathways and, therefore, inflammation in immune complex-mediated syndromes; to reproductive immunologists on account of its expression on sperm and at the maternal-fetal interface; and to tumor immunologists because of its high expression on malignant cells. The availability of monoclonal and polyclonal antibodies and molecular probes will be helpful in addressing questions about the biology of MCP in these and other areas.

SummaryThe chemokine eotaxin is unusual in that it appears to be a highly specific chemoattractant for eosinophils. Ligand-binding studies with radiolabeled eotaxin demonstrated a receptor on eosinophils distinct from the known chemokine receptors CKR-1 and -2. The distinct eotaxinbinding site on human eosinophils also bound RANTES (regulated on activation T expressed and secreted) and monocyte chemotactic protein (MCP)3. We have now isolated a cDNA from eosinophils, termed CKR-3, with significant sequence similarity to other well characterized chemokine receptors. Cells transfected with CKR-3 cDNA bound radiolabeled eotaxin specifically and with high affinity, comparable to the binding affinity observed with eosinophils. This receptor also bound R.ANTES and MCP-3 with high affinity, but not other CC or CXC chemokines. Furthermore, receptor transfectants generated in a murine B cell lymphoma cell line migrated in transwell chemotaxis assays to eotaxin, RANTES, and MCP-3, but not to any other chemokines. A monoclonal antibody recognizing CKR-3 was used to show that eosinophils, but not other leukocyte types, expressed this receptor. This pattern of expression was confirmed by Northern blot with RNA from highly purified leukocyte subsets. The restricted expression of CKR-3 on eosinophils and the fidelity of eotaxin binding to CKR-3, provides a potential mechanism for the selective recruitment and migration of eosinophils within tissues.

SummaryMembrane cofactor protein (MCP) is a complement regulatory protein that is expressed on human cells and cell lines as two relatively broad species with M, of 58,000-68,000 and 48,000-56,000. The structure of a previously reported cDNA clone indicated that MCP was a type 1 membrane glycoprotein and a member of the regulators of complement activation gene/protein cluster. However, it did not provide an explanation for the unusual phenotypic pattern ofMCP. Therefore, in parallel with an analysis of the gene, additional cDNAs were cloned and characterized . Six different MCP cDNA classes were identified. All encode the same 5' untranslated signal peptide, four SCRs, transmembrane domain, and basic amino acid anchor. However, they differ in the length and composition of an extracellular serine/threonine/proline (STP)-rich area, a site of heavy O-glycosylation, and cytoplasmic tail. Analysis of the MCP gene demonstrated that the variation in cDNA structure was a result of alternative splicing. Peripheral blood cells and cell lines predominantly expressed four of the six isoforms. These varied by the presence or absence of an STP-rich segment of 15 amino acids (STPB) and by the use of one of two cytoplasmic domains . Analysis by polymerase chain reaction, Northern blots, and transfection indicated that the predominance of MCP cDNA isoforms with STPB correlated with the high molecular weight protein phenotype, while the predominance of isoforms without STPB correlated with the lower molecular weight phenotype . The expression in a single cell of four distinct protein species with variable STP-rich regions and cytoplasmic tails represents an interesting example of the use of alternative splicing to provide variability in a mammalian protein .

Membrane cofactor protein (MCP), a regulatory molecular of the complement system with cofactor activity for the factor I-mediated inactivation of C3b and C4b, is widely distributed, being present on leukocytes, platelets, endothelial cells, epithelial cells, and fibroblasts. MCP was purified from a human T cell line (HSB2) and the NH2-terminal 24-amino acid sequence obtained by Edman degradation. An oligonucleotide probe based on this sequence was used to identify a clone from a human monocytic (U937) cDNA library. Nucleotide sequencing showed a 43-bp 5'-untranslated region, an open reading frame of 1,152 bp, and a 335-bp 3'-untranslated region followed by a 16-bp poly(A) track. The deduced full-length MCP protein consists of a 34-amino acid signal peptide and a 350-amino acid mature protein. The protein has, beginning at the NH2 terminus, four approximately 60-amino acid repeat units that match the consensus sequence found in a multigene family of complement regulatory proteins (C3b-receptor or CR1, C3d-receptor or CR2, decay-accelerating factor, C4-binding protein, and factor H), as well as several other complement and non-complement proteins. The remainder of the MCP protein consists of 25 amino acids that are rich in serine and threonine (probable site of heavy O-linked glycosylation of MCP), 17 amino acids of unknown significance, and a 23-amino acid transmembrane hydrophobic region followed by a 33-amino acid cytoplasmic tail. The MCP gene was localized to human chromosome 1, bands 1q31-41, by analysis of human x rodent somatic cell hybrid clones and by in situ hybridization. This same genetic region contains the multigene family of complement-regulatory proteins, which is thereby enlarged to include the functionally and structurally related MCP.

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