Vicki L. Schweickart

Seven-transmembrane receptors.

Seven-transmembrane receptors, which constitute the largest, most ubiquitous and most versatile family of membrane receptors, are also the most common target of therapeutic drugs. Recent findings indicate that the classical models of G-protein coupling and activation of second-messenger-generating enzymes do not fully explain their remarkably diverse biological actions.

Molecular cloning and functional characterization of a novel human CC chemokine receptor (CCR5) for RANTES, MIP-1beta, and MIP-1alpha

Chemokines affect leukocyte chemotactic and activation activities through specific G protein-coupled receptors. In an effort to map the closely linked CC chemokine receptor genes, we identified a novel chemokine receptor encoded 18 kilobase pairs downstream of the monocyte chemoattractant protein-1 (MCP-1) receptor (CCR2) gene on human chromosome 3p21. The deduced amino acid sequence of this novel receptor, designated CCR5, is most similar to CCR2B, sharing 71% identical residues. Transfected cells expressing the receptor bind RANTES (regulated on activation normal T cell expressed), MIP-1β, and MIP-1α with high affinity and generate inositol phosphates in response to these chemokines. This same combination of chemokines has recently been shown to potently inhibit human immunodeficiency virus replication in human peripheral blood leukocytes (Cocchi, F., DeVico, A. L., Garzino-Demo, A., Arya, S. K., Gallo, R. C., and Lusso, P. (1995) Science 270, 1811-1815). CCR5 is expressed in lymphoid organs such as thymus and spleen, as well as in peripheral blood leukocytes, including macrophages and T cells, and is the first example of a human chemokine receptor that signals in response to MIP-1β.

New members of the chemokine receptor gene family

Chemokines are relatively small peptides with potent chemoattractant and activation activities for leukocytes. Several chemokine receptors have been cloned and characterized and all are members of the G protein‐coupled receptor superfamily. Using degenerate oligonucleotides and polymerase chain reaction, we have identified seven novel receptors with significant homology to chemokine receptors. Two of these sequences are presented here for the first time. We have shown, with gene mapping studies, that receptors with the highest sequence similarity are closely linked on human chromosomes. This close genetic association suggests a functional relationship as well.

The orphan G-protein-coupled receptor-encoding gene V28 is closely related to genes for chemokine receptors and is expressed in lymphoid and neural tissues

A polymerase chain reaction (PCR) strategy with degenerate primers was used to identify novel G-protein-coupled receptor-encoding genes from human genomic DNA. One of the isolated clones, termed V28, showed high sequence similarity to the genes encoding human chemokine receptors for monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 1 alpha (MIP-1 alpha)/RANTES, and to the rat orphan receptor-encoding gene RBS11. When RNA was analyzed by Northern blot, V28 was found to be most highly expressed in neural and lymphoid tissues. Myeloid cell lines, particularly THP.1 cells, showed especially high expression of V28. We have mapped V28 to human chromosome 3p21-3pter, near the MIP-1 alpha/RANTES receptor-encoding gene.

Monocyte chemotactic protein-4: tissue-specific expression and signaling through CC chemokine receptor-2.

Chemokines constitute a family of low‐molecular‐weight proteins that attract or activate a variety of cell types, including leukocytes, endothelial cells, and fibroblasts. An electronic search of the GenBank Expressed Sequence Tags database uncovered a partial cDNA sequence with homology to the chemokine monocyte chemotactic protein‐1 (MCP‐1). Isolation of the full‐length clone revealed that it encodes the chemokine MCP‐4, an eosinophil chemoattractant recently described by Uguccioni et al. [J. Exp. Med. 183, 2379–2384]. Recombinant MCP‐4 was expressed in mammalian cells and purified by heparin‐Sepharose chromatography. Sequencing the amino terminus of this protein corroborated the reported sequence of recombinant MCP‐4 produced in insect cells. As shown by calcium flux assays, MCP‐4 activated the cloned G protein‐coupled receptor CCR‐2, which also recognizes MCP‐1 and MCP‐3. Northern hybridization indicated that MCP‐4 is constitutively expressed at high levels in the small intestine, colon, and lung. This expression profile is consistent with its role as a chemoattractant for eosinophils, which can be rapidly mobilized to the lung or intestine in response to invading pathogens. In marked contrast to MCP‐1, MCP‐4 was not induced in cell lines treated with pro‐inflammatory stimuli such as lipopolysaccharide or tumor necrosis factor α. J. Leukoc. Biol. 61: 353–360; 1997.

The human platelet-activating factor receptor gene (PTAFR) contains no introns and maps to chromosome 1

Platelet-activating factor (PAF), a phospholipid, exhibits a variety of potent inflammatory bioactivities that are mediated by a specific cell surface receptor. The gene for the human PAF receptor (PTAFR) has been isolated by hybridization with a guinea pig probe. The coding sequence contains no intervening sequences. The encoded protein is highly homologous to the guinea pig PAF receptor (82% identity) and contains seven putative transmembrane domains. The PAF receptor therefore appears to be a member of the G protein coupled family of receptors and exhibits significant similarity to many members of the family. Analysis of somatic cell hybrids suggests that the PAF receptor is encoded by a single gene on human chromosome 1.

Profile of human macrophage transcripts: insights into macrophage biology and identification of novel chemokines.

High throughput partial sequencing of randomly selected cDNA clones has proven to be a powerful tool for examining the relative abundance of mRNAs and for the identification of novel gene products. Because of the important role played by macrophages in immune and inflammatory responses, we sequenced over 3000 randomly selected cDNA clones from a human macrophage library. These sequences represent a molecular inventory of mRNAs from macrophages and provide a catalog of highly expressed transcripts. Two of the most abundant clones encode recently identified CC chemokines. Macrophage‐derived chemokine (MDC) plays a complex role in immunoregulation and is a potent chemoattractant for dendritic cells, T cells, and natural killer cells. The chemokine receptor CCR4 binds MDC with high affinity and also responds by calcium flux and chemotaxis. CCR4 has been shown to be expressed by Th2 type T cells. Recent studies also implicate MDC as a major component of the host defense against human immunodeficiency virus. J. Leukoc. Biol. 64: 49–54; 1998.

The Chemokine Gene Family

Our understanding of the function of chemokines has been reshaped over the past few years because of the large number of new chemokines recently discovered. Earlier reviews on chemokine structure and function presented a relatively simple picture of the chemokine family: a handful of CXC and CC chemokines, whose genes clustered on chromosomes 4 and 17, respectively, involved in the recruitment of leukocyte subsets to sites of inflammation (1,2). Over the past few years, however, the use of computer technology to search vast libraries of randomly sequenced cDNAs has brought to light many novel chemokines that complicate the picture described above, but also enrich our understanding of the functional diversity of chemokines. Not only have new chemokine families and chromoobtained, and how they have broadened our understanding of the structure and function of chemokines.