Chandrika Kumar

Molecular Cloning and Characterization of the Human Anaphylatoxin C3a Receptor

In a human neutrophil cDNA library, an orphan G-protein-coupled receptor, HNFAG09, with 37% nucleotide identity to the C5a receptor (C5a-R, CD88) was identified. A novel feature of this gene, unlike C5a-R and other G-protein-coupled receptors, is the presence of an extraordinarily large predicted extracellular loop comprised of in excess of 160 amino acid residues between transmembrane domains 4 and 5. Northern blot analysis revealed that expression of mRNA for this receptor in human tissues, while similar, was distinct from C5a-R expression. Although there were differences in expression, transcripts for both receptors were detected in tissues throughout the body and the central nervous system. Mammalian cells stably expressing HNFAG09 specifically bound 125I-C3a and responded to a C3a carboxyl-terminal analogue synthetic peptide and to human C3a but not to rC5a with a robust calcium mobilization response. HNFAG09 encodes the human anaphylatoxin C3a receptor.

CLONING AND CHARACTERIZATION OF A HUMAN ANGIOTENSIN II TYPE 1 RECEPTOR

A human liver cDNA library was screened using a rat type 1 angiotensin II receptor cDNA coding sequence as a probe. cDNA clones were isolated which encoded a protein of 359 amino acids that shared 94.4% and 95.3% identify to rat and bovine type 1 angiotensin II receptors, respectively. Ligand binding studies of the cloned receptor expressed in COS cells suggested that it is pharmacologically a type 1 angiotensin II receptor subtype. Electrophysiological studies of the receptor expressed in Xenopus laevis oocytes revealed that it could functionally couple to a second messenger system leading to the mobilization of intracellular stores of calcium. Southern and Northern blot analyses indicated that the cloned receptor is represented as a single copy in the human genome and is expressed in many tissues of different histogenic origin with the exception of brain, where mRNA transcripts were barely detectable.

Cloning and Characterization of a Novel Integrin β3Subunit

We have identified a novel integrin β3 subunit, termed β3C, from a human osteoclast cDNA library. The COOH-terminal sequence and 3′-untranslated region of the β3C subunit differs from the previously reported β3A (platelet) and β3B (placenta) sequences, while the regions coding for the transmembrane and extracellular domains are identical. The β3C cytoplasmic domain contains 37 amino acids, the last 17 of which are encoded by a novel exon located about 6 kilobase pairs downstream of exon 14 of the β3A gene. HEK 293 cells were stably co-transfected with αV and either β3C (HEKβ3C) or β3A(HEKβ3A). The viability of HEKβ3C cells was lower than that of HEKβ3A cells, and HEKβ3Ccells in culture grew as clusters rather than as a monolayer. The novel cytoplasmic domain did not affect receptor binding affinity; both αVβ3A and αVβ3Cisoforms exhibited high affinity binding to 125I-echistatin and cyclic and linear RGD peptides. However, in contrast to HEKβ3A, HEKβ3C cells failed to adhere to osteopontin, an αVβ3 matrix protein. The data provide further support for the key role of the cytoplasmic domain of the β3 integrin in cell adhesion and suggest a potential role for the β3C integrin subunit in modulating cell-matrix interactions.

IN XENOPUS OOCYTES THE HUMAN C3A AND C5A RECEPTORS ELICIT A PROMISCUOUS RESPONSE TO THE ANAPHYLATOXINS

The Xenopus laevis oocyte has been widely utilized for cloning and functional expression of G‐protein coupled receptors (GPCR). This system was used for the functional expression and characterization of the recently identified human C3a receptor. Complementary RNA from the human C3a receptor was transcribed in vitro and microinjected into Xenopus oocytes for functional characterization. A positive response to a synthetic C3a peptide agonist and to C3a, but not to platelet activating factor or fMetLeuPhe was detected. In addition, a response of approximately one third the amplitude obtained with C3a was obtained with rC5a. Conversely, oocytes co‐injected with the C5a receptor and total RNA isolated from U937 cells responded to C5a as well as to C3a and the C3a synthetic peptide. A functional response with the anaphylatoxin C3a receptor in oocytes was dependent on co‐injection of a pertussis toxin sensitive complementary human factor which could be supplied by co‐injection of total RNA isolated from U937 cells. Oocytes expressing the anaphylatoxin C3a and C5a receptors responded to both agonists, in each case the response to the cognate ligand was substantially more robust than the response elicited by the other anaphylatoxin.

Absence of endothelin receptors and receptor mRNA in mammalian fibroblasts transformed with SV40 or ras oncogene

Endothelin-1 (ET-1), a peptide isolated from the culture medium of endothelial cells, mediates a variety of physiological and pathological responses including mitogenesis. We have compared the expression of ET receptors in untransformed versus ras-transformed NIH-3T3 murine fibroblasts and in untransformed versus SV40-transformed Wl38 (VA13) human fibroblasts by ligand binding and Northern analysis. NIH-3T3 and Wl38 cells displayed high affinity (200 and 220 pM) and high density (23,000 sites/cell and 14,000 sites/cell for NIH-3T3 and Wl38 cells, respectively) ET receptors. Competition binding experiments using subtype-selective ligands identified these receptors as the ETA subtype. Addition of ET-1 to the cells produced a concentration-dependent increase in intracellular calcium release. Both ras-transformed NIH-3T3 cells and SV40-transformed Wl38 cells (VA13) completely lacked [125I]ET-1 binding and failed to release calcium when exposed to ET-1. Northern analysis of the polyadenylat ed RNA (polyA RNA) isolated from untransformed and transformed cells revealed that the steady-state level of ETA receptor RNA was 90-95% less in transformed cells compared to untransformed cells. Thus, the loss of ET receptors as well as the receptor-mediated responses in transformed cells can be explained by down-regulation of ET receptor mRNA.