SummaryAlthough there is a mounting body of evidence that eosinophils are recruited to sites of allergic inflammation by a number of ~-chemokines, particularly eotaxin and RANTES, the receptor that mediates these actions has not been identified. We have now cloned a G protein-coupled receptor, CC CKR3, from human eosinophils which, when stably expressed in AML14.3D10 cells bound eotaxin, MCP-3 and R_ANTES with Kas of 0.1, 2.7, and 3.1 nM, respectively. CC CKR3 also bound MCP-1 with lower affinity, but did not bind MIP-10~ or MIP-I[~. Eotaxin, RANTES, and to a lessor extent MCP-3, but not the other chemokines, activated CC CKR3 as determined by their ability to stimulate a CaZ+-flux. Competition binding studies on primary eosinophils gave binding at~inities for the different chemokines which were indistinguishable from those measured with CC CKR3. Since CC CKR3 is prominently expressed in eosinophils we conclude that CC CKR3 is the eosinophil eotaxin receptor. Eosinophils also express a much lower level of a second chemokine receptor, CC CKR1, which appears to be responsible for the effects of MIP-llx.
StllnllllaryTransgenic mice expressing human major histocompatibility complex (MHC) class II molecules would provide a valuable model system for studying human immunology. However, attempts to obtain human class II-restricted T cell responses in such transgenic mice have had only limited success, possibly due to an inability of mouse CD4 to interact efficiently with human MHC class II molecules. To circumvent this problem, we constructed recombinant MHC class II genes in which the peptide-binding domain was derived from human DR sequences whereas the CD4-binding domain was derived from mouse I-E sequences. Purified chimeric human/mouse MHC class II molecules were capable of specifically binding DR-restricted peptides. Human B cell transformants that expressed these chimeric MHC class II molecules could present peptide antigens to human T cell clones. Expression of these chimeric class II molecules in transgenic mice led to the intrathymic deletion of T cells expressing superantigen-reactive V/3 gene segments, indicating that the chimeric class II molecules could influence the selection of the mouse T cell repertoire. These transgenic mice were fully capable of mounting human DR-restricted immune responses after challenge with peptide or whole protein antigens. Thus, the chimeric class II molecules can serve as functional antigen presentation molecules in vivo. In addition, transgenic mice expressing chimeric class II molecules could be used to generate antigen-specific mouse T cell hyhridomas that were capable of interacting with human antigen-presenting cells.
A congenic, non-obese diabetic (NOD) mouse strain that contains a segment of chromosome 3 from the diabetes-resistant mouse strain B6.PL-Thy-1a was less susceptible to diabetes than NOD mice. A fully penetrant immunological defect also mapped to this segment, which encodes the high-affinity Fc receptor for immunoglobulin G (IgG), Fc gamma RI. The NOD Fcgr1 allele, which results in a deletion of the cytoplasmic tail, caused a 73 percent reduction in the turnover of cell surface receptor-antibody complexes. The development of congenic strains and the characterization of Mendelian traits that are specific to the disease phenotype demonstrate the feasibility of dissecting the pathophysiology of complex, non-Mendelian diseases.
We describe the development and characterization of substance P labeled at Lys3 with fluorescein ([fluorescein Lys3]SP) as a fluorescent probe for the neurokinin 1 (NK1) receptor. [fluorescein Lys3]SP is an agonist at the human NK1 receptor, with an affinity for both the high-affinity and low-affinity binding states of the receptor approximately 6-fold lower than that of substance P. Binding of the probe to the human NK1 receptor expressed in Sf9 insect cells was observed directly by monitoring either a decrease in fluorescence intensity or an increase in anisotropy of the [fluorescein Lys3]SP. Detection by anisotropy gave the larger signal and thus was used to characterize the interaction of [fluorescein Lys3]SP with the receptor. The anisotropy of the bound ligand was 0.17, compared to 0.04 for the free ligand. The fluorescence was quenched by about 15% upon binding to the receptor. Bound [fluorescein Lys3]SP was displaced by unlabeled SP and by the quinuclidine antagonist L-703,606. As expected for an agonist, binding was also reduced by the addition of the nonhydrolyzable guanine nucleotide analog GppNHp. [fluorescein Lys3]SP should provide a useful structural and kinetic probe for the NK1 receptor.
Glucagon is a 29-amino acid peptide produced by proteolytic cleavage of the proglucagon gene product in the A cells of the pancreas. The peptide acts at the liver to increase the rate of gluconeogenesis and glycogenolysis, in this regard serving as the major counterregulatory hormone of insulin (for review, see Ref. 1). Glucagon binds to specific receptors on the surface of hepatocytes to stimulate increases in cyclic AMP, inositol phosphate, and intracellular calcium. The rat and human glucagon receptors have been cloned (2-4) and shown to contain seven putative transmembrane domains characteristic of the G protein-coupled family of receptors (5). The glucagon receptor shares significant sequence homology with the subfamily of G protein-coupled receptors that includes receptors for glucagonlike peptide-1 and parathyroid hormone (for review, see Ref. 6). This subclass of G protein-coupled receptors bears little sequence homology to the well characterized -adrenergic/rhodopsin subfamily, and relatively little is known about the molecular interactions of ligands with receptors in this class.Glucagon itself has been the subject of a wide variety of physical studies such as x-ray crystallography (7), NMR (8), and circular dichroism (9). However, these studies have been performed in various lipid or detergent solutions, which affect the conformation of the peptide. In addition, it has been observed that the secondary structure of glucagon is dependent on the concentration of peptide used in the experiment (9). For these reasons, the structure determined by these techniques may not reflect the physiological state of glucagon as it interacts with its receptor.As is the case for most G protein-coupled receptors, biophysical and structural characterization of the glucagon receptor has been hampered by an inability to produce sufficient quantities of receptor protein. In the present study, we have expressed the glucagon receptor at high density using the Drosophila Schneider 2 (S2) cell system (10). [fluoresceinTrp 25 ]Glucagon (11) has been used as a tool for obtaining kinetic and structural information on the human glucagon receptor. The high levels of expression of human glucagon receptor obtained in S2 cells have allowed us to directly monitor changes in the fluorescence properties of this ligand as it binds to the receptor. This system has proven useful in understanding the environment of the ligand binding site of the human glucagon receptor and in monitoring conformational changes in both the receptor and the ligand during the binding interaction.
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