Quantitative analysis of the formation and diffusion of A1-adenosine receptor-antagonist complexes in single living cells.

The A1-adenosine receptor (A1-AR) is a G protein-coupled receptor that mediates many of the physiological effects of adenosine in the brain, heart, kidney, and adipocytes. Currently, ligand interactions with the A1-AR can be quantified on large cell populations only by using radioligand binding. To increase the resolution of these measurements, we have designed and characterized a previously undescribed fluorescent antagonist for the A1-AR, XAC-BY630, based on xanthine amine congener (XAC). This compound has been used to quantify ligand-receptor binding at a single cell level using fluorescence correlation spectroscopy (FCS). XAC-BY630 was a competitive antagonist of A1-AR-mediated inhibition of cAMP accumulation [log10 of the affinity constant (pKb) = 6.7)] and stimulation of inositol phosphate accumulation (pKb = 6.5). Specific binding of XAC-BY630 to cell surface A1-AR could also be visualized in living Chinese hamster ovary (CHO)-A1 cells by using confocal microscopy. FCS analysis of XAC-BY630 binding to the membrane of CHO-A1 cells revealed three components with diffusion times (tauD) of 62 micros (tauD1, free ligand), 17 ms (tauD2, A1-AR-ligand), and 320 ms (tauD3). Confirmation that tauD2 resulted from diffusion of ligand-receptor complexes came from the similar diffusion time observed for the fluorescent A1-AR-Topaz fusion protein (15 ms). Quantification of tauD2 showed that the number of receptor-ligand complexes increased with increasing free ligand concentration and was decreased by the selective A1-AR antagonist, 8-cyclopentyl-1,3-dipropylxanthine. The combination of FCS with XAC-BY630 will be a powerful tool for the characterization of ligand-A1-AR interactions in single living cells in health and disease.

IFN-gamma exposes a cryptic cytotoxic T lymphocyte epitope in HIV-1 reverse transcriptase.

The proteasome, an essential component of the ATP-dependent proteolytic pathway in eukaryotic cells, is responsible for the degradation of most cellular proteins and is believed to be the main source of MHC class I-restricted antigenic peptides for presentation to CTL. Inhibition of the proteasome by lactacystin or various peptide aldehydes can result in defective Ag presentation, and the pivotal role of the proteasome in Ag processing has become generally accepted. However, recent reports have challenged this observation. Here we examine the processing requirements of two HLA A*0201-restricted epitopes from HIV-1 reverse transcriptase and find that they are produced by different degradation pathways. Presentation of the C-terminal ILKEPVHGV epitope is impaired in ME275 melanoma cells by treatment with lactacystin, and is independent of expression of the IFN-gamma-inducible proteasome beta subunits LMP2 and LMP7. In contrast, both lactacystin treatment and expression of LMP7 induce the presentation of the N-terminal VIYQYMDDL epitope. Consistent with these observations we show that up-regulation of LMP7 by IFN-gamma enhances presentation of the VIYQYMDDL epitope. Hence interplay between constitutive and IFN-gamma-inducible beta-subunits of the proteasome can qualitatively influence Ag presentation. These observations may have relevance to the patterns of immunodominance during the natural course of viral infection.