RESUMO
We present complementary flow cytometric and microscopic imaging methods, both utilizing a membrane-targeted cAMP sensor protein ICUE3, to examine hormone-dependent signaling by the luteinizing hormone (LH) receptor in individual cells. This receptor, a seven transmembrane domain protein belonging to the GPCR family, signals by activating adenylate cyclase to increase cAMP levels. The ICUE3 sensor protein exhibits fluorescence energy transfer between its CFP and YFP moieties and the ratio of CFP emission to YFP sensitized emission (YFPSE) increases with cAMP concentration. We used multichannel flow cytometry to compare CFP emission and YFPSE from each cell and hence measure that cell's cAMP level. This technique measured changes in cAMP levels in CHO cells expressing LH receptors and stimulated by forskolin or the hormone human chorionic gonadotropin (hCG) and showed that significant cell-to-cell variations exist in such cAMP responses. Because LH receptor behavior may reflect receptor expression levels, we developed a procedure to measure numbers of particular fluorescent cell proteins from measurements of MESF bead standards for slightly different fluorophores. We find that basal cAMP levels increase substantially in cells expressing high numbers mCherry-LH receptors per cell. This suggests activation through increased inter-receptor interactions at high concentrations. We then explored a microscope-based method for single cell measurements so that responses could be correlated with specific cell morphology and with time after treatments. This showed that cell responses to hCG are fully-developed after ~100 s. Taken together, these results demonstrate the utility of fluorescence methods in exploring cAMP signaling in individual cells.