ORIGINAL ARTICLE Fluorescence Observation of Single-Cell cAMP Signaling by G Protein-Coupled Receptors Dongmei Zhang 1 & Giuma E. Hadhoud 2 & Karen Helm 3 & Deborah A. Roess 2,4 & B. George Barisas 1,2 Received: 6 August 2018 /Accepted: 14 October 2018 # Springer Science+Business Media, LLC, part of Springer Nature 2018 Abstract 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 in- creased inter-receptor interactions at high concentrations. We then explored a microscope-based method for single cell measure- ments 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. Keywords Fluorescence . Cytometry . Microscopy . ICUE . Beads . CFP . YFP . Single cell . G protein coupled . Receptor . Signaling Introduction The LH receptor is a seven-transmembrane domain receptor in the G protein-coupled receptors (GPCR) superfamily [1]. Signal transduction by LH receptors in both males and fe- males is important for successful reproduction. Loss-of- function LH receptor mutations cause a number of diseases including Leydig cell hypoplasia [2]. Familial male-limited precocious puberty (FMPP) is caused by a genetic mutation in the LH receptor resulting in a constitutively-active LH re- ceptor which causes early puberty in males who have high levels of testosterone and low levels of gonadotropin [3]. It has been shown that specific LH receptors can be detected in human endometrial cancer where their expression levels are related to the cancer grade [4]. Activation of the LH receptor by binding human chorionic gonadotropin (hCG) leads to an increase in intracellular levels of adenosine 3′ ,5′ -cyclic monophosphate (cAMP). Of specific interest is whether high levels of LH receptor expression are associated with reduced cell response to ligands that function through the cAMP sec- ond messenger. Reduced cAMP response has been previously observed in cells expressing constitutively active LH receptors Dongmei Zhang and Giuma E. Hadhoud contributed equally to the project. * B. George Barisas george.barisas@colostate.edu 1 Department of Chemistry, Colorado State University, Fort Collins, CO 80523, USA 2 Cell and Molecular Biology Program, Colorado State University, Fort Collins, CO 80523, USA 3 Flow Cytometry Shared Resource, University of Colorado Cancer Center, Aurora, CO 80045, USA 4 Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA Journal of Fluorescence https://doi.org/10.1007/s10895-018-2309-1