G protein-coupled receptors (GPCRs) constitute important targets for drug discovery against a wide range of ailments including cancer, inflammatory, and cardiovascular diseases. Efforts are underway to screen selective modulators of GPCRs and also to deorphanize GPCRs with unidentified natural ligands. Most GPCR-based cellular screens depend on labeling or recombinant expression of receptor or reporter proteins, which may not capture the true physiology or pharmacology of the GPCRs. In this paper, we describe a noninvasive and label-free assay for GPCRs that can be used with both engineered and nonengineered cell lines. The assay is based on using cell-electrode impedance to measure minute changes in cellular morphology as a result of ligand-dependent GPCR activation. We have used this technology to assay the functional activation of GPCRs coupled to different signaling pathways and have compared it to standard assays. We have used pharmacological modulators of GPCR signaling pathways to demonstrate the specificity of impedance-based measurements. Our data indicate that cell-electrode impedance measurements offer a convenient, sensitive, and quantitative method for assessing GPCR function. Moreover, the noninvasive nature of the readout offers the added advantage of performing multiple treatments in the same well to study events such as desensitization and receptor cross-talk.

IL-15 and IL-2 are two structurally and functionally related cytokines whose high affinity receptors share the IL-2R β-chain and γ-chain in association with IL-15R α-chain (IL-15Rα) or IL-2R α-chain, respectively. Whereas IL-2 action seems restricted to the adaptative T cells, IL-15 appears to be crucial for the function of the innate immune responses, and the pleiotropic expression of IL-15 and IL-15Rα hints at a much broader role for the IL-15 system in multiple cell types and tissues. In this report, using a highly sensitive radioimmunoassay, we show the existence of a soluble form of human IL-15Rα (sIL-15Rα) that arises from proteolytic shedding of the membrane-anchored receptor. This soluble receptor is spontaneously released from IL-15Rα-expressing human cell lines as well as from IL-15Rα transfected COS-7 cells. This release is strongly induced by PMA and ionomycin, and to a lesser extent by IL-1β and TNF-α. The size of sIL-15Rα (42 kDa), together with the analysis of deletion mutants in the ectodomain of IL-15Rα, indicates the existence of cleavage sites that are proximal to the plasma membrane. Whereas shedding induced by PMA was abrogated by the synthetic matrix metalloproteinases inhibitor GM6001, the spontaneous shedding was not, indicating the occurrence of at least two distinct proteolytic mechanisms. The sIL-15Rα displayed high affinity for IL-15 and behaved as a potent and specific inhibitor of IL-15 binding to the membrane receptor, and of IL-15-induced cell proliferation (IC50 in the range from 3 to 20 pM). These results suggest that IL-15Rα shedding may play important immunoregulatory functions.

Background: TLQP-21 is a bioactive peptide for which the receptor(s) are unknown. Results: We demonstrate that C3AR1 is a receptor for TLQP-21. Conclusion: Many of the effects of TLQP-21 can be explained by C3AR1 activation. Significance: These results provide a bridge linking the regulation of metabolism and the activation of complement in rodents.

We report the existence of eight different interleukin-15 receptor ␣-chain (IL-15R␣) transcripts resulting from exon-splicing mechanisms within the IL-15R␣ gene. Two main classes of transcripts can be distinguished that do or do not (⌬2 isoforms) contain the exon 2-coding sequence. Both classes were expressed in numerous cell lines and tissues (including peripheral blood lymphocytes) at comparable levels and could be transcribed in COS-7 cells, and the proteins were expressed at the cell surface. Both receptor forms displayed numerous glycosylation states, reflecting differential usage of a single N-glycosylation site as well as extensive O-glycosylations. Whereas IL-15R␣ bound IL-15 with high affinity, ⌬2IL-15R␣ was unable to bind IL-15, thus revealing the indispensable role of the exon 2-encoded domain in cytokine binding. A large proportion of IL-15R␣ was expressed at the nuclear membrane with some intranuclear localization, supporting a potential direct action of the IL-15⅐IL-15R␣ complex at the nuclear level. In sharp contrast, ⌬2IL-15R␣ was found only in the non-nuclear membrane compartments, indicating that the exon 2-encoded domain (which is shown to contain a potential nuclear localization signal) plays an important role in receptor post-translational routing. Together, our data indicate that exon 2 splicing of human IL-15R␣ is a natural process that might play regulatory roles at different levels.