Figure 4.

FLP-34 neuropeptides of the NPY/NPF neuropeptide family activate NPR-11 in vitro. A, Schematic of the calcium-based receptor activation assay to identify ligands of NPR-11. The receptor is expressed in CHO cells that stably coexpress the promiscuous Gα₁₆ subunit and the luminescent calcium indicator aequorin. Upon receptor activation, NPR-11 signaling via Gα₁₆ activates phospholipase C_β (PLC_β), resulting in intracellular Ca²⁺ mobilization monitored by the luminescent biosensor aequorin. B, Two peptides of the FLP-34 precursor dose-dependently activate NPR-11 with nanomolar EC₅₀. The Ca²⁺ responses of CHO cells expressing NPR-11 are shown as percentage of the highest peptide-evoked response (100% activation). Error bars indicate SEM (n ≥ 7). C, FLP-34, but not NLP-1, peptides dose-dependently activate NPR-11. Bar graphs represent the ratio of total calcium response of CHO cells expressing NPR-11 (top) or pcDNA3.1 empty vector (bottom) challenged with five 10-fold serial dilutions of peptides (from 10⁴ to 1 nm), BSA (negative control), or ATP (positive control). Peptide sequences are as follows: FLP-34-1 (ADISTFASAINNAGRLRYa), FLP-34-2 (ALNRDSLVASLNNAERLRFa), NLP-1-1 (MDANAFRMSFa), NLP-1-2 (MDPNAFRMSFa), NLP-1-3 (VNLDPNSFRMSFa), NLP-1-4 (STVGYNLDARNYFVGL). Ratio of total calcium response is calculated as peptide-evoked response normalized to the total calcium response. Data were analyzed by Kruskal–Wallis test, pcDNA3.1::npr-11 (KW(31) = 176.4, p < 0.0001), and pcDNA3.1 empty vector (KW(31) = 78.69, p < 0.0001), and Dunn's post hoc test. Error bars indicate SEM (n = 8). D, Structure of the FLP-34 neuropeptide precursor. Green represents regions of the precursor encoding the mature peptides. The peptide sequences (before amidation) are shown. Top, Cleavage sites for proprotein convertases. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.