Contents lists available at ScienceDirect Neuropeptides journal homepage: www.elsevier.com/locate/npep PK2β ligand, a splice variant of prokineticin 2, is able to modulate and drive signaling through PKR1 receptor Roberta Lattanzi a , Daniela Maftei a , Lucia Negri a , Ilaria Fusco a , Rossella Miele b, ⁎ a Dipartimento di Fisiologia e Farmacologia “Vittorio Erspamer”, Sapienza Università di Roma, Piazzale Aldo Moro 5, I-00185 Rome, Italy b Dipartimento di Scienze Biochimiche A. Rossi Fanelli, CNR Istituto di Biologia e Patologia Molecolare, Sapienza Università di Roma, Piazzale Aldo Moro 5, I-00185 Rome, Italy ARTICLE INFO Keywords: Prokineticin 2β Prokineticin receptors G protein coupled receptor Yeast-STAT3 Thermal hyperalgesia Tactile allodynia ABSTRACT Prokineticin-2 (PK2) is a secreted bioactive peptide that signals through two GPCRs, the prokineticin receptors (PKRs), and regulates a variety of biological processes including angiogenesis, immunity and nociception. The PK2 primary transcript has two alternative splice variants, PK2 and PK2L (a Long form) which is cleaved in an active peptide, named PK2β that preferentially binds to PKR1 receptor. The aim of this study was to characterize the PK2β. Using different Saccharomyces cerevisiae strains, we examined the specificity of PKR1 and PKR2 G-protein coupling following PK2β binding. Data obtained in yeast confirmed that PK2 binds both receptors, inducing a comparable response throughout a promiscuous coupling of G protein subtypes. Conversely, we demonstrated, for the first time, that PK2β preferentially binding to PKR1, activates a signaling cascade that not depends on Gα i/o coupling. The binding specificity of PK2β for PKR1 was evaluated by the analysis of PKR mutant in yeast and GST pull-down experiments, suggesting an important role of PKR1 amino-terminal region. We also eval- uated the ability of PK2β to differentially activate PKR1 and/or PKR2 by in vivo nociceptive experiments and we showed that PK2β induces intense sensitization of peripheral nociceptors to painful stimuli through the acti- vation of PKR1. To analyze PK2β-induced signal transduction, we demonstrated the inability of PK2β to induce STAT3 protein phosphorylation in organotypic primary explants from mice Dorsal Root Ganglion (DRG), an important pain station. The control of the concentration ratio between PK2β and PK2 could be one of the keys to allow the specificity of the cell response of prokineticin signaling pathway. 1. Introduction Prokineticin-2 (PK2) is a secreted bioactive peptide that is highly conserved across species. The PK2 primary transcript has two alter- native splice variants, PK2 and PK2 Long (PK2L). PK2L presents an insertion of 21 basic amino acids sequence containing a site recognized by Furin, which generates the smaller active form of PK2 protein, called PK2β (Chen et al., 2005; Giannini et al., 2009; LeCouter et al., 2003; Martucci et al., 2006; Negri et al., 2006; Wechselberger et al., 1999). The binding of PK2 to their cognate GPCRs, prokineticin receptor 1 (PKR1) and prokineticin receptor 2 (PKR2), activates Gα q/11 signaling leading to the mobilization of intracellular calcium (Lin et al., 2002a; Masuda et al., 2002; Soga et al., 2002). PK2-induced ERK phosphor- ylation and chemotaxis of human monocytes, mainly expressing PKR2, are inhibited by pertussis toxin (Lecouter et al., 2004; Lin et al., 2002b; Martucci et al., 2006), suggesting the involvement of the Gα i proteins. However, PKRs can also activate other pathways, including the mod- ulation of intracellular cAMP levels via Gα s (Chen et al., 2005). Re- cently, it was demonstrated that PKR2 may also couple Gα 12 in cor- onary endothelial cells (Guilini et al., 2010). Receptor activation has been shown to mediate different biological processes including noci- ception, feeding behavior, the control of physiological and neoplastic angiogenesis, the regulation of circadian rhythms and the modulation of cell functions, growth and survival in the immune and central ner- vous system (Gordon et al., 2016; Negri and Ferrara, 2018; Severini et al., 2015). We already demonstrated that in rodents, the activation of PKR1, widely distributed at peripheral level, and PKR2, highly ex- pressed in the central nervous system by Bv8 (the amphibian homo- logue of PK2), induces intense sensitization to thermal and mechanical stimuli (Negri et al., 2002; Negri and Lattanzi, 2012; Negri and Maftei, https://doi.org/10.1016/j.npep.2018.06.005 Received 1 February 2018; Received in revised form 27 June 2018; Accepted 27 June 2018 ⁎ Corresponding author. E-mail address: rossella.miele@uniroma1.it (R. Miele). Abbreviations: GPCR, G Protein-Coupled Receptor; PK, Prokineticin; PKR, Prokineticin Receptor; PK2β, Prokineticin 2β; ERK, Extracellular signal-Regulated Kinase; DRG, Dorsal Root Ganglion; STAT3, Signal Transducer and Activator of Transcription 3; MAPK, Mitogen-Activated Protein Kinase; GST, Glutathione S-Transferase; WT, Wild Type; KO, Knock Out Neuropeptides xxx (xxxx) xxx–xxx 0143-4179/ © 2018 Elsevier Ltd. All rights reserved. Please cite this article as: Lattanzi, R., Neuropeptides (2018), https://doi.org/10.1016/j.npep.2018.06.005