Analysis of receptor and processing enzyme of pain regulated peptide, nocistatin using bioluminescence.

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We identified a neuropeptide and named it "nocistatin (NST)". NST is derived from the same precursor protein as nociceptin/orphanin FQ (N/OFQ), while NST exhibits antagonism against Noc/OFQ-actions. Both NST and N/OFQ are co-localized in the superficial laminae of mouse spinal dorsal horn, but these peptides induce several functions via the respective receptors. We carried out a photaffiniiy labeling approach for identification of the NST receptor, and we attempt an intra-molecular BRET system for monitoring dynamic biological process of the production of NST and N/OFQ in the living cells.1. NST receptor: Mouse spinal cord membranes were incubated with a ^<125>I-labeled photoaffinity NST analogue, and this analogue bound approximately 33-kDa protein. The 33-kDa protein labeling was decreased by NST but not by N/OFQ. The photoaffinity-labeled protein disappeared after GTPγS treatment, suggesting that the NST-specific photolabeled protein may be associated with a GTP-binding protein.2. P … More eptide-production: We constructed a fusion protein (Rluc-GFP) covalently linking luciferase (Renilla luciferase; Rluc) to Aequorea GFP as an intra-molecular BRET partner, and we inserted constructs of NST and Noc/OFQ containing a proteolytic cleavage motif (Lys-Arg) within Rluc-GFP. The bioluminescence spectrum of the fusion protein with DeepBlueC as a substrate was bimodal (λmax= 400 nm (Rluc) and 510 nm (GFP)), indicating that the excited-state energy of Rluc transfers to GFP (in short, BRET). Furthermore, we developed a novel BRET system using a secreated luciferase, which enabled us to follow a real-time and continuous BRET in culture medium. The BRET had the desired profile for monitoring the protein processing of NST and N/OFQ in intact cells, and the change in BRET signals quantified the processing of the protein. Co-transfection of the BRET probe with proprotein convertases showed that PC1 was involved in the processing of Lys-Arg between NST and N/OFQ and furin in that of Lys-Arg upstream of NST. Less