Abstract
Prokineticin 2 (PROK2) is a secreted bioactive peptide that regulates a variety of biological responses via two GPCRs, the prokineticin receptors (PROKRs). The aim of this study was to characterize a new alternatively spliced product of the prok2 gene consisting of four exons. The 40-amino acid peptide, designated PROK2C, is encoded by exon 1 and exon 4, and its expression was detected in the hippocampus and spinal cord of mice. PROK2C was expressed in a heterologous system, Pichia pastoris, and its binding specificity to the amino-terminal regions of PROKR1 and PROKR2 was investigated by GST pull-down experiments. In addition, the introduction of the unnatural amino acid p-benzoyl-L-phenylalanine using amber codon suppression technology demonstrated the role of tryptophan at position 212 of PROKR2 for PROK2C binding by photoactivatable cross-linking. The functional significance of this new isoform was determined in vivo by nociceptive experiments, which showed that PROK2C elicits strong sensitization of peripheral nociceptors to painful stimuli. In order to analyze the induction of PROK2C signal transduction, STAT3 and ERK phosphorylation levels were determined in mammalian CHO cells expressing PROKR1 and PROKR2. Our data show by in vivo and in vitro experiments that PROK2C can bind and activate both prokineticin receptors.
Highlights
Alternative splicing (AS) is an important mechanism in gene modulation
PROK2C was expressed in a heterologous system, Pichia pastoris, and its binding specificity to the amino-terminal regions of PROKR1 and PROKR2 was investigated by glutathione S-transferase (GST) pull-down experiments
In order to analyze the induction of PROK2C signal transduction, STAT3 and ERK phosphorylation levels were determined in mammalian CHO cells expressing PROKR1 and PROKR2
Summary
Alternative splicing (AS) is an important mechanism in gene modulation. It allows a single gene to produce several different mRNAs with different exon compositions and lengths that can code for multiple forms of the corresponding proteins.Most eukaryotic protein-coding genes involved in developmental processes and regulation of cell proliferation have multiple transcriptional isoforms [1,2]. Alternative splicing (AS) is an important mechanism in gene modulation. It allows a single gene to produce several different mRNAs with different exon compositions and lengths that can code for multiple forms of the corresponding proteins. Most eukaryotic protein-coding genes involved in developmental processes and regulation of cell proliferation have multiple transcriptional isoforms [1,2]. AS can affect mRNA localization, stability, and translation or change the reading frame, resulting in different protein isoforms with diverse functions, localizations, or both [3,4]. Any alteration in the splicing mechanism can affect the maturation of mRNA and the formation of functional proteins, causing various pathological states such as neurodegenerative diseases and inflammatory, immune, and metabolic disorders [5,6]
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