Identification of two prokineticin cDNAs: Recombinant proteins potently contract gastrointestinal smooth muscle

The motility of gastrointestinal tract is regulated by classical neurotrans mitters, neuropeptides, and humoral agents. Two novel human cDNAs have been cloned based on their sequence similarity to a frog skin secretion protein , Bv8, and a nontoxic protein of mamba snake venom. These human cDNAs encod e two secreted proteins of 86 and 81 amino acids. Northern blot hybridizati on has revealed that these cDNAs are expressed in gastrointestinal tract, p articularly the stomach. Recombinant proteins with authentic N-terminal seq uences have been produced in Escherichia coli and refolded into functional proteins by careful control of protein aggregation. Mass spectrometry has c onfirmed the formation of five pairs of disulfide bonds. The refolded recom binant proteins potently contract gastrointestinal smooth muscle with EC50 values in the subnanomolar range. The contractile effects of the recombinan t proteins are specific for gastrointestinal smooth muscle, because they ha ve no effect on vascular or respiratory smooth muscle. To reflect their pot ent and specific effects on gastrointestinal smooth muscle cells, we have n amed these recombinant proteins prokineticins. Ligand binding studies with iodinated prokineticin revealed the presence of a high-affinity site in ile al smooth muscle. The displacement of specific binding by GTP gammaS sugges ts that the prokineticin receptor may belong to the family of G protein-cou pled receptors. Experiments with verapamil and nifedipine revealed that cal cium influx is essential for the contractile activity of prokineticins on g astrointestinal smooth muscle. In summary, we have identified two novel end ogenous regulators of gastrointestinal motility. The availability of recomb inant prokineticins should provide novel therapeutic agents for disorders i nvolving impaired gastrointestinal motility.