Identification of Two Prokineticin cDNAs: Recombinant Proteins Potently Contract Gastrointestinal Smooth Muscle

Abstract

The motility of gastrointestinal tract is regulated by classical neurotransmitters, 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 encode two secreted proteins of 86 and 81 amino acids. Northern blot hybridization has revealed that these cDNAs are expressed in gastrointestinal tract, particularly the stomach. Recombinant proteins with authentic N-terminal sequences have been produced in Escherichia coli and refolded into functional proteins by careful control of protein aggregation. Mass spectrometry has confirmed the formation of five pairs of disulfide bonds. The refolded recombinant proteins potently contract gastrointestinal smooth muscle with EC(50) values in the subnanomolar range. The contractile effects of the recombinant proteins are specific for gastrointestinal smooth muscle, because they have no effect on vascular or respiratory smooth muscle. To reflect their potent and specific effects on gastrointestinal smooth muscle cells, we have named these recombinant proteins prokineticins. Ligand binding studies with iodinated prokineticin revealed the presence of a high-affinity site in ileal smooth muscle. The displacement of specific binding by GTP gamma S suggests that the prokineticin receptor may belong to the family of G protein-coupled receptors. Experiments with verapamil and nifedipine revealed that calcium influx is essential for the contractile activity of prokineticins on gastrointestinal smooth muscle. In summary, we have identified two novel endogenous regulators of gastrointestinal motility. The availability of recombinant prokineticins should provide novel therapeutic agents for disorders involving impaired gastrointestinal motility.

The endocrine-gland-derived VEGF homologue Bv8 promotes angiogenesis in the testis: Localization of Bv8 receptors to endothelial cells

“…Although our principal focus has been the role of Bv8 in the testis, the major site of expression, we and others (16)(17)(18)(19)(20), have noted expression of both Bv8 and its two receptors in the brain. We did not detect Bv8 expression in the human brain by either Northern blot analysis or ISH studies.…”

“…EG-VEGF (80% similar to VPRA) and VPRA are most closely related (83% and 79% similarity, respectively) to the Bv8 peptide. Mouse and human orthologues of Bv8, also known as prokineticin-2 (16), have been recently described, and a variety of activities for these proteins have been reported, including effects on neuronal survival (17), gastrointestinal smooth muscle contraction (16), and circadian locomotor rhythm (18).…”

Abnormal development of the olfactory bulb and reproductive system in mice lacking prokineticin receptor PKR2

“…and PK2 (also called Bv8), are secreted bioactive proteins possessing 10 conserved cysteines that form five disulfide bonds (1). The mature forms of PK1 and PK2 consist of 86 and 81 amino acids, respectively, and share Ϸ40% amino acid identity.…”

Characterization of Endocrine Gland-derived Vascular Endothelial Growth Factor Signaling in Adrenal Cortex Capillary Endothelial Cells

“…include the secreted protein from the frog Bombina variegata, Bv8 (21) and its mammalian orthologues (22,23), the digestive enzyme colipase (24), and the Xenopus head-organizer protein dickkopf (25). EG-VEGF/VPRA shows a high degree of homology (70 -76%) to the Bv8 protein and its murine and human orthologues.…”