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652206 
Journal Article 
Structural determinants required for the bioactivities of prokineticins and identification of prokineticin receptor antagonists 
Bullock, CM; Li, JD; Zhou, QY 
2004 
1 
Molecular Pharmacology
ISSN: 0026-895X
EISSN: 1521-0111 
65 
3 
582-588 
English 
Prokineticins are cysteine-rich secreted proteins that regulate diverse biological processes, including gastrointestinal motility, angiogenesis, and circadian rhythms. Two closely related G protein-coupled receptors that mediate signal transduction of prokineticins have recently been cloned. The structural elements required for prokineticins' bioactivities are still unknown. We show here that both the N-terminal hexapeptide (AVITGA) and C-terminal cysteine-rich domains are critical for the bioactivities of prokineticins. Substitutions, deletions, and insertions to the conserved N-terminal hexapeptides result in the loss of agonist activity. Mutant prokineticins with the substitution of the first N-terminal alanine with methionine or the addition of a methionine to the N terminus inhibit the activation of prokineticin receptors and thus are considered as antagonists of prokineticin receptors. We have further shown that mutations in selected cysteine residues in the C-terminal domain result in prokineticins without biological activity. The essential role of C-terminal domain is reinforced by two observations: that peptides without the carboxyl domain and proteins with the N-terminal hexapeptide fused to the carboxyl domains of colipase or dickkopf are devoid of biological activity. We demonstrate that limited structural changes of C-terminal cysteine-rich regions of prokineticins are tolerable because chimeric prokineticins with swapped cysteine-rich domains between prokineticin 1 and prokineticin 2, as well as a splice variant of prokineticin 2 that contains extra 21 residue insertion in its C-terminal domain, are biologically active. 
*Alternative Splicing; Animals; CHO Cells; Carrier Proteins/chemistry/metabolism; Cricetinae; Cysteine/chemistry; Gastrointestinal Hormones/chemistry/genetics/*metabolism; Mutagenesis, Site-Directed; Neuropeptides/chemistry/drug effects/genetics/*metabolism; Peptide Fragments/chemistry/genetics/metabolism/pharmacology; Protein Structure, Tertiary; Recombinant Fusion Proteins/chemistry/*metabolism