Functional and binding characterizations of urotensin II-related peptides in human and rat urotensin II-receptor assay.
Urotensin II (U-II; cyclo5-10[H-Glu-Thr-Pro-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val-OH]) is a potent vasoconstrictor in mammals, and it is postulated that it plays a central role in cardiovascular homeostasis. Thus, we initiated a structure-to-function analysis of this peptide characterized by a N-terminal tail and a cyclic core formed through a disulfide bridging. A total of 41 analogs focusing on these characteristics were developed and evaluated using a binding assay on membranes from a stable HEK-293 cell line containing the human or rat U-II receptor, a functional assay for Ca2+ mobilization on transiently transfected CHO-K1 cells with the human or rat U-II receptor, and a rat thoracic aorta bioassay. At first, the focus was applied on peptide compounds containing exocyclic modifications. From this series, it appeared that only valine-11 played a significant role although it is not an essential amino acid. Similarly, endocyclic and ring transformations of hU-II were also studied. In most cases, a detrimental effect on affinity and biological activity was observed. However, two compounds, [Tyr6]hU-II and [Phe9]hU-II, retained affinity and activity. So far, our binding, functional, and pharmacological data clearly demonstrated the minor contribution of the N-terminal segment and the essential role of the cyclic structure. More particularly, three residues within the loop, i.e., Trp-7, Lys-8, and Tyr-9, are required for receptor recognition and activation. This three-pole feature, kept by the disulfide bond in a correct spatial arrangement, appears as the key pharmacophore for the U-II receptor.
Brkovic
Alexandre
A
Institut national de la recherche scientifique, Université du Québec, INRS, Institut Armand-Frappier, Pointe-Claire, Montréal, Quebec, Canada.
Hattenberger
Almut
A
Kostenis
Evi
E
Klabunde
Thomas
T
Flohr
Stefanie
S
Kurz
Michael
M
Bourgault
Steve
S
Fournier
Alain
A
D000818:Animals; D001011:Aorta / Q000187:drug effects / Q000502:physiology; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D006801:Humans; D010446:Peptide Fragments / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled; D013045:Species Specificity; D014579:Urotensins / Q000494:pharmacology*; D014661:Vasoconstriction / Q000187:drug effects*
D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C121697:UTS2R protein, human; D014579:Urotensins; C473142:Uts2r protein, rat; C029677:urotensin II; D002118:Calcium