Urotensin-II (U-II) is a vasoactive 'somatostatin-like' cyclic peptide which was originally isolated from fish spinal cords, and which has recently been cloned from man. Here we describe the identification of an orphan human G-protein-coupled receptor homologous to rat GPR14 and expressed predominantly in cardiovascular tissue, which functions as a U-II receptor. Goby and human U-II bind to recombinant human GPR14 with high affinity, and the binding is functionally coupled to calcium mobilization. Human U-II is found within both vascular and cardiac tissue (including coronary atheroma) and effectively constricts isolated arteries from non-human primates. The potency of vasoconstriction of U-II is an order of magnitude greater than that of endothelin-1, making human U-II the most potent mammalian vasoconstrictor identified so far. In vivo, human U-II markedly increases total peripheral resistance in anaesthetized non-human primates, a response associated with profound cardiac contractile dysfunction. Furthermore, as U-II immunoreactivity is also found within central nervous system and endocrine tissues, it may have additional activities.

dehydrogenase by phosphorylation involves no long-range conformational change in the free enzyme.

Formation of the covalently stabilized complex of a1-antitrypsin (a,-AT) with neutrophil elastase, the archetype of serine proteinase inhibitor serpin-enzyme complexes, is associated with structural rearrangement of the a,-AT molecule and hydrolysis of a reactive-site peptide bond.An =4-kDa carboxyl-terminal cleavage fragment is generated. a1-AT-elastase complexes are biologically active, possessing chemotactic activity and mediating increases in expression of the a,-AT gene in human monocytes and macrophages. This suggested that structural rearrangement of the a,-AT molecule, during formation of a complex with elastase, exposes a domain that is recognized by a specific cell surface receptor or receptors. To test this hypothesis, the known three-dimensional structure of al-AT and comparisons of the primary structures of the serpins were used to select a potentially exteriorly exposed and highly conserved region in the complexed form of Several recent studies suggest that a1-AT-elastase complexes have intrinsic functional activities. These complexes stimulate neutrophil chemotaxis (5) and mediate increases in expression of the a1-AT gene in human monocytes and macrophages (6, 7). a1-AT-elastase complexes are more rapidly cleared from the circulation than the corresponding native proteins. In fact, in vivo clearance of a1-AT-elastase complexes is blocked by other serpin-enzyme complexes (8, 9), suggesting the presence of a common recognition system. Taken together, these observations suggest that structural rearrangement of the a1-AT molecule, during formation of a complex with elastase, exposes a domain that is recognized by a specific cell surface receptor, or receptors. In the following study we used synthetic peptides based on the sequence of the carboxyl-terminal fragment of a1-AT as candidate mediators for regulation of a1-AT synthesis and as candidate ligands for cell surface binding. This region of the a1-AT molecule was selected because it had been previously implicated in the chemotactic activity of a1-AT-elastase complexes (5) and because crystal structure analysis (10) predicted that a domain within this region was exteriorly exposed after formation of a complex. EXPERIMENTAL PROCEDURESMaterials. Peptides were synthesized by the solid-phase method (11, 12), purified, and subjected to amino acid composition and sequence analysis. Peptides were dissolved in water and then added to cell culture fluid. Preparation of purified human plasma a1-AT, leukocyte elastase, and leukocyte cathepsin G has been previously described (6). Purified human plasma AT III and a,-ACT were purchased from Athens Research and Technology (Athens, GA). Purified human thrombin was purchased from Boehringer-MannAbbreviations: a1-AT, a1-antitrypsin; AT III, antithrombin III; a,-ACT, a1-antichymotrypsin; serpin, serine proteinase inhibitor; SEC, serpin-enzyme complex.tTo whom reprint requests should be addressed at:

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