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.

Uridine 5-diphosphoglucose (UDP-glucose) has a well established biochemical role as a glycosyl donor in the enzymatic biosynthesis of carbohydrates. It is less well known that UDP-glucose may possess pharmacological activity, suggesting that a receptor for this molecule may exist. Here, we show that UDP-glucose, and some closely related molecules, potently activate the orphan G protein-coupled receptor KIAA0001 heterologously expressed in yeast or mammalian cells. Nucleotides known to activate P2Y receptors were inactive, indicating the distinctly novel pharmacology of this receptor. The receptor is expressed in a wide variety of human tissues, including many regions of the brain. These data suggest that some sugar-nucleotides may serve important physiological roles as extracellular signaling molecules in addition to their familiar role in intermediary metabolism.

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

No abstract

Amyloid 39 -42 ␤ -peptides are the main components of amyloid plaques found in the brain of Alzheimer's disease patients. Amyloid 39 -42 ␤-peptide is formed from amyloid precursor protein by the sequential action of ␤-and ␥-secretases. Asp-2 is a transmembrane aspartic protease expressed in the brain, shown to have ␤-secretase activity. Mature Asp-2 has four N-glycosylation sites. In this report we have characterized the carbohydrate structures in this glycoprotein expressed in three different cell lines, namely Chinese hamster ovary, CV-1 origin of SV40, and baculovirus-infected SF9 cells. Biantennary and triantennary oligosaccharides of the "complex" type were released from glycoprotein expressed in the mammalian cells, whereas mannose-rich glycans were identified from glycoprotein synthesized in the baculovirus-infected cells. Site-directed mutagenesis of the asparagine residues at amino acid positions 153, 172, 223, and 354 demonstrate that the protease activity of Asp-2 is dependent on its glycosylation.One of the key pathological features of Alzheimer's disease is the formation of brain plaques primarily due to the fibrilization of amyloid 39 -42 ␤-peptides (A␤) 1 (1, 2). The formation of these peptides by the action of proteases on amyloid precursor protein (APP) has been the subject of several studies (3, 4), as the identification of these proteases could lead to the discovery of inhibitors useful in the treatment of Alzheimer's disease. Over the last year there have been a number of reports detailing the discovery, purification, and characterization of a transmembrane aspartic proteinase (Asp-2). This glycoprotein has been shown to cleave the APP at the ␤-secretase site (5-9). High levels of this proteinase were identified in the human brain, and the enzyme activity was found in cells associated with the central nervous system (7) and in cell lines known to produce A␤ via cleavage of APP (5).Asp-2 contains four potential N-linked glycosylation sites at the following asparagine residues: Asn 153 , Asn 172 , Asn 223 , and Asn 354 . The close proximity of some of these glycosylated sites to the three intramolecular disulfide linkages and the catalytic site of Asp-2 has been the subject of more recent interest (10), especially because the type and extent glycosylation of a protein can have a profound effect on its physico-chemical properties (11). For instance, it is well known that N-linked oligosaccharides can influence glycoprotein folding in the endoplasmic reticulum (12) and can protect a protein from protease attack (13).The N-linked oligosaccharide structures on Asp-2 have not yet been characterized in detail, although it has been reported that all four asparagine sites have a heterogeneous mixture of carbohydrate attached (10). Another report has indicated that the glycans in mature Asp-2 are of the complex type, as removal of this carbohydrate is not possible by endoglycosidase H treatment (14).In this study we have released and analyzed the N-linked glycans from Asp-2 expressed as an Fc fusion in ...