In vitro cell culture model systems for investigating the biochemical mechanisms involved in the neurodegenerative actions of β‐amyloid peptide (β‐AP) have been established. Using rat pheochromocytoma PC12 or human epitheloid HeLa cell lines, submicromolar concentrations of the β‐AP fragments β1–40, β1–39, and β25–35, but not β1–28, were found to inhibit the reduction of the redox dye 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT). In both cell lines, the β‐AP‐sensitive component represented ∼70% of total cellular MTT reduction. When the reduction of a series of structurally related dyes was compared with that of MTT, the reduction of 3α‐naphthyl‐2‐phenyl‐5‐(4‐nitrophenyl)‐2H‐tetrazolium chloride (NTV) was also found to be sensitive to β25–35, but that of seven other redox dyes was not. A property common to MTT and NTV is that they are both readily taken up into PC12 and HeLa cells and do not require an artificial electron coupling agent to be reduced. Microscopic analysis of MTT‐formazan product formation in PC12 and HeLa cells following β25–35 treatment revealed that it was the intracellular component of the reduction of this dye that was abolished. These results support the hypothesis that the cellular reduction of MTT represents a specific indicator of the initial events underlying the mechanism of β‐AP toxicity.

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Many hormones that are central to normal reproductive functioning mediate their physiological effects by activating a receptor which belongs to the large family of G-protein-coupled receptors (GPCR). Members of this family of receptor proteins are usually glycosylated on extracellular domains. In recent years the role of this glycosylation in cell surface expression/protein folding, ligand recognition and receptor-effector coupling has been investigated. This review summarises current knowledge of the role of glycosylation in the functioning of the receptors for gonadotrophin-releasing hormone (GnRH), luteinizing hormone/human chorionic gonadotrophin (LH/HCG), follicle stimulating hormone (FSH), oxytocin (OT) and vasopressin (AVP).

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For G-protein-coupled receptors (GPCRs) in general, the roles of extracellular residues are not well defined compared with residues in transmembrane helices (TMs). Nevertheless, extracellular residues are important for various functions in both peptide-GPCRs and amine-GPCRs. In this study, the V 1a vasopressin receptor was used to systematically investigate the role of extracellular charged residues that are highly conserved throughout a subfamily of peptide-GPCRs, using a combination of mutagenesis and molecular modeling. Of the 13 conserved charged residues identified in the extracellular loops ( G-protein-coupled receptors (GPCRs)4 exhibit a common tertiary structure comprising seven transmembrane helices (TMs) linked by extracellular loops (ECLs) and intracellular loops. The atomic detail of this general GPCR fold has been elucidated for bovine rhodopsin (bRho) using x-ray crystallography (1). This confirmed that the chromophore 11-cis-retinal is covalently linked to Lys 296(7.43) in transmembrane helix VII (TMVII) via a protonated Schiff-base and projects into a binding pocket formed within the TM bundle where it interacts with amino acid side chains and water molecules (1, 2).5 Likewise, the binding pocket for small biogenic amine neurotransmitters such as acetylcholine and norepinephrine is buried deep within the TM bundle (3). Nevertheless, it is known from the bRho x-ray structure that the extracellular domains possess defined structure and are orientated to interact with each other and with the TM helices. Indeed ECL2 of bRho forms a ␤-hairpin that plunges down into the helical bundle to form a plug over the chromophore. Furthermore, the orientation of ECL2 in the majority of GPCRs is restrained by a conserved disulfide bond between ECL2 and the top of TMIII (1, 2).The neurohypophysial peptide hormones vasopressin (AVP) and oxytocin (OT) generate a wide range of physiological effects, including vasopressor and antidiuretic and uterotonic actions (4, 5). The effects of AVP/OT are mediated by a family of receptors (V 1a R, V 1b R, V 2 R, and OTR), which together with the vasotocin receptor (VTR), mesotocin receptor, and isotocin receptor from lower vertebrates constitute a subfamily of the rhodopsin/␤-adrenergic receptor class of GPCRs (family A). The V 1a R, V 1b R, and OTR couple to phospholipase C thereby generating inositol 1,4,5-trisphosphate and diacylglycerol as second messengers, whereas the V 2 R stimulates adenylyl cyclase. The V 1a R is widely distributed and mediates nearly all of the actions of AVP with the exceptions of antidiuresis (V 2 R) * This work was supported by grants (to M. W.) from the Wellcome Trust, the Biotechnology and Biological Sciences Research Council, and Ferring Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.

The effects of the peptide hormone oxytocin (OT) are mediated by the oxytocin receptor, which is a member of the G-protein-coupled receptor family. Defining differences between the binding of agonists and antagonists to the OTR, at the molecular level, is of fundamental importance to understanding OTR activation and to rational drug design. Previous reports have indicated that the N-terminus of the OTR is required for OT binding. The aim of this study was to identify which individual residues within the N-terminal domain of the human OTR provided these OT binding epitopes. A series of truncated OTRs and mutant receptor constructs with systematic alanine substitution were characterized with respect to their pharmacological profile and intracellular signaling capability. Although a number of residues within the OTR will be required for optimal OT-OTR interaction, our data establish that Arg(34) within the N-terminal domain contributes to high-affinity OT binding. Removal of Arg(34) by truncation or substitution resulted in a 2000-fold decrease in OT affinity. In addition, we show that the arginyl at this locus is required for high-affinity binding of agonists in general. However, the importance of Arg(34) is restricted to agonist interaction with the OTR, as it was not required for binding peptide antagonist or non-peptide antagonist. It is noteworthy that the corresponding Arg in the related rat V(1a) vasopressin receptor is also required for high-affinity agonist binding. This study defines, at the molecular level, the role of the N-terminus of the OTR in high-affinity agonist binding and identifies a key residue for this function.

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A fundamental issue in molecular pharmacology is to define how agonist:receptor interaction differs from that of antagonist:receptor. The V(1a) receptor (V(1a)R) is a member of a family of related G-protein-coupled receptors that are activated by the neurohypophysial peptide hormone arginine-vasopressin (AVP). Here we define a short subdomain of the N-terminus of the V(1a)R from Glu(37) to Asn(47) that is an absolute requirement for binding AVP and other agonists. In marked contrast to the situation for agonists, deleting this segment has little or no effect on the binding of either peptide or non-peptide antagonists. In addition, we established that this subdomain was crucial for receptor activation and second messenger generation. The oxytocin receptor (OTR) also binds AVP with high affinity but exhibits a different pharmacological profile to the V(1a)R. Substitution of the N-terminus of the V(1a)R with the corresponding sequence from the OTR generated a chimeric receptor (OTR(N)-V(1a)R). The presence of the OTR N-terminus recovered high affinity agonist binding such that the OTR(N)-V(1a)R possessed almost wild-type V(1a)R pharmacology and signaling. Consequently, a domain within the N-terminus is required for agonist binding but it does not provide the molecular discriminator for subtype-selective agonist recognition. Cotransfection and peptide mimetic studies demonstrated that this N-terminal subdomain had to be contiguous with the receptor polypeptide to be functional. This study establishes that a segment of the V(1a)R N-terminus has a pivotal role in the mechanism of agonist binding and provides molecular insight into key differences between the interaction of agonists and antagonists with a peptide receptor family.