Cloning and characterization of a vasopressin V2 receptor and possible link to nephrogenic diabetes insipidus.

The antidiuretic effect of arginine vasopressin (AVP) is mediated by renal-type (V2) receptors linked to adenylyl cyclase. We report here the cloning of the rat kidney V2 AVP receptor complementary DNA that encodes a 370-amino-acid protein with a transmembrane topography characteristic of G protein-coupled receptors, and with similarity to the V1a (hepatic) AVP receptor in its seven membrane-spanning domains. Expression of the cloned cDNA in mammalian cells showed specific ligand binding and activity characteristic of the native V2 AVP receptor. The receptor messenger RNA is detected only in the kidney. The human V2 receptor gene has been localized to the long arm of the X chromosome close to the locus for nephrogenic diabetes insipidus, an X-linked recessive disorder characterized by renal resistance to the antidiuretic action of AVP.     

The cytoplasmic carboxy terminus of M13 procoat is required for the membrane insertion of its central domain

The M13 coat protein spans the Escherichia coli plasma membrane with its amino-terminus facing the periplasm. It is made as a precursor--the procoat--with a typical leader peptide. Mutations which destroy the basic character of the carboxy-terminal domain of procoat, a domain which is oriented towards the cytoplasm, block membrane assembly, while insertion of three lysyl residues near the carboxy terminus partially restores assembly. Thus the information specifying membrane insertion of M13 procoat protein is found in its mature region as well as the leader and is not simply decoded in an amino to carboxy direction.     

Central target for the behavioural effects of vasopressin neuropeptides

The neurohypophysial hormone vasopressin exerts antidiuretic, vasopressor and behavioural effects (for example, facilitation of memory processes). Vasopressin may alter animal behaviour via direct effect on brain processes. Recently, however, it has been suggested that vasopressin acts mainly at peripheral receptor systems and influences behavioural mechanisms by altering visceral afferent signals. We now present data showing that (1) central administration of [Arg8]vasopressin (AVP) and more potently [pGlu, Cyt]AVP(4-8), the desglycinamide derivative of a peptide generated from AVP by brain synaptic membranes, produce the behavioural effect (promotion of passive avoidance behaviour) without the pressor effect; (2) central administration of a vasopressor antagonist blocks the behavioural but not the pressor effect of systemically administered AVP; and (3) [pGlu, Cyt]AVP(4-8) induces the behavioural effect in the absence of the pressor effect. The results indicate that AVP and related peptides affect passive avoidance behaviour by a direct central action and that the structural requirement for activation of central vasopressin receptors differs from that of the peripheral cardiovascular receptors, although both can be blocked by the same vasopressor antagonist.     

A modular switch for spatial Ca2+ selectivity in the calmodulin regulation of CaV channels

Ca2+/calmodulin-dependent regulation of voltage-gated CaV1-2 Ca2+ channels shows extraordinary modes of spatial Ca2+ decoding and channel modulation, vital for many biological functions. A single calmodulin (CaM) molecule associates constitutively with the channel's carboxy-terminal tail, and Ca2+ binding to the C-terminal and N-terminal lobes of CaM can each induce distinct channel regulations. As expected from close channel proximity, the C-lobe responds to the roughly 100-microM Ca2+ pulses driven by the associated channel, a behaviour defined as 'local Ca2+ selectivity'. Conversely, all previous observations have indicated that the N-lobe somehow senses the far weaker signals from distant Ca2+ sources. This 'global Ca2+ selectivity' satisfies a general signalling requirement, enabling a resident molecule to remotely sense cellular Ca2+ activity, which would otherwise be overshadowed by Ca2+ entry through the host channel. Here we show that the spatial Ca2+ selectivity of N-lobe CaM regulation is not invariably global but can be switched by a novel Ca2+/CaM-binding site within the amino terminus of channels (NSCaTE, for N-terminal spatial Ca2+ transforming element). Native CaV2.2 channels lack this element and show N-lobe regulation with a global selectivity. On the introduction of NSCaTE into these channels, spatial Ca2+ selectivity transforms from a global to local profile. Given this effect, we examined CaV1.2/CaV1.3 channels, which naturally contain NSCaTE, and found that their N-lobe selectivity is indeed local. Disruption of this element produces a global selectivity, confirming the native function of NSCaTE. Thus, differences in spatial selectivity between advanced CaV1 and CaV2 channel isoforms are explained by the presence or absence of NSCaTE. Beyond functional effects, the position of NSCaTE on the channel's amino terminus indicates that CaM can bridge the amino terminus and carboxy terminus of channels. Finally, the modularity of NSCaTE offers practical means for understanding the basis of global Ca2+ selectivity.     

No requirements of cyclic conformation of antagonists in binding to vasopressin receptors

Early reports that acyclic analogues of oxytocin and vasopressin (AVP) have drastically reduced agonistic activities established as dogma that an intact hexapeptide ring structure is essential for the pharmacological activities of analogues of neurohypophysial hormones. Thus, virtually all the many hundreds of agonistic and antagonistic analogues of the neurohypophysial peptides that have been reported contain an intact ring. Here we report that an intact ring is not essential for binding of antagonistic AVP analogues to vasopressor (V1) or antidiuretic (V2) AVP receptors. In fact, one acyclic AVP analogue seems to be about as potent as any previously reported cyclic V2 antagonist. This finding suggests new possibilities for the design of AVP analogues as pharmacological probes and for therapeutic use. Similar modifications might be useful in the design of analogues of other cyclic peptides, such as calcitonin, somatostatin and the atrial natriuretic factors.     

抗利尿素和催产素对家兔尿分泌的影响及无尿期形成原因初探

垂体后叶素的抗利尿作用顺序表现为无尿期、多尿期和少尿期。前文已报导,无尿期是在注射垂体后叶素后、输尿管下部二分之一处生理状态改变所致,而少尿期才是垂体后叶素的抗利尿作用。由于垂体后叶素的主要组成成份为抗利尿素和催产素,本实验进一步研究两者分别对家兔泌尿的影响,并进一步探讨尿期的形成原因。     

Nucleotide sequence of cloned cDNA encoding bovine arginine vasopressin-neurophysin II precursor

The sequence of a cDNA encoding the nonapeptide arginine vasopressin (AVP) and its carrier protein, neurophysin II (NpII) from bovine hypothalamus, proves that the 166-amino acid precursor molecule contains a signal peptide of 19 amino acids followed directly by AVP connected to NpII by a Gly-Lys-Arg sequence. The carboxy-terminal region of the precursor contains a naturally occurring glycopolypeptide of 39 amino acids which is separated from NpII by a single arginine residue.     

Enterotoxin residues determining T-cell receptor V beta binding specificity.

Superantigens such as the staphylococcal enterotoxins bind to major histocompatibility complex (MHC) class II molecules and activate T cells through a specific interaction between the V beta region of the T-cell antigen receptor (TCR) and the toxin. The TCR beta-chain alone is sufficient to produce the interaction with the enterotoxin-class II complex. Identification of the regions of enterotoxins that interact with TCR has so far proved equivocal because of difficulties in distinguishing between direct effects on T-cell recognition and indirect effects resulting from alteration of binding to class II. For example, amino-terminal truncations of SEB abrogated T-cell stimulation whereas carboxy-terminal truncation of SEA stopped its mitogenic activity. The most comprehensive study to date, accounting for both enterotoxin binding to class II and enterotoxin interactions with the TCR, identified two functionally important regions for SEB binding to TCR. Although the amino-acid sequences of staphylococcal enterotoxins A and E are 82% identical, they activate T cells bearing different V beta elements. We have assayed the binding of cells coated with these enterotoxins to soluble secreted TCR beta-chain protein and find that V beta 3 binds enterotoxin A but not E, whereas V beta 11 binds enterotoxin but not A. To map the amino-acid residues responsible for these different binding specificities, we prepared a series of hybrids between the two staphylococcal enterotoxins. We report that just two amino-acid residues near the carboxy terminus of the enterotoxins are responsible for the discrimination between these molecules by V beta 3 and V beta 11.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural identification of beta- and gamma-human atrial natriuretic polypeptides

Atrial natriuretic polypeptides (ANPs) of varying chain length have been identified recently in human and rat atrial tissue. Their potent natriuretic-diuretic activities indicate their key role in the regulation of extracellular fluid volume and electrolyte balance. Furthermore, human and rat cDNAs encoding their precursor have been cloned and identified. Natriuretic-diuretic activity in human atrial extract comprises three distinct components (alpha, relative molecular mass (Mr) approximately 3,000; beta, Mr approximately 6,000; gamma, Mr approximately 13,000). However, only the 3,000-Mr peptide, alpha-human atrial polypeptide (alpha-hANP), comprising 28 amino acids, has so far been identified. We report here the purification and sequence analysis of two novel hANPs of higher Mr, beta- and gamma-hANP, both of which exhibit natriuretic and hypotensive activity. gamma-hANP, composed of 126 amino acids, carries the alpha-hANP sequence at its carboxy terminus. The identification of gamma-hANP reveals that the peptide, being the largest form of hANP, is processed directly from a 151-residue precursor by removal of a 26-residue signal peptide. In contrast, beta-hANP (56 residues) comprises an anti-parallel dimer of alpha-hANP; such a dimeric peptide possessing bioactivity has never been found in the tissue as an endogenous entity.     

Structure of the dengue virus envelope protein after membrane fusion

Dengue virus enters a host cell when the viral envelope glycoprotein, E, binds to a receptor and responds by conformational rearrangement to the reduced pH of an endosome. The conformational change induces fusion of viral and host-cell membranes. A three-dimensional structure of the soluble E ectodomain (sE) in its trimeric, postfusion state reveals striking differences from the dimeric, prefusion form. The elongated trimer bears three 'fusion loops' at one end, to insert into the host-cell membrane. Their structure allows us to model directly how these fusion loops interact with a lipid bilayer. The protein folds back on itself, directing its carboxy terminus towards the fusion loops. We propose a fusion mechanism driven by essentially irreversible conformational changes in E and facilitated by fusion-loop insertion into the outer bilayer leaflet. Specific features of the folded-back structure suggest strategies for inhibiting flavivirus entry.     

The structural basis of agonist-induced activation in constitutively active rhodopsin

G-protein-coupled receptors (GPCRs) comprise the largest family of membrane proteins in the human genome and mediate cellular responses to an extensive array of hormones, neurotransmitters and sensory stimuli. Although some crystal structures have been determined for GPCRs, most are for modified forms, showing little basal activity, and are bound to inverse agonists or antagonists. Consequently, these structures correspond to receptors in their inactive states. The visual pigment rhodopsin is the only GPCR for which structures exist that are thought to be in the active state. However, these structures are for the apoprotein, or opsin, form that does not contain the agonist all-trans retinal. Here we present a crystal structure at a resolution of 3 ? for the constitutively active rhodopsin mutant Glu 113 Gln in complex with a peptide derived from the carboxy terminus of the α-subunit of the G protein transducin. The protein is in an active conformation that retains retinal in the binding pocket after photoactivation. Comparison with the structure of ground-state rhodopsin suggests how translocation of the retinal β-ionone ring leads to a rotation of transmembrane helix 6, which is the critical conformational change on activation. A key feature of this conformational change is a reorganization of water-mediated hydrogen-bond networks between the retinal-binding pocket and three of the most conserved GPCR sequence motifs. We thus show how an agonist ligand can activate its GPCR.     

Site-directed mutation affecting polyomavirus capsid self-assembly in vitro

Nonequivalent bonding of identical protein subunits occurs in the polyomavirus capsid were identical pentameric capsomeres occupy both hexavalent and pentavalent positions in the icosahedral surface lattice. The polyomavirus major capsid protein VP1, purified after expression of the recombinant gene in Escherichia coli, has been isolated as capsomeres that self-assemble into capsid-like structures in vitro. The ability to switch bonding specificity in different symmetry environments therefore must be intrinsic to the VP1 molecule. In vitro self-assembly provides an assay for VP1 mutations affecting capsomere and capsid formation. We report here that a directed mutation in the VP1 expression vector, leading to a protein truncated at the carboxy terminus, results in a mutant VP1 that forms capsomeres, but not capsids, in the in vitro assembly assay. The carboxy terminus of VP1 therefore appears to be involved in the specific bonding responsible for the non-equivalent association of capsomeres.     

Transvascular delivery of small interfering RNA to the central nervous system

A major impediment in the treatment of neurological diseases is the presence of the blood-brain barrier, which precludes the entry of therapeutic molecules from blood to brain. Here we show that a short peptide derived from rabies virus glycoprotein (RVG) enables the transvascular delivery of small interfering RNA (siRNA) to the brain. This 29-amino-acid peptide specifically binds to the acetylcholine receptor expressed by neuronal cells. To enable siRNA binding, a chimaeric peptide was synthesized by adding nonamer arginine residues at the carboxy terminus of RVG. This RVG-9R peptide was able to bind and transduce siRNA to neuronal cells in vitro, resulting in efficient gene silencing. After intravenous injection into mice, RVG-9R delivered siRNA to the neuronal cells, resulting in specific gene silencing within the brain. Furthermore, intravenous treatment with RVG-9R-bound antiviral siRNA afforded robust protection against fatal viral encephalitis in mice. Repeated administration of RVG-9R-bound siRNA did not induce inflammatory cytokines or anti-peptide antibodies. Thus, RVG-9R provides a safe and noninvasive approach for the delivery of siRNA and potentially other therapeutic molecules across the blood-brain barrier.     

A discrete sequence in a platelet integrin is involved in ligand recognition

Platelet membrane glycoprotein IIb-IIIa (gpIIb-IIIa; alpha IIb-beta 3), the most prominent member of the integrin family of adhesion receptors on these cells, mediates platelet aggregation by binding fibrinogen and is critical in thrombosis and haemostasis. A short amino-acid sequence at the carboxy terminus of the gamma chain of fibrinogen is recognized by gpIIb-IIIa and peptides containing this sequence are selectively crosslinked to residues 294-314 of gpIIb. Here we show that an 11-residue peptide from this region of gpIIb inhibits platelet aggregation and binding of fibrinogen to platelets and to purified gpIIb-IIIa, and that it interacts directly with fibrinogen. These results implicate this segment of gpIIb-IIIa in the ligand-binding function of the receptor. Moreover, as this region is highly conserved among integrins, it may have a general function in ligand recognition by this broadly distributed family of adhesion receptors.     

Crystal structure of opsin in its G-protein-interacting conformation

Opsin, the ligand-free form of the G-protein-coupled receptor rhodopsin, at low pH adopts a conformationally distinct, active G-protein-binding state known as Ops*. A synthetic peptide derived from the main binding site of the heterotrimeric G protein-the carboxy terminus of the alpha-subunit (GalphaCT)-stabilizes Ops*. Here we present the 3.2 A crystal structure of the bovine Ops*-GalphaCT peptide complex. GalphaCT binds to a site in opsin that is opened by an outward tilt of transmembrane helix (TM) 6, a pairing of TM5 and TM6, and a restructured TM7-helix 8 kink. Contacts along the inner surface of TM5 and TM6 induce an alpha-helical conformation in GalphaCT with a C-terminal reverse turn. Main-chain carbonyl groups in the reverse turn constitute the centre of a hydrogen-bonded network, which links the two receptor regions containing the conserved E(D)RY and NPxxY(x)(5,6)F motifs. On the basis of the Ops*-GalphaCT structure and known conformational changes in Galpha, we discuss signal transfer from the receptor to the G protein nucleotide-binding site.     

Identification of a receptor mediating Nogo-66 inhibition of axonal regeneration

Nogo has been identified as a component of the central nervous system (CNS) myelin that prevents axonal regeneration in the adult vertebrate CNS. Analysis of Nogo-A has shown that an axon-inhibiting domain of 66 amino acids is expressed at the extracellular surface and at the endoplasmic reticulum lumen of transfected cells and oligodendrocytes. The acidic amino terminus of Nogo-A is detected at the cytosolic face of cellular membranes and may contribute to inhibition of axon regeneration at sites of oligodendrocyte injury. Here we show that the extracellular domain of Nogo (Nogo-66) inhibits axonal extension, but does not alter non-neuronal cell morphology. In contrast, a multivalent form of the N terminus of Nogo-A affects the morphology of both neurons and other cell types. Here we identify a brain-specific, leucine-rich-repeat protein with high affinity for soluble Nogo-66. Cleavage of the Nogo-66 receptor and other glycophosphatidylinositol-linked proteins from axonal surfaces renders neurons insensitive to Nogo-66. Nogo-66 receptor expression is sufficient to impart Nogo-66 axonal inhibition to unresponsive neurons. Disruption of the interaction between Nogo-66 and its receptor provides the potential for enhanced recovery after human CNS injury.     

Heat-shock proteins DnaK and GroEL facilitate export of LacZ hybrid proteins in E. coli

The use of lacZ gene fusions, producing a hybrid protein containing an amino terminus specified by a target gene fused to the functional carboxy terminus of beta-galactosidase, has facilitated the study of protein targeting in various organisms. One of the best characterized fusions in Escherichia coli is phi(lamB-lacZ)42-1(Hyb), which produces a hybrid protein with the signal sequence and 181 N-terminal amino acids of the exported protein LamB, attached to LacZ. In common with other LacZ hybrids, the LamB-LacZ(42-1) protein is poorly exported from E. coli, conferring a Lac+ phenotype. beta-Galactosidase activity decreases markedly when cells producing the LamB-LacZ protein are grown at 42 degrees C or when a heat-shock response is induced at lower temperatures by overproducing heat-shock factor RpoH3, indicating the LacZ hybrids are being efficiently targeted to the cell envelope. We now report that the heat-shock proteins DnaK and GroEL can, in sufficient amounts, decrease beta-galactosidase activity and facilitate the export of lacZ-hybrid proteins.