Functional architecture of vasopressin/oxytocin receptors.

Three-dimensional models of G protein-coupled receptors (GPCR) have been defined using most experimental data available and protein modeling techniques. The endogenous ligand binding sites have been qualitatively described and putative receptor activation mechanisms have been proposed. The model has been recently refined to take into account recent crystallographic data. Most experimental results published are in excellent qualitative agreement with the initial model. We have undertaken to study more systematically by site directed mutagenesis the vasopressin/oxytocin receptor binding domain as a prototype of neuropeptide receptors. The experimental results are in very good agreement with the models. The residues responsible for the neuropeptide binding have been identified and confirm the predicted localization of the neuromediator in the transmembrane domain of the receptors. The side chain of the 8th residue of vasopressin interacts with a non-conserved receptor residue located in the first extracellular loop. As predicted from the model, this interaction is completely responsible for the selectivity of the ligand-receptor interaction. Finally, aromatic residues which allow the modulation of the efficacy of agonists have been identified.

Functional studies of twelve mutant V2 vasopressin receptors related to nephrogenic diabetes insipidus: molecular basis of a mild clinical phenotype.

X-linked nephrogenic diabetes insipidus (NDI) is a rare disease with defective renal and extrarenal arginine vasopressin V2 receptor responses due to mutations in the AVPR2 gene in Xq28. To study the cause of loss of function of mutant V2 receptors, we expressed 12 mutations (N55H, L59P, L83Q, V88M, 497CC-->GG, deltaR202, I209F, 700delC, 908insT, A294P, P322H, P322S) in COS-7 cells. Eleven of these, including P322H, were characterized by a complete loss of function, but the mutation P322S demonstrated a mild clinical and in vitro phenotype. This was characterized by a late diagnosis without any growth or developmental delay and a significant increase in urine osmolality after intravenous 1-deamino[D-Arg8]AVP administration. In vitro, the P322S mutant was able to partially activate the Gs/adenylyl cyclase system in contrast to the other V2R mutants including P322H, which were completely inactive in this regard. This showed not only that Pro 322 is important for proper V2R coupling, but also that the degree of impairment is strongly dependent on the identity of the substituting amino acid. Three-dimensional modeling of the P322H and P322S mutant receptors suggested that the complete loss of function of the P322H receptor could be due, in part, to hydrogen bond formation between the His 322 side chain and the carboxyl group of Asp 85, which does not occur in the P322S receptor.

Mapping peptide-binding domains of the human V1a vasopressin receptor with a photoactivatable linear peptide antagonist.

The study of antagonist-binding domains of the human V1a vasopressin receptor was performed using a radioiodinated photoreactive peptide antagonist. This ligand displayed a high affinity for the receptor expressed in Chinese hamster ovary cell membranes, and specifically labeled two protein bands with apparent molecular mass at 85-90 and 46 kDa. Our results clearly show that the V1a receptor is degraded during incubation with the ligand and that the 46-kDa species is probably the result of the 85-90-kDa species proteolytic cleavage. Truncation of the receptor was then confirmed by deglycosylation with N-glycosidase F. A monoclonal antibody directed against a c-Myc epitope added at the receptor NH2 terminus allowed immunoprecipitation of the 85-90-kDa photolabeled species. The 46-kDa photolabeled protein never immunoprecipitated, indicating that the truncated form of the receptor lacks the NH2 terminus region. To localize photolabeled domains of the receptor, the 46-kDa protein was cleaved with V8 and/or Lys-C endoproteinases. The identity of the smallest photolabeled fragment, observed at approximately 6 kDa, was then confirmed by mutation of the potential V8 cleavage sites. Our results indicate that covalent labeling of the vasopressin V1a receptor with the photoreactive antagonist occurs in a region including transmembrane domain VII (residues Asn327-Lys370).

Properties of a new radioiodinated antagonist for human vasopressin V2 and V1a receptors.

A vasopressin receptor antagonist, [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid), 2-o-ethyl-D-tyrosine, 4-valine, 9-tyrosylamide] arginine vasopressin (d(CH2)5[o-ethyl-D-Tyr2,Val4,Tyr-NH9(2)]AVP), has been prepared. This antagonist is a potent antiantidiuretic, antivasopressor and antioxytocic peptide with pA2 values of 7.69-7.94 and affinities of 1.12-11.0 nM. When radioiodinated at the phenyl moiety of the tyrosylamide residue at position 9, this peptide was demonstrated to bind to vasopressin V2 and V1a receptors with a dissociation constant of 0.22-0.75 nM. This ligand is a good tool for further studies on human vasopressin V2 receptor localization and characterization, when used in combination with a selective vasopressin V1a ligand.

Characterization of SR 121463A, a highly potent and selective, orally active vasopressin V2 receptor antagonist.

SR 121463A, a potent and selective, orally active, nonpeptide vasopressin V2 receptor antagonist, has been characterized in several in vitro and in vivo models. This compound displayed highly competitive and selective affinity for V2 receptors in rat, bovine and human kidney (0.6 < or = Ki [nM] < or = 4.1). In this latter preparation, SR 121463A potently antagonized arginine vasopressin (AVP)-stimulated adenylyl cyclase activity (Ki = 0.26+/-0.04 nM) without any intrinsic agonistic effect. In autoradiographic experiments performed in rat kidney sections, SR 121463A displaced [3H]AVP labeling especially in the medullo-papillary region and confirmed that it is a suitable tool for mapping V2 receptors. In comparison, the nonpeptide V2 antagonist, OPC-31260, showed much lower affinity for animal and human renal V2 receptors and lower efficacy to inhibit vasopressin-stimulated adenylyl cyclase (Ki in the 10 nanomolar range). Moreover, OPC-31260 exhibited a poor V2 selectivity profile and can be considered as a V2/V1a ligand. In normally hydrated conscious rats, SR 121463A induced powerful aquaresis after intravenous (0.003-0.3 mg/kg) or oral (0.03-10 mg/kg) administration. The effect was dose-dependent and lasted about 6 hours at the dose of 3 mg/kg p.o. OPC-31260 had a similar aquaretic profile but with markedly lower oral efficacy. The action of SR 121463A was purely aquaretic with no changes in urine Na+ and K+ excretions unlike that of known diuretic agents such as furosemide or hydrochlorothiazide. In addition, no antidiuretic properties have been detected with SR 121463A in vasopressin-deficient Brattleboro rats. Thus, SR 121463A is the most potent and selective, orally active V2 antagonist yet described and could be a powerful tool for exploring V2 receptors and the therapeutical usefulness of V2 blocker aquaretic agents in water-retaining diseases.

Two aromatic residues regulate the response of the human oxytocin receptor to the partial agonist arginine vasopressin.

We investigated the mechanisms that regulate the efficacy of agonists in the arginine-vasopressin (AVP)/oxytocin (OT) receptor system. In this paper, we present evidence that AVP, a full agonist of the vasopressin receptors, acts as a partial agonist on the oxytocin receptor. We also found that AVP becomes a full agonist when two aromatic residues of the oxytocin receptor are replaced by the residues present at equivalent positions in the vasopressin receptor subtypes. Our results indicate that these two residues modulate the response of the oxytocin receptor to the partial agonist AVP.

The binding site of neuropeptide vasopressin V1a receptor. Evidence for a major localization within transmembrane regions.

To identify receptor functional domains underlying binding of the neurohypophysial hormones vasopressin (AVP) and oxytocin (OT), we have constructed a three-dimensional (3D) model of the V1a vasopressin receptor subtype and docked the endogenous ligand AVP. To verify and to refine the 3D model, residues likely to be involved in agonist binding were selected for site-directed mutagenesis. Our experimental results suggest that AVP, which is characterized by a cyclic structure, could be completely buried into a 15-20-A deep cleft defined by the transmembrane helices of the receptor and interact with amino acids located within this region. Moreover, the AVP-binding site is situated in a position equivalent to that described for the cationic neurotransmitters. Since all mutated residues are highly conserved in AVP and OT receptors, we propose that the same agonist-binding site is shared by all members of this receptor family. In contrast, the affinity for the antagonists tested, including those with a structure closely related to AVP, is not affected by mutations. This indicates a different binding mode for agonists and antagonists in the vasopressin receptor.

Characterization of a novel, linear radioiodinated vasopressin antagonist: an excellent radioligand for vasopressin V1a receptors.

We report on the pharmacological properties of a potent and selective linear vasopressin (AVP) V1a receptor antagonist HO-Phenylacetyl1-D-Tyr(Me)2-Phe3-Gln4-Asn5-Arg6-Pro7-Arg8-NH2 (HO-LVA). Iodinated on the phenolic substituent at position 1, [125I]-HO-LVA displayed the highest affinity for rat liver V1a receptors (8 pM) ever reported. Furthermore, affinities of HO-LVA and I-HO-LVA for V1b, V2 and oxytocin (OT) receptors was 400- to 1,000-fold lower than for V1a receptors, rendering it a highly selective ligand. Both HO-LVA and its iodinated derivative are V1 antagonists, they potently inhibited AVP-induced inositol-phosphate accumulation in WRK1 cells, and also, although with a much lower potency, the AVP-induced ACTH release from freshly prepared pituitary cells. Using autoradiography [125I]-HO-LVA appeared to be the first radioligand to successfully identify and localize the presence of V1a receptors in rat liver and blood vessel walls. Moreover, several new brain regions expressing V1a receptors could be identified, in addition to those brain regions that were previously identified with other radiolabelled AVP analogues.

Close association of the alpha subunits of Gq and G11 G proteins with actin filaments in WRK1 cells: relation to G protein-mediated phospholipase C activation.

A selective polyclonal antibody directed toward the C-terminal decapeptide common to the alpha subunits of Gq and G11 G proteins (G alpha q/G alpha 11) was prepared and used to investigate the subcellular distribution fo these proteins in WRK1 cells, a rat mammary tumor cell line. In immunoblots, the antibody recognized purified G alpha q and G alpha 11 proteins and labeled only two bands corresponding to these alpha subunits. Functional studies indicated that this antibody inhibited vasopressin- and guanosine 5'-[alpha-thio]triphosphate-sensitive phospholipase C activities. Immunofluorescence experiments done with this antibody revealed a filamentous labeling corresponding to intracytoplasmic and perimembranous actin-like filament structures. Colocalization of G alpha q/G alpha 11 and F-actin filaments (F-actin) was demonstrated by double-labeling experiments with anti-G alpha q/G alpha 11 and anti-actin antibodies. Immunoblot analysis of membrane, cytoskeletal, and F-actin-rich fractions confirmed the close association of G alpha q/G alpha 11 with actin. Large amounts of G alpha q/G alpha 11 were recovered in the desmin- and tubulin-free F-actin-rich fraction obtained by a double depolymerization-repolymerization cycle. Disorganization of F-actin filaments with cytochalasin D preserved G alpha q/G alpha 11 and F-actin colocalization but partially inhibited vasopressin- and fluoroaluminate-sensitive phospholipase C activity, suggesting that actin-associated G alpha q/G alpha 11 proteins play a role in signal transduction.

Tyr115 is the key residue for determining agonist selectivity in the V1a vasopressin receptor.

Using a three-dimensional model of G protein-coupled receptors (GPCR), we have previously succeeded in docking the neurohypophysial hormone arginine-vasopressin (AVP) into the V1a receptor. According to this model, the hormone is completely embedded in the transmembrane part of the receptor. Only the side chain of the Arg residue at position 8 projects outside the transmembrane core of the receptor and possibly interacts with a Tyr residue located in the first extracellular loop at position 115. Residue 8 varies in the two natural neurohypophysial hormones, AVP and oxytocin (OT); similarly, different residues are present at position 115 in the different members of the AVP/OT receptor family. Here we show that Arg8 is crucial for high affinity binding of AVP to the rat V1a receptor. Moreover, when Tyr115 is replaced by an Asp and a Phe, the amino acids naturally occurring in the V2 and in the OT receptor subtypes, the agonist selectivity of the V1a receptor switches accordingly. Our results indicate that the interaction between peptide residue 8 and the receptor residue at position 115 is not only crucial for agonist high affinity binding but also for receptor selectivity.

Identification of agonist binding sites of vasopressin and oxytocin receptors.

The present study aims at delineating residues in the vasopressin/oxytocin receptor family responsible for the high affinity binding of the hormone. Therefore, we have constructed a computer-generated 3 dimensional model of the rat V1a vasopressin receptor subtype which allowed us to propose residues likely to be involved in agonist binding. Among these residues, several are highly conserved in the receptor family. They were selected for site-directed mutagenesis on the basis of putative direct interaction with bound ligands. The present model and experimental results led us to conclude that the hormone is docked in a pocket completely buried in the transmembrane core of the receptor. Large polar residues, such as glutamine and lysine, located in transmembrane regions 2,3,4 and 6 are involved in the binding of the neurohypophysial hormone. Since all the mutated residues are highly conserved in AVP and OT receptors, we propose that the agonist binding site is similar in all members of the receptor family; only minor changes were found in antagonist potencies, suggesting that agonist and antagonist binding sites do not completely overlap.

Localization and characterization of vasopressin binding sites in the rat brain using an iodinated linear AVP antagonist.

The binding characteristics and central distribution of 125I-Linear AVP antagonist, a new ligand for vasopressin binding sites, are described in the following studies. Saturation studies performed on rat brain septal membranes demonstrated that 125I-Linear AVP antagonist binds to a single class of sites with high affinity (55 pM) and limited capacity (88 fmol/mg protein). In autoradiographic studies, 125I-Linear AVP antagonist labeled brain areas known to contain vasopressin receptors without binding to neurophysins. 125I-Linear AVP antagonist also labeled sites in cortex, hypothalamus, ventral tegmental area and substantia nigra. In competition studies, 125I-Linear AVP antagonist binding was most readily blocked by AVP and a selective V1a agonist. Oxytocin and a selective V2 ligand were effective only in micromolar concentrations. A selective oxytocin agonist was virtually ineffective in blocking 125I-Linear AVP antagonist binding. In regions that contain a high density of oxytocin binding sites, however, oxytocin-displaceable binding was observed. In agreement with studies on peripheral tissues, the binding profile generated from these studies indicates that 125I-Linear AVP antagonist binds to vasopressin receptors of the V1a subtype. These results suggest that 125I-Linear AVP antagonist is a valuable ligand for the study of central AVP receptors.

Deglycosylation and fragmentation of purified rat liver angiotensin II receptor: application to the mapping of hormone-binding domains.

We report new structural data about the rat liver angiotensin II receptor, which belongs to the AT1 subclass. This receptor has been purified at analytical or semi-preparative levels by a previously described strategy involving its photolabelling with a biotinylated azido probe and selective adsorption of the covalent probe-receptor complexes to immobilized streptavidin [Marie, Seyer, Lombard, Desarnaud, Aumelas, Jard and Bonnafous (1990) Biochemistry 29, 8943-8950]. Chemical or enzymic deglycosylation of the purified receptor has shown a shift in its molecular mass from 65 kDa to 40 kDa. Fragmentation of the purified receptor was carried out with V8 protease from Staphylococcus aureus, CNBr and trypsin. It was possible to find trypsin-treatment conditions which allowed production of a 6 kDa probe-fragment complex with a satisfactory yield. Attempts to localize this small fragment (5 kDa after subtraction of the probe contribution) in the recently published rat AT1 receptor sequence are reported. As expected, this fragment is not glycosylated; moreover, its further fragmentation by CNBr induces a very slight decrease in its size. These data support the hypothesis that a receptor sequence comprising the third transmembrane domain and adjacent portions of extra- and intracellular loops is involved in photolabelling by the C-terminal azidophenylalanine of the angiotensin-derived probe. These preliminary results are discussed in terms of future prospects for the characterization of hormone-binding domains of angiotensin II receptors.

Novel approach to the design of synthetic radioiodinated linear V1A receptor antagonists of vasopressin.

We report the solid phase synthesis of six analogs of the potent and selective linear AVP vasopressor (V1a receptor) antagonist: Phaa1-D-Tyr(Et)2-Phe3-Gln4-Asn5-Lys6-Pro7-Arg-NH(8)2(A) (where Phaa = phenylacetyl) in which the Phaa1 residue is replaced by hydroxyphenylacetyl (HO-Phaa), hydroxyphenylpropionyl (HO-Phpa) and phenylpropionyl (Phpa) and the D-Tyr(Et)2 and Lys6 residues by D-Tyr(Me)2 and Arg6 substituents. The phenolic-containing peptides were synthesized to test the feasibility of using this approach for the design of high affinity selective ligands for AVP V1a receptors. The following analogs of A were synthesized: 11 [(HO)Phaa1]; 2. [(HO)Phaa1,D-Tyr(Me)2]; 3. [(HO)Phaa1,D-Tyr(Me)2, Arg6]; 4. [(HO)Phaa1,Arg6]; 5. [Phpa1]; 6. [(HO)Phpa1]. All six peptides were examined for agonistic and antagonistic potencies in vasopressor (V1a-receptor) and antidiuretic (V2-receptor) and in vitro oxytocic assays in rats. The affinities of the phenolic-containing peptides for hepatic V1a and uterine receptors were also determined. The phenolic-containing peptides all exhibit potent V1a antagonism. Their anti-V1a pA2 values range from 8.23 to 8.63 (the anti-V1a pA2 value of A = 8.69). Their inhibition constants (Ki in nM) range 0.4 to 1.0. They are weak antidiuretic agonists with activities ranging from 0.022 U/mg to 0.13 U/mg (A = 0.033 U/mg). They all exhibit OT antagonism in vitro. Their anti-OT pA2 values range from 7.28 to 7.71 (A = 7.62). All five phenolic compounds were iodinated using iodine chloride and tested in the same in vivo and in vitro assay system.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein purification using combined streptavidin (or avidin)-Sepharose and thiopropyl-Sepharose affinity chromatography.

The major problem usually encountered in the application of the (strept)avidin-biotin system to the purification of proteins (or other biological molecules) lies in the difficult reversion of the interaction between immobilized (strept)avidin and the adsorbed biotinylated protein. Among the proposed solutions is the selective biotinylation of the entity to be purified by a disulphide-containing biotinylated reagent which allows its recovery from (strept)avidin gels by dithiothreitol (DTT) treatment. As emphasized by the example of angiotensin II receptor purification, achieved using this strategy, optimum reduction of this disulphide bridge may require improvement of its accessibility using denaturating agents such as sodium dodecyl sulphate or urea. However, these agents release important amounts of (strept)avidin. Two general ways of solving this problem are proposed. One solution takes advantage of the absence of cysteine in the streptavidin sequence: the protein to be purified is selectively readsorbed to thiopropyl-Sepharose through the thiol function generated on DTT cleavage of the biotinylated reagent. The other solution is an empirical approach to make possible the use of avidin, which possesses cysteine residues: combined avidin-Sepharose and thiopropyl-Sepharose chromatography proved efficient when carried out in the presence of urea as denaturing agent.