Cardiomyopathy is a major cause of morbidity and mortality. Ventricular conduction delay, as shown by prolonged deflections in the electrocardiogram caused by delayed ventricular contraction (wide QRS complex), is a common feature of cardiomyopathy and is associated with a poor prognosis. Although the G i-signaling pathway is up-regulated in certain cardiomyopathies, previous studies suggested this up-regulation was compensatory rather than a potential cause of the disease. Using the tetracycline transactivator system and a modified G i-coupled receptor (Ro1), we provide evidence that increased Gi signaling in mice can result in a lethal cardiomyopathy associated with a wide QRS complex arrhythmia. Induced expression of Ro1 in adult mice resulted in a >90% mortality rate at 16 wk, whereas suppression of Ro1 expression after 8 wk protected mice from further mortality and allowed partial improvement in systolic function. Results of DNA-array analysis of over 6,000 genes from hearts expressing Ro1 are consistent with hyperactive G i signaling. DNA-array analysis also identified known markers of cardiomyopathy and hundreds of previously unknown potential diagnostic markers and therapeutic targets for this syndrome. Our system allows cardiomyopathy to be induced and reversed in adult mice, providing an unprecedented opportunity to dissect the role of G i signaling in causing cardiac pathology.G proteins ͉ signal transduction ͉ gene expression ͉ genome ͉ bioinformatics I diopathic dilated cardiomyopathy (IDC) is a major cause of heart failure characterized by cardiac dilation and reduced systolic function. In the United States, about half of the cases of dilated cardiomyopathy are associated with myocarditis or coronary artery disease, and half are considered idiopathic (1-4). Ventricular conduction delay, as shown by a prolonged depolarization in the electrocardiogram (wide QRS complex), is associated with up to 70% of IDC cases (5) and is an independent risk factor for death among IDC patients (6, 7). Several lines of evidence implicate altered G i signaling in the development of cardiomyopathies such as IDC, but a direct relationship between G i signaling and cardiomyopathy has not been demonstrated in vivo. We recently created a system that utilizes a specifically designed G i -coupled receptor and inducible gene expression techniques to control G i signaling in the adult mouse heart (8).With over 2

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 NH 2 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 NH 2 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 Asn 327 -Lys 370 ).Neurohypophysial hormones, arginine-vasopressin (AVP) 1 and oxytocin, exert a wide range of physiological effects through binding to specific membrane receptors belonging to the G protein-coupled receptor (GPCR) superfamily. To date, three AVP receptor subtypes and one oxytocin receptor have been pharmacologically and functionally described (1). V1a, V1b, and oxytocin receptors activate phospholipase C, resulting in the production of inositol 1,4,5-trisphosphate and diacylglycerol, mobilization of intracellular calcium and activation of protein kinase C. V2 receptors stimulate adenylyl cyclase, resulting in the accumulation of cyclic AMP and activation of protein kinase A. All receptor subtypes from several mammalian species have been recently cloned (2-5), as well as closely related receptors from bony fishes and invertebrates (6, 7).Analysis of the primary sequence of these receptors suggests that they possess the same general architecture with seven transmembrane helices as other well characterized G proteincoupled receptors. Moreover, the comparison of their amino acid sequence reveals significant homology within the putative transmembrane regions (TM) and within the first and second extracellular loops as well. The natural ligands for the receptors of the AVP/oxytocin family are also closely structurally related. All are nonapeptides composed of a 6-amino acid disulfide-linked ring and a COOH terminus tripeptide.Peptides of the AVP/oxytocin series were subjected to an extensive analysis of structure-activity relationships. These studies (for review, see Refs. 8 -11) led to the production of a profusion of valuable pharmacological probes for asse...

Phpa-LVA (covalent attachment to transmembrane domain VII), threedimensional models of the antagonist-bound receptors were constructed and then verified by site-directed mutagenesis studies. Strikingly, these two linear peptide antagonists, when bound to the V 1a receptor, could adopt a pseudocyclic conformation similar to that of the cyclic agonists. Despite divergent functional properties, these peptide antagonists could interact with a transmembrane-binding site significantly overlapping that of the natural hormone vasopressin.Over the past few years, interest in locating ligand-binding sites in G protein-coupled receptors has increased exponentially. Indeed, identification of these binding sites is of prime importance both for a better understanding of the structure and the function of the G protein-coupled receptor superfamily and for facilitating rational design of potential therapeutic agents. Extensive mutational analysis and receptor three-dimensional molecular modeling have led to valuable information concerning "small ligand" and peptide/protein ligand receptor-binding sites (for review, see Refs. 1-6).In 1995, we published the mapping of arginine-vasopressin (AVP) 1 -binding site in the V 1a receptor subtype and described a major localization within transmembrane regions (TMR) in a position equivalent to that defined for the cationic neurotransmitters (7). Because all receptor residues potentially interacting with AVP are conserved in the different members of the AVP/oxytocin (OT) receptor family, we proposed that the binding pocket identified in the V 1a might be common to V 2 , V 1b , and OT receptor subtypes. Extracellular residues responsible for receptor-selective and species-selective binding have also been identified (8 -10). Unfortunately, these first analyses of AVP receptor structure/function relationships did not provide much information on AVP receptor antagonist-binding domains (Refs. 11 and 12, and for review see Ref. 13). The photoaffinity labeling technique is an essential complement to modeling and mutagenesis approaches and allows direct unambiguous identification of the contact regions between a receptor and its specific photoactivatable ligands (for review see Ref. 14). At the present time, very few photoaffinity labeling studies have led to the direct determination of labeled amino acid residues in peptide G protein-coupled receptor; remarkable results with bovine V 2 receptor (15), human NK1 tachykinin receptor (16), and rat type A cholecystokinin receptor (17) allowed identification of covalently labeled residues with photoactivatable agonist analogues of AVP, substance P, and cholecystokinin, respectively.Very recently, a first radioiodinated photoreactive linear peptide antagonist has been used in our laboratory to photolabel the human and rat V 1a receptors (12,18,19). Our results have clearly indicated that covalent attachment of the [ 125 I]3N 3 Phpa-LVA occurs in a restricted domain of the human receptor including TMR VII. Based both on this photolabeling result and on th...

To improve our understanding of the functional architecture of G protein-coupled receptors, we have taken advantage of differences among mammalian species in ligand binding to search for the rat versus human selectivity determinants of the V2 vasopressin receptor and of its peptide ligands. Our data indicate that residue 2 of species-selective peptide antagonists such as d(CH 2 ) 5 -[DIle 2 ,Ile 4 ,Tyr-NH 2 9 ]arginine vasopressin controls their rat versus human selectivity. For species-selective agonists such as desmopressin, residues 1 and 8 modulate the binding selectivity. Among residues different between rat and human V2 receptors, those localized in the upper part of the human V2 receptor have been substituted with their rat V2 homologs. Pharmacological analysis of mutant receptors revealed that residues 202 and 304 fully control the species selectivity of the discriminating antagonists in an independent and additive manner. A third residue (position 100) is necessary to observe an equivalent phenomenon for the discriminating agonists. The substitution of these three residues does not modify the affinity of the nonselective agonists and antagonists. In conclusion, extracellular loops and the top of the transmembrane domains of V2 vasopressin receptors may provide the molecular basis for peptide ligand-binding species selectivity. Very few residues in these regions may control the binding mode of both agonists and antagonists. The arginine vasopressin (AVP)1 /oxytocin receptor family represents a suitable system to investigate structure-function relationships among G protein-coupled receptors. Indeed, all four receptor subtypes for these neurohypophyseal hormones, namely the V1a, V1b, and V2 AVP receptors and the unique oxytocin receptor, have been well characterized by pharmacological and molecular cloning studies (see Ref. 1 for review). These peptide receptors share a high primary sequence homology, but display a great diversity in their functional properties. Moreover, these receptors interact with many potent selective agonists and several classes of antagonist ligands, such as cyclic and linear peptides (2) as well as nonpeptides (3,4). By a combination of receptor three-dimensional molecular modeling and site-directed mutagenesis approaches, our preceding study has led to the mapping of the AVP agonist-binding domain in the V1a receptor subtype (5). The hormone-binding site is localized in a narrow cleft delimited by most of the transmembrane regions ϳ15 Å away from the extracellular surface. Because of the high conservation of residues involved in the interaction with the hormone, this binding site has been proposed to be common to the V1b, V2, and oxytocin receptors. An extracellular residue responsible for receptor subtype agonist selectivity has also been identified (6 -8).These first analyses did not provide much information on the definition of AVP/oxytocin receptor antagonist-binding domains. To date, only the upper part of transmembrane region VII and a hydrophobic cluster of aromatic residue...

The substitution, in the human V 2 vasopressin receptor, of the aspartate at position 136 by alanine leads to agonist-independent activation of this mutant V 2 receptor.