G-protein-coupled receptors for catecholamines and some other small ligands are activated when agonists bind to the transmembrane region of the receptor. The docking interactions through which peptide agonists activate their receptors are less well characterized. The thrombin receptor is a specialized peptide receptor. It is activated by binding its tethered ligand domain, which is unmasked upon receptor cleavage by thrombin. Human and Xenopus thrombin receptor homologues are each selectively activated by the agonist peptide representing their respective tethered ligand domains. Here we identify receptor domains that confer this agonist specificity by replacing the Xenopus receptor's aminoterminal exodomain and three extracellular loops with the corresponding human structures. This switches receptor specificity from Xenopus to human. The specificity of these thrombin receptors for their respective peptide agonists is thus determined by their extracellular surfaces. Our results indicate that agonist interaction with extracellular domains is important for thrombin receptor activation.

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

Constitutively active thrombin receptors were generated while constructing chimeric receptors to identify the structural basis for thrombin receptor agonist specificity. Substitution of eight amino acids from the Xenopus receptor's second extracellular loop (XECL2B) for the cognate sequence in the human thrombin receptor was sufficient to confer robust constitutive activity. Smaller substitutions within the XECL2B site yielded less constitutive activation, and substitution of several unrelated sequences at this site caused no activation. Expression of the XECL2B receptor caused high basal 45 Ca efflux in Xenopus oocytes and high basal phosphoinositide hydrolysis and reporter gene induction in COS cells. Of note, a mutant receptor in which all four of the Xenopus thrombin receptor's extracellular segments replaced the cognate human sequences showed much less constitutive activity than XECL2B and preserved responsiveness to agonist. This partial complementation of the XECL2B phenotype by addition of other Xenopus extracellular structures suggests that the XECL2B mutation causes constitutive activation by altering interactions among the human receptor's extracellular domains. Thus, a change in an extracellular loop of a G protein-coupled receptor can transmit information across the cell membrane to cause signaling, perhaps via a conformational change similar to that caused by agonist binding. Indeed, the site of the activating mutation in XECL2B coincides with a putative agonist-docking site, supporting the hypothesis that agonist interactions with the thrombin receptor's extracellular loops contribute to receptor activation.A variety of naturally occurring and engineered mutations in G protein-coupled receptors have been found to cause constitutive activation. Their importance is underscored by the observation that activating mutations in G protein-coupled receptors underlie a variety of human diseases. The locations of activating mutations both within a single receptor and across receptors are widespread, with activating mutations reported in transmembrane domains 2, 3, 6, and 7; in cytoplasmic loops 1 and 3; and at the junction of transmembrane domain 2 and extracellular loop 1 (reviewed in Refs. 1-4; see also Refs. 5-9). This diversity suggests that specific interactions maintain G protein-coupled receptors in their off-state(s) and that these interactions can be disrupted in a variety of ways. We recently generated a constitutively active thrombin receptor in the course of studying chimeric thrombin receptors designed to identify the receptor domains that distinguish the human from Xenopus thrombin receptor agonist peptides. Building human thrombin receptor sequence into the Xenopus receptor (specifically small regions of the receptor's amino-terminal exodomain near transmembrane domain 1 and its second extracellular loop) conferred human receptor-like agonist specificity (10, 11). These same receptor regions, particularly receptor residues 260 -268 in the second extracellular loop, were identified by a sec...

Seven transmembrane domain G protein-coupled receptors respond to a structurally diverse set of ligands to regulate a host of biological processes. Catecholamines and certain other small ligands elicit responses by binding to their receptors' transmembrane regions (1). The docking interactions by which peptide agonists activate their receptors are less well-characterized (2-7). The thrombin receptor can be viewed as a specialized peptide receptor that contains its own "tethered ligand." Thrombin activates its receptor by cleaving the receptor's amino-terminal exodomain. This limited proteolysis unmasks a new amino terminus which then functions as a tethered peptide agonist, binding to the body of the receptor to cause signaling (8 -10). Synthetic peptides which mimic the tethered ligand domain behave as peptide agonists, activating the receptor independent of thrombin. Identification of the interactions by which the thrombin receptor's tethered ligand domain triggers transmembrane signaling is central to understanding signaling by this and perhaps other peptide receptors. Such information may also aid the development of novel pharmaceuticals for inhibiting thrombotic, inflammatory, and proliferative actions of thrombin (11,12).Three-dimensional structures for seven transmembrane domain G protein-coupled receptors are currently of low resolution, especially in the extramembranous regions (13). Thus structures that reveal the details of agonist docking, particularly for agonists that interact with the receptor's extracellular loops, are unlikely to be available in the near future. Mutagenesis studies which examine functional end points remain a valuable approach that provides constraints for model building and analysis of future structural data.We exploited the specificity of the human and Xenopus thrombin receptor homologues for their respective agonist peptides to identify the receptor domains which distinguish between these agonists (14). A chimeric receptor in which the Xenopus receptor's extracellular surface (its amino-terminal exodomain and three extracellular loops) was replaced with that of the human receptor showed a remarkable gain of responsiveness to the human agonist and loss of responsiveness to the Xenopus agonist, resulting in human receptor-like agonist specificity (14). More limited replacement of Xenopus with human receptor sequence suggested that two regions accounted for this change in specificity: residues 244 -268 in the second extracellular loop and residues 76 -93 located in the amino-terminal exodomain near the start of transmembrane domain 1 (14). An antiserum which recognized the latter region blocked receptor activation by agonist peptide, consistent with a role for this region in agonist function (15,16).With the long term goal of defining the agonist-receptor interactions which mediate receptor activation, we analyzed progressively finer chimeras to identify the specific amino acids in the human and Xenopus thrombin receptors which distinguish their cognate agonists. Ultimately, two...

Heart failure causes a significant medical, social, and economic burden within the U.S. The escalating health resource utilization for heart failure has led to the development of strategies devised to optimize outpatient treatment and prevent rehospitalization. Programs for improving outpatient care of heart failure patients have included approaches that emphasize compliance with recommended treatment options, patient adherence to self-care recommendations, and careful surveillance for signs of worsening symptomatology. Several studies have shown improvement in patient functional status, reduced hospital admissions, and possibly lower medical costs with use of comprehensive, multidisciplinary heart failure management programs. 1-4 Further research is needed, however, to elucidate approaches that will have the most clinical and economic impact. In addition, it remains to be seen how applicable these management programs will be to a diverse population of patients, such as those treated in large inner city hospitals. However, due to limited hospital staff and resources, language barriers, travel difficulties, and limited health literacy, indigent patients are the least likely to gain access to-and remain compliant withcomplex disease management programs.A promising strategy for addressing these problems is to communicate with heart failure patients between outpatient visits using telecommunication services with nurse/physician follow up as needed. There are a number of communication devices available, which use specialized computer technology to collect patient information between outpatient encounters, and can be used by providers managing patient care. Clinicians can also use such systems to identify patients who need targeted reinforcement of behavioral changes. Moreover, these services can increase patients' access to language-appropriate health services, provide a surveillance mechanism for early detection of patients who are getting into difficulties, and focus scarce clinical resources on subpopulations who have the greatest need for intervention. 5-7