Local membrane charge regulates ß2 adrenergic receptor coupling to Gi3.

The ß2 adrenergic receptor (ß2AR) signals through both Gs and Gi in cardiac myocytes, and the Gi pathway counteracts the Gs pathway. However, Gi coupling is much less efficient than Gs coupling in most cell-based and biochemical assays, making it difficult to study ß2AR-Gi interactions. Here we investigate the role of phospholipid composition on Gs and Gi coupling. While negatively charged phospholipids are known to enhance agonist affinity and stabilize an active state of the ß2AR, we find that they impair coupling to Gi3 and facilitate coupling to Gs. Positively charged Ca2+ and Mg2+, known to interact with the negative charge on phospholipids, facilitates Gi3 coupling. Mutational analysis suggests that Ca2+ coordinates an interaction between phospholipid and the negatively charged EDGE motif on the amino terminal helix of Gi3. Taken together, our observations suggest that local membrane charge modulates the interaction between ß2AR and competing G protein subtypes.

Behavioral and neurochemical characterization of alpha(2A)-adrenergic receptor knockout mice.

Genetic manipulation of mice now provides new tools to evaluate the biological functions of the alpha(2)-adrenergic receptor (alpha(2)-AR) subtypes (alpha(2A), alpha(2B), and alpha(2C)). To investigate the role of the alpha(2A)-AR in the modulation of mouse primary behavioral characteristics and brain neurochemistry, mice with targeted inactivation of the gene for the alpha(2A)-AR were compared with wild-type C57BL/6 control animals. First, a comprehensive behavioral screen was employed to provide a detailed characterization of basic neurologic functions. Thereafter, the mice were analyzed in three models of anxiety, i.e. the elevated-plus maze test, the marble burying test and the open field test. The diurnal activity pattern of the mice was assessed in a 24-h locomotor activity test. Furthermore, receptor autoradiography of the brain was performed using the subtype-non-selective alpha(2)-AR antagonist radioligand [(3)H]RS-79948-197. Lack of the alpha(2A)-AR was associated with alterations in autonomic functions, including increased heart rate and piloerection. The mutant mice also exhibited impaired motor coordination skills, increased anxiety-like behavior and an abnormal diurnal activity pattern. In addition, neurochemical analysis of monoamine neurotransmitters revealed a considerable increase in brain norepinephrine turnover in mice lacking alpha(2A)-AR. Our results provide further support for the crucial role of the alpha(2A)-AR in modulating brain noradrenergic neurotransmission and many aspects of mouse behavior and physiology.

Differential distribution of beta-adrenergic receptor subtypes in blood vessels of knockout mice lacking beta(1)- or beta(2)-adrenergic receptors.

beta-Adrenergic receptors (beta-AR) are essential regulators of cardiovascular homeostasis. In addition to their prominent function in the heart, beta-AR are located on vascular smooth muscle cells, where they mediate vasodilating effects of endogenous catecholamines. In this study, we have investigated in an isometric myograph different types of blood vessels from mice lacking beta(1)- and/or beta(2)-adrenergic receptor subtypes (beta(1)-KO, beta(2)-KO, beta(1)beta(2)-KO). In wild-type mice, isoproterenol induced relaxation of segments from thoracic aorta, carotid, femoral and pulmonary arteries, and portal vein. The relaxant effect of beta-receptor stimulation was absent in femoral and pulmonary arteries from beta(1)-KO mice. In aortic and carotid arteries and in portal veins, the vasodilating effect of isoproterenol was reduced in mice lacking beta(1)- or beta(2)-receptors. However, in these vessels the vasodilating effect was only abolished in double KO mice lacking both beta(1)- and beta(2)-receptors. Vessel relaxation induced by forskolin did not differ between wild-type and KO mice. Similar contributions of beta(1)- and beta(2)-receptors to isoproterenol-induced vasorelaxation were found when vessels from KO mice were compared with wild-type arteries in the presence of subtype-selective beta-receptor antagonists. These studies demonstrate that beta(1)-adrenergic receptors play a dominant role in the murine vascular system to mediate vasodilation. Surprisingly, beta(2)-receptors contribute to adrenergic vasodilation only in a few major blood vessels, suggesting that differential distribution of beta-adrenergic receptor subtypes may play an important role in redirection of tissue perfusion.

A genetically engineered cell-based biosensor for functional classification of agents.

Cell-based biosensors (CBBs) utilize whole cells to detect biologically active agents. Although CBBs have shown success in detecting the presence of biological agents, efforts to classify the type of agent based on functional activity have proven difficult because multiple biochemical pathways can lead to the same cellular response. However, a new approach using a genetically-engineered cell-based biosensor (GECBB) described in this paper translates this cross-talk noise into common-mode noise that can be rejected. The GECBB operates by assaying for an agent's ability to differentially activate two populations of cells, wild-type (WT) cells and cells genetically engineered to lack a specific receptor, knockout (KO) cells. Any biological agent that targets the knocked out receptor will evoke a response in the WT but not in the KO. Thus, the GECBB is exquisitely sensitive to agents that effect the engineered pathway. This approach provides the benefits of an assay for specific functional activity while simplifying signal analysis. The GECBB implemented was designed to be sensitive to agents that activate the beta 1-adrenergic receptor (beta 1-AR). This was achieved by using mouse cardiomyocytes in which the beta 1-AR had been knocked out. The cellular signal used in the GECBB was the spontaneous beat rate of the two cardiomyocyte syncitia as measured with microelectrode arrays. The GECBB was able to detect the beta-AR agonist isoproterenol (ISO) at a concentration of 10 microM (P<0.005).

Single-molecule spectroscopy of the beta(2) adrenergic receptor: observation of conformational substates in a membrane protein.

Single-molecule studies of the conformations of the intact beta(2) adrenergic receptor were performed in solution. Photon bursts from the fluorescently tagged adrenergic receptor in a micelle were recorded. A photon-burst algorithm and a Poisson time filter were implemented to characterize single molecules diffusing across the probe volume of a confocal microscope. The effects of molecular diffusion and photon number fluctuations were deconvoluted by assuming that Poisson distributions characterize the molecular occupation and photon numbers. Photon-burst size histograms were constructed, from which the source intensity distributions were extracted. Different conformations of the beta(2) adrenergic receptor cause quenching of the bound fluorophore to different extents and hence produce different photon-burst sizes. An analysis of the photon-burst histograms shows that there are at least two distinct substates for the native adrenergic membrane receptor. This behavior is in contrast to one peak observed for the dye molecule, rhodamine 6G. We test the reliability and robustness of the substate number determination by investigating the application of different binning criteria. Conformational changes associated with agonist binding result in a marked change in the distribution of photon-burst sizes. These studies provide insight into the conformational heterogeneity of G protein-coupled receptors in the presence and absence of a bound agonist.

Functional differences between full and partial agonists: evidence for ligand-specific receptor conformations.

The interaction of an agonist-bound G-protein-coupled receptor (GPCR) with its cognate G-protein initiates a sequence of experimentally quantifiable changes in both the GPCR and G-protein. These include the release of GDP from G(alpha), the formation of a ternary complex between the nucleotide-free G-protein and the GPCR, which has a high affinity for agonist, followed by the binding of GTP to G(alpha), the dissociation of the GPCR/G-protein complex, and the hydrolysis of GTP. The efficacy of an agonist is a measure of its ability to activate this cascade. It has been proposed that efficacy reflects the ability of the agonist to stabilize the active state of the GPCR. We examined a series of beta(2)-adrenoceptor (beta(2)AR) agonists (weak partial agonists to full agonists) for their efficacy at promoting two different steps of the G-protein activation/deactivation cycle: stabilizing the ternary complex (high-affinity, GTP-sensitive agonist binding), and steady-state GTPase activity. We obtained results for the wild-type beta(2)AR and a constitutively active mutant of the beta(2)AR (beta(2)AR(CAM)) using fusion proteins between the GPCRs and G(salpha) to facilitate GPCR/G-protein interactions. There was no correlation between efficacy of ligands in activating GTPase and their ability to stabilize the ternary complex at beta(2)AR(CAM). Our results suggest that the GPCR state that optimally promotes the GDP release and GTP binding is different from the GPCR state that stabilizes the ternary complex. By strongly stabilizing the ternary complex, certain partial agonists may reduce the rate of G-protein turnover relative to a full agonist.

Agonist-induced conformational changes in the G-protein-coupling domain of the beta 2 adrenergic receptor.

The majority of extracellular physiologic signaling molecules act by stimulating GTP-binding protein (G-protein)-coupled receptors (GPCRs). To monitor directly the formation of the active state of a prototypical GPCR, we devised a method to site specifically attach fluorescein to an endogenous cysteine (Cys-265) at the cytoplasmic end of transmembrane 6 (TM6) of the beta(2) adrenergic receptor (beta(2)AR), adjacent to the G-protein-coupling domain. We demonstrate that this tag reports agonist-induced conformational changes in the receptor, with agonists causing a decline in the fluorescence intensity of fluorescein-beta(2)AR that is proportional to the biological efficacy of the agonist. We also find that agonists alter the interaction between the fluorescein at Cys-265 and fluorescence-quenching reagents localized to different molecular environments of the receptor. These observations are consistent with a rotation and/or tilting of TM6 on agonist activation. Our studies, when compared with studies of activation in rhodopsin, indicate a general mechanism for GPCR activation; however, a notable difference is the relatively slow kinetics of the conformational changes in the beta(2)AR, which may reflect the different energetics of activation by diffusible ligands.

Functionally different agonists induce distinct conformations in the G protein coupling domain of the beta 2 adrenergic receptor.

G protein-coupled receptors represent the largest class of drug discovery targets. Drugs that activate G protein-coupled receptors are classified as either agonists or partial agonists. To study the mechanism whereby these different classes of activating ligands modulate receptor function, we directly monitored ligand-induced conformational changes in the G protein-coupling domain of the beta(2) adrenergic receptor. Fluorescence lifetime analysis of a reporter fluorophore covalently attached to this domain revealed that, in the absence of ligands, this domain oscillates around a single detectable conformation. Binding to an antagonist does not change this conformation but does reduce the flexibility of the domain. However, when the beta(2) adrenergic receptor is bound to a full agonist, the G protein coupling domain exists in two distinct conformations. Moreover, the conformations induced by a full agonist can be distinguished from those induced by partial agonists. These results provide new insight into the structural consequence of antagonist binding and the basis of agonism and partial agonism.

Dual modulation of cell survival and cell death by beta(2)-adrenergic signaling in adult mouse cardiac myocytes.

The goal of this study was to determine whether beta(1)-adrenergic receptor (AR) and beta(2)-AR differ in regulating cardiomyocyte survival and apoptosis and, if so, to explore underlying mechanisms. One potential mechanism is that cardiac beta(2)-AR can activate both G(s) and G(i) proteins, whereas cardiac beta(1)-AR couples only to G(s). To avoid complicated crosstalk between beta-AR subtypes, we expressed beta(1)-AR or beta(2)-AR individually in adult beta(1)/beta(2)-AR double knockout mouse cardiac myocytes by using adenoviral gene transfer. Stimulation of beta(1)-AR, but not beta(2)-AR, markedly induced myocyte apoptosis, as indicated by increased terminal deoxynucleotidyltransferase-mediated UTP end labeling or Hoechst staining positive cells and DNA fragmentation. In contrast, beta(2)-AR (but not beta(1)-AR) stimulation elevated the activity of Akt, a powerful survival signal; this effect was fully abolished by inhibiting G(i), G(beta gamma), or phosphoinositide 3 kinase (PI3K) with pertussis toxin, beta ARK-ct (a peptide inhibitor of G(beta gamma)), or LY294002, respectively. This indicates that beta(2)-AR activates Akt via a G(i)-G(beta gamma)-PI3K pathway. More importantly, inhibition of the G(i)-G(beta gamma)-PI3K-Akt pathway converts beta(2)-AR signaling from survival to apoptotic. Thus, stimulation of a single class of receptors, beta(2)-ARs, elicits concurrent apoptotic and survival signals in cardiac myocytes. The survival effect appears to predominate and is mediated by the G(i)-G(beta gamma)-PI3K-Akt signaling pathway.

beta 2-adrenergic receptor-induced p38 MAPK activation is mediated by protein kinase A rather than by Gi or gbeta gamma in adult mouse cardiomyocytes.

Increasing evidence shows that stimulation of beta-adrenergic receptor (AR) activates mitogen-activated protein kinases (MAPKs), in addition to the classical G(s)-adenylyl cyclase-cAMP-dependent protein kinase (PKA) signaling cascade. In the present study, we demonstrate a novel beta(2)-AR-mediated cross-talk between PKA and p38 MAPK in adult mouse cardiac myocytes expressing beta(2)-AR, with a null background of beta(1)beta(2)-AR double knockout. beta(2)-AR stimulation by isoproterenol increased p38 MAPK activity in a time- and dose-dependent manner. Inhibiting G(i) with pertussis toxin or scavenging Gbetagamma with betaARK-ct overexpression could not prevent beta(2)-AR-induced p38 MAPK activation. In contrast, a specific peptide inhibitor of PKA, PKI (5 microm), completely abolished the stimulatory effect of beta(2)-AR, suggesting that beta(2)-AR-induced p38 MAPK activation is mediated via a PKA-dependent mechanism, rather than by G(i) or Gbetagamma. This conclusion was further supported by the ability of forskolin (10 microm), an adenylyl cyclase activator, to elevate p38 MAPK activity in a PKI-sensitive manner. Furthermore, inhibition of p38 MAPK with SB203580 (10 microm) markedly enhanced the beta(2)-AR-mediated contractile response, without altering base-line contractility. These results provide the first evidence that cardiac beta(2)-AR activates p38 MAPK via a PKA-dependent signaling pathway, rather than by G(i) or Gbetagamma, and reveal a novel role of p38 MAPK in regulating cardiac contractility.

Spontaneous activation of beta(2)- but not beta(1)-adrenoceptors expressed in cardiac myocytes from beta(1)beta(2) double knockout mice.

Although ligand-free, constitutive beta(2)-adrenergic receptor (AR) signaling has been demonstrated in naive cell lines and in transgenic mice overexpressing cardiac beta(2)-AR, it is unclear whether the dominant cardiac beta-AR subtype, beta(1)-AR, shares the ability of spontaneous activation. In the present study, we expressed human beta(1)- or beta(2)-AR via recombinant adenoviral infection in ventricular myocytes isolated from beta(1)beta(2)-AR double knockout mice, creating pure beta(1)-AR and beta(2)-AR systems with variable receptor densities. A contractile response to a nonselective beta-AR agonist, isoproterenol, was absent in double knockout mouse myocytes but was fully restored after adenoviral beta(1)-AR or adenoviral beta(2)-AR infection. Increasing the titer of adenoviral vectors (multiplicity of infection 10-1000) led to a dose-dependent expression of beta(1)- or beta(2)-AR with a maximal density of 1207 +/- 173 (36-fold over the wild-type control value) and 821+/-38 fmol/mg protein (69-fold), respectively. Using confocal immunohistochemistry, we directly visualized the cellular distribution of beta(1)-AR and beta(2)-AR and found that both subtypes were distributed on the cell surface membrane and transverse tubules, resulting in a striated pattern. In the absence of ligand, beta(2)-AR expression resulted in graded increases in baseline cAMP and contractility up to 428% and 233% of control, respectively, at the maximal beta(2)-AR density. These effects were specifically reversed by a beta(2)-AR inverse agonist, ICI 118,551 (10(-7) M). In contrast, overexpression of beta(1)-AR, even at a greater density, failed to enhance either basal cAMP or contractility; the alleged beta(1)-AR inverse agonist, CGP 20712A (10(-6) M), had no significant effect on basal contraction in these cells. Thus, we conclude that acute beta(2)-AR overexpression in cardiac myocytes elicits significant physiological responses due to spontaneous receptor activation; however, this property is beta-AR subtype specific because beta(1)-AR does not exhibit agonist-independent spontaneous activation.

Culture and adenoviral infection of adult mouse cardiac myocytes: methods for cellular genetic physiology.

Rapid development of transgenic and gene-targeted mice and acute genetic manipulation via gene transfer vector systems have provided powerful tools for cardiovascular research. To facilitate the phenotyping of genetically engineered murine models at the cellular and subcellular levels and to implement acute gene transfer techniques in single mouse cardiomyocytes, we have modified and improved current enzymatic methods to isolate a high yield of high-quality adult mouse myocytes (5.3 +/- 0.5 x 10(5) cells/left ventricle, 83.8 +/- 2.5% rod shaped). We have also developed a technique to culture these isolated myocytes while maintaining their morphological integrity for 2-3 days. The high percentage of viable myocytes after 1 day in culture (72.5 +/- 2.3%) permitted both physiological and biochemical characterization. The major functional aspects of these cells, including excitation-contraction coupling and receptor-mediated signaling, remained intact, but the contraction kinetics were significantly slowed. Furthermore, gene delivery via recombinant adenoviral infection was highly efficient and reproducible. In adult beta(1)/beta(2)-adrenergic receptor (AR) double-knockout mouse myocytes, adenovirus-directed expression of either beta(1)- or beta(2)-AR, which occurred in 100% of cells, rescued the functional response to beta-AR agonist stimulation. These techniques will permit novel experimental settings for cellular genetic physiology.

The effect of pH on beta(2) adrenoceptor function. Evidence for protonation-dependent activation.

The transition of rhodopsin from the inactive to the active state is associated with proton uptake at Glu(134) (1), and recent mutagenesis studies suggest that protonation of the homologous amino acid in the alpha(1B) adrenergic receptor (Asp(142)) may be involved in its mechanism of activation (2). To further explore the role of protonation in G protein-coupled receptor activation, we examined the effects of pH on the rate of ligand-induced conformational change and on receptor-mediated G protein activation for the beta(2) adrenergic receptor (beta(2)AR). The rate of agonist-induced change in the fluorescence of NBD-labeled, purified beta(2)AR was 2-fold greater at pH 6.5 than at pH 8, even though agonist affinity was lower at pH 6.5. This biophysical analysis was corroborated by functional studies; basal (agonist-independent) activation of Galpha(s) by the beta(2)AR was greater at pH 6.5 compared with pH 8.0. Taken together, these results provide evidence that protonation increases basal activity by destabilizing the inactive state of the receptor. In addition, we found that the pH sensitivity of beta(2)AR activation is not abrogated by mutation of Asp(130), which is homologous to the highly conserved acidic amino acids that link protonation to activation of rhodopsin (Glu(134)) and the alpha(1B) adrenergic receptor (Asp(142)).

Antinociceptive action of nitrous oxide is mediated by stimulation of noradrenergic neurons in the brainstem and activation of [alpha]2B adrenoceptors.

Although nitrous oxide (N(2)O) has been used to facilitate surgery for >150 years, its molecular mechanism of action is not yet defined. Having established that N(2)O-induced release of norepinephrine mediates the analgesic action at alpha(2) adrenoceptors in the spinal cord, we now investigated whether activation of noradrenergic nuclei in the brainstem is responsible for this analgesic action and which alpha(2) adrenoceptor subtype mediates this property. In rats, Fos immunoreactivity was examined in brainstem noradrenergic nuclei after exposure to nitrous oxide. After selective lesioning of noradrenergic nuclei by intracerebroventricular application of the mitochondrial toxin saporin, coupled to the antibody directed against dopamine beta hydroxylase (DbetaH-saporin), the analgesic and sedative actions of N(2)O were determined. Null mice for each of the three alpha(2) adrenoceptor subtypes (alpha(2A), alpha(2B), and alpha(2C)), and their wild-type cohorts, were tested for their antinociceptive and sedative response to N(2)O. Exposure to N(2)O increased expression of Fos immunoreactivity in each of the pontine noradrenergic nuclei (A5, locus coeruleus, and A7). DbetaH-saporin treatment eliminated nearly all of the catecholamine-containing neurons in the pons and blocked the analgesic but not the sedative effects of N(2)O. Null mice for the alpha(2B) adrenoceptor subtype exhibited a reduced or absent analgesic response to N(2)O, but their sedative response to N(2)O was intact. Our results support a pivotal role for noradrenergic pontine nuclei and alpha(2B) adrenoceptors in the analgesic, but not the sedative effects of N(2)O. Previously we demonstrated that the analgesic actions of alpha(2) adrenoceptor agonists are mediated by the alpha(2A) subtype; taken together with these data we propose that exogenous and endogenous alpha(2) adrenoceptor ligands activate different alpha(2) adrenoceptor subtypes to produce their analgesic action.

Genetic alteration of the alpha2-adrenoceptor subtype c in mice affects the development of behavioral despair and stress-induced increases in plasma corticosterone levels.

alpha2-Adrenoceptors (alpha2-AR) modulate many central nervous system functions, such as regulation of sympathetic tone, vigilance, attention, and reactivity to environmental stressors. Three alpha2-AR subtypes (alpha2A, alpha2B, and alpha2C) with distinct tissue-distribution patterns are known to exist, but the functional significance of each subtype is not clear. Since specific, alpha2-AR subtype-selective pharmacological probes are not available, mice with genetically altered alpha2C-AR expression were studied in order to investigate the possible involvement of the alpha2C-AR in physiological and behavioral responses to acute and repeated stress. A modified version of Porsolt's forced swimming test was used to assess the possible effects of altered alpha2C-AR expression on the development of behavioral despair. alpha2C-Overexpression increased and the lack of alpha2C-AR (alpha2C-KO) decreased the immobility of mice in the forced swimming test, ie alpha2C-AR expression appeared to promote the development of behavioral despair. In addition, alpha2C-KO was associated with attenuated elevation of plasma corticosterone after different stressors, and overexpression of alpha2C-ARs was linked with increased corticosterone levels after repeated stress. Moreover, the brain dopamine and serotonin balance, but not norepinephrine turnover, was dependent on alpha2C-AR expression, and the expression of c-fos and junB mRNA was increased in alpha2C-KO mice. Since alpha2C-KO produced stress-protective effects, and alpha2C-AR overexpression seemed to promote the development of changes related to depression, it is suggested that a yet-to-be developed subtype-selective alpha2C-AR antagonist might have therapeutic value in the treatment of stress-related neuropsychiatric disorders.

Restricting the mobility of Gs alpha: impact on receptor and effector coupling.

The alpha-subunit of the stimulatory G protein, Gs, has been shown to dissociate from the plasma membrane into the cytosol following activation by G protein-coupled receptors (GPCR) in some experimental systems. This dissociation may involve depalmitoylation of an amino-terminal cysteine residue. However, the functional significance of this dissociation is not known. To investigate the functional consequence of Gs alpha dissociation, we constructed a membrane-tethered Gs alpha (tetGs alpha), expressed it in Sf9 insect cells, and examined its ability to couple with the beta(2) adrenoceptor and to activate adenylyl cyclase. Compared to wild-type Gs alpha, tetGs alpha coupled much more efficiently to the beta 2 adrenoceptor and the D1 dopamine receptor as determined by agonist-stimulated GTP gamma S binding and GTPase activity. The high coupling efficiency was abolished when Gs )alpha was proteolytically cleaved from the membrane tether. The membrane tether did not prevent the coupling of tetGS alpha to adenylyl cyclase. These results demonstrate that regulating the mobility of Gs alpha relative to the plasma membrane, through fatty acylation or perhaps interactions with cytoskeletal proteins, could have a significant impact on receptor-G protein coupling. Furthermore, by enabling the use of more direct measures of receptor-G protein coupling (GTPase activity, GTP gamma S binding), tetGS alpha can facilitate the study for receptor-G protein interactions.

GPCR-Galpha fusion proteins: molecular analysis of receptor-G-protein coupling.

The efficiency of interactions between G-protein-coupled receptors (GPCRs) and heterotrimeric guanine nucleotide-binding proteins (G proteins) is greatly influenced by the absolute and relative densities of these proteins in the plasma membrane. The study of these interactions has been facilitated by the use of GPCR-Galpha fusion proteins, which are formed by the fusion of GPCR to Galpha. These fusion proteins ensure a defined 1:1 stoichiometry of GPCR to Galpha and force the physical proximity of the signalling partners. Thus, fusion of GPCR to Galpha enhances coupling efficiency can be used to study aspects of receptor-G-protein coupling that could not otherwise be examined by co-expressing GPCRs and G proteins as separate proteins. The results of studies that have made use of GPCR-Galpha fusion proteins will be discussed in this article, along with the strengths and limitations of this approach.

Gene substitution/knockout to delineate the role of alpha 2-adrenoceptor subtypes in mediating central effects of catecholamines and imidazolines.

Adrenergic receptors form the interface between the sympathetic nervous system and the cardiovascular system as well as many endocrine and parenchymal tissues. For the three alpha 2-adrenergic receptors (alpha 2A, alpha 2B, and alpha 2C), genetic mouse models have been developed that can be used to elucidate the physiologic function of each receptor subtype in vivo. Different strategies for homologous recombination in embryonic stem cells were applied to generate lines of mice with gene knockouts of the individual alpha 2-receptor subtypes (alpha 2A-KO, alpha 2B-KO, and alpha 2C-KO) or with a substitution of a mutant receptor at the wild-type locus (alpha 2-D79N). In these transgenic mice, the cardiovascular effects of alpha 2-agonists and imidazoline receptor agonists were tested. Stimulation of alpha 2B receptors in vascular smooth muscle produces hypertension and counteracts the clinically beneficial hypotensive effect of stimulating alpha 2A receptors in the central nervous system.

Effects of guanine, inosine, and xanthine nucleotides on beta(2)-adrenergic receptor/G(s) interactions: evidence for multiple receptor conformations.

The aim of our study was to examine the effects of different purine nucleotides [GTP, ITP, and xanthosine 5'-triphosphate (XTP)] on receptor/G protein coupling. As a model system, we used a fusion protein of the beta(2)-adrenergic receptor and the alpha subunit of the G protein G(s). GTP was more potent and efficient than ITP and XTP at inhibiting ternary complex formation and supporting adenylyl cyclase (AC) activation. We also studied the effects of several beta(2)-adrenergic receptor ligands on nucleotide hydrolysis and on AC activity in the presence of GTP, ITP, and XTP. The efficacy of agonists at promoting GTP hydrolysis correlated well with the efficacy of agonists for stimulating AC in the presence of GTP. This was, however, not the case for ITP hydrolysis and AC activity in the presence of ITP. The efficacy of ligands at stimulating AC in the presence of XTP differed considerably from the efficacies of ligands in the presence of GTP and ITP, and there was no evidence for receptor-regulated XTP hydrolysis. Our findings support the concept of multiple ligand-specific receptor conformations and demonstrate the usefulness of purine nucleotides as tools to study conformational states of receptors.