The putative beta4-adrenergic receptor is a novel state of the beta1-adrenergic receptor

The atypical beta3-adrenergic receptor (AR) agonist CGP-12177 has been used to define a novel atypical beta-AR subtype, the putative beta4-AR. Recent evaluation of recombinant beta-AR subtypes and beta-AR-deficient mice, however, has established the identity of the pharmacological beta4-AR as a novel state of the beta1-AR protein. The ability of aryloxypropanolamine ligands like CGP-12177 to independently interact with agonist and antagonist states of the beta1-AR has important implications regarding receptor classification and the potential development of tissue-specific beta-AR agonists.     

Cross-regulation between G-protein-mediated pathways. Acute activation of the inhibitory pathway of adenylylcyclase reduces beta 2-adrenergic receptor phosphorylation and increases beta-adrenergic responsiveness.

Cross-regulation from the stimulatory to the inhibitory adenylylcyclase pathways has been described (Hadcock, J. R., Ros, M., Watkins, D. C., and Malbon, C. C. (1990) J. Biol. Chem. 265, 14784-14790). More recently, persistent activation (48 h) of the inhibitory adenylylcyclase pathway has been shown to cross-regulate the stimulatory pathway (i) enhancing the maximal response of beta-adrenergic agonits, (ii) increasing the expression of beta-adrenergic receptor, and (iii) reducing the ED50 for the isoproterenol-stimulated response by 50-fold (Hadcock, J. R., Port, J. D., and Malbon, C. C. (1991) J. Biol. Chem. 266, 11915-11922). Here, we report that short term activation (60 min) of the inhibitory adenylylcyclase pathway of hamster smooth muscle DDT1MF-2 cells with the A1-adenosine receptor agonist N6-phenylisopropyladenosine (PIA) likewise enhances the stimulatory adenylylcyclase response to the beta-adrenergic agonist isoproterenol. The PIA effect was exerted at the level of the receptor, i.e., the beta-adrenergic receptor-mediated response was enhanced, whereas the guanosine 5'-O-(thiotriphosphate)- and forskolin-stimulated adenylylcyclase activities were largely unaffected. In contrast to longer term persistent activation of the inhibitory pathway, receptor number and affinity for 125I-labeled cyanopindolol were unaffected. Metabolic labeling of cells with [32P]orthophosphate and immuneprecipitation of beta-adrenergic receptors detected phosphorylation of the receptor in unstimulated cells and marked phosphorylation in cells challenged with epinephrine. When cells were challenged short term with PIA, the basal state of beta-adrenergic receptor phosphorylation was reduced by 75%. Treating cells with PIA in combination with the cAMP analog 8-(4-chlorophenylthio)adenosine cyclic AMP attenuated the enhanced receptor-mediated adenylylcyclase response observed in cells treated with PIA alone. These data suggest that short term cross-regulation from the inhibitory to stimulatory adenylylcyclase pathways results in the following: (i) decreased intracellular cAMP levels and protein kinase A activity, (ii) reduced phosphorylation of the beta 2-adrenergic receptor in the "basal" (i.e. unstimulated) state, and (iii) enhanced receptor-mediated activation of Gs.     

Genetic variation of the beta2-adrenergic receptor is associated with differences in lung fluid accumulation in humans.

The beta2-adrenergic receptors (beta2AR) play an important role in lung fluid regulation. Previous research has suggested that subjects homozygous for arginine at amino acid 16 of the beta2AR (Arg16) may have attenuated receptor function relative to subjects homozygous for glycine at the same amino acid (Gly16). We sought to determine if the Arg16Gly polymorphism of the beta2AR influenced lung fluid balance in response to rapid saline infusion. We hypothesized that subjects homozygous for Arg at amino acid 16 (n=14) would have greater lung fluid accumulation compared with those homozygous for Gly (n=15) following a rapid intravenous infusion of isotonic saline (30 ml/kg over 17 min). Changes in lung fluid were determined using measures of lung density and tissue volume (computerized tomography imaging) and measures of pulmonary capillary blood volume (Vc) and alveolar-capillary conductance (DM, determined from the simultaneous assessment of the diffusing capacities of the lungs for carbon monoxide and nitric oxide). The saline infusion resulted in elevated catecholamines in both genotype groups (Arg16 283+/-117% vs. Gly16 252+/-118%, P>0.05). The Arg16 group had a larger decrease in DM and increase in lung tissue volume and lung water after saline infusion relative to the Gly16 group (DM -13+/-14 vs. 0+/-26%, P<0.05; lung tissue volume 13+/-11 vs. 3+/-11% and lung water +90+/-66 vs. +48+/-144 ml, P=0.10, P<0.05, for Arg vs. Gly16, respectively, means+/-SD). These data suggest that subjects homozygous for Arg at amino acid 16 of the beta2AR have a greater susceptibility for lung fluid accumulation relative to subjects homozygous for Gly at this position.     

Functional receptor coupling to Gi is a mechanism of agonist-promoted desensitization of the beta2-adrenergic receptor

The beta2-adrenergic receptor (beta2AR) couples to Gs activating adenylyl cyclase (AC) and increasing cAMP. Such signaling undergoes desensitization with continued agonist exposure. Beta2AR also couple to Gi after receptor phosphorylation by the cAMP dependent protein kinase A, but the efficiency of such coupling is not known. Given the PKA dependence of beta2AR-Gi coupling, we explored whether this may be a mechanism of agonist-promoted desensitization. HEK293 cells were transfected to express beta2AR or beta2AR and Gialpha2, and then treated with vehicle or the agonist isoproterenol to evoke agonist-promoted beta2AR desensitization. Membrane AC activities showed that Gialpha2 overexpression decreased basal levels, but the fold-stimulation of the AC over basal by agonist was not altered. However, with treatment of the cells with isoproterenol prior to membrane preparation, a marked decrease in agonist-stimulated AC was observed with the cells overexpressing Gialpha2. In the absence of such overexpression, beta2AR desensitization was 23+/-7%, while with 5-fold Gialpha2 overexpression desensitization was 58+/-5% (p<0.01, n=4). The effect of Gi on desensitization was receptor-specific, in that forskolin responses were not altered by G(i)alpha2 overexpression. Thus, acquired beta2AR coupling to Gi is an important mechanism of agonist-promoted desensitization, and pathologic conditions that increase Gi levels contribute to beta2AR dysfunction.     

Hierarchy of polymorphic variation and desensitization permutations relative to beta 1- and beta 2-adrenergic receptor signaling

Agonist-promoted desensitization of G-protein-coupled receptors results in partial uncoupling of receptor from cognate G-protein, a process that provides for rapid adaptation to the signaling environment. This property plays important roles in physiologic and pathologic processes as well as therapeutic efficacy. However, coupling is also influenced by polymorphic variation, but the relative impact of these two mechanisms on signal transduction is not known. To determine this we utilized recombinant cells expressing the human beta(1)-adrenergic receptor (beta(1)AR) or a gain-of-function polymorphic variant (beta(1)AR-Arg(389)), and the beta(2)-adrenergic receptor (beta(2)AR) or a loss-of-function polymorphic receptor (beta(2)AR-Ile(164)). Adenylyl cyclase activities were determined with multiple permutations of the possible states of the receptor: genotype, basal, or agonist stimulated and with or without agonist pre-exposure. For the beta(1)AR, the enhanced function of the Arg(389) receptor underwent less agonist-promoted desensitization compared with its allelic counterpart. Indeed, the effect of polymorphic variation on absolute adenylyl cyclase activities was such that desensitized beta(1)AR-Arg(389) signaling was equivalent to non-desensitized wild-type beta(1)AR; that is, the genetic component had as much impact as desensitization on receptor coupling. In contrast, the enhanced signaling of wild-type beta(2)AR underwent less desensitization compared with beta(2)AR-Ile(164), thus the heterogeneity in absolute signaling was markedly broadened by this polymorphism. Inverse agonist function was not affected by polymorphisms of either subtype. A general model is proposed whereby up to 10 levels of signaling by G-protein-coupled receptors can be present based on the influences of desensitization and genetic variation on coupling.     

Agonist-dependent phosphorylation of the alpha 2-adrenergic receptor by the beta-adrenergic receptor kinase

Desensitization of the beta-adrenergic receptor, a receptor which is coupled to the stimulation of adenylate cyclase, may be regulated via phosphorylation by a unique protein kinase. This recently discovered enzyme, known as the beta-adrenergic receptor kinase, only phosphorylates the agonist-occupied form of the beta-adrenergic receptor. To assess whether receptors coupled to the inhibition of adenylate cyclase might also be substrates, we examined the effects of beta-adrenergic receptor kinase on the partially purified human platelet alpha 2-adrenergic receptor. Phosphorylation of the reconstituted alpha 2-adrenergic receptor was dependent on agonist occupancy and was completely blocked by coincubation with alpha 2-antagonists. The time course of phosphorylation of the alpha 2-adrenergic receptor was virtually identical to that observed with the beta-adrenergic receptor with maximum stoichiometries of 7-8 mol of phosphate/mol of receptor in each case. In contrast, the alpha 1-adrenergic receptor, which is coupled to stimulation of phosphatidylinositol hydrolysis, is not a substrate for the beta-adrenergic receptor kinase. These results suggest that receptors coupled to either stimulation or inhibition of adenylate cyclase may be regulated by an agonist-dependent phosphorylation mediated by the beta-adrenergic receptor kinase.     

Light-driven activation of beta 2-adrenergic receptor signaling by a chimeric rhodopsin containing the beta 2-adrenergic receptor cytoplasmic loops

Structure-function studies of rhodopsin indicate that both intradiscal and transmembrane (TM) domains are required for retinal binding and subsequent light-induced structural changes in the cytoplasmic domain. Further, a hypothesis involving a common mechanism for activation of G-protein-coupled receptor (GPCR) has been proposed. To test this hypothesis, chimeric receptors were required in which the cytoplasmic domains of rhodopsin were replaced with those of the beta(2)-adrenergic receptor (beta(2)-AR). Their preparation required identification of the boundaries between the TM domain of rhodopsin and the cytoplasmic domain of the beta(2)-AR necessary for formation of the rhodopsin chromophore and its activation by light and subsequent optimal activation of beta(2)-AR signaling. Chimeric receptors were constructed in which the cytoplasmic loops of rhodopsin were replaced one at a time and in combination. In these replacements, size of the third cytoplasmic (EF) loop critically determined the extent of chromophore formation, its stability, and subsequent signal transduction specificity. All the EF loop replacements showed significant decreases in transducin activation, while only minor effects were observed by replacements of the CD and AB loops. Light-dependent activation of beta(2)-AR leading to Galphas signaling was observed only for the EF2 chimera, and its activation was further enhanced by replacements of the other loops. The results demonstrate coupling between light-induced conformational changes occurring in the transmembrane domain of rhodopsin and the cytoplasmic domain of the beta(2)-AR.     

Downregulation of caveolin by chronic beta -adrenergic receptor stimulation in mice

Caveolae, flask-shaped invaginations of cell membranes, arebelieved to play pivotal roles in transmembrane transportation ofmolecules and cellular signaling. Caveolin, a structural component ofcaveolae, interacts directly with G proteins and regulates theirfunction. We investigated the effect of chronic -adrenergic receptorstimulation on the expression of caveolin subtypes in mouse hearts byimmunoblotting and Northern blotting. Caveolin-1 and -3 were abundantlyexpressed in the heart and skeletal muscles, but not in the brain.Continuous ()-isoproterenol, but not (+)-isoproterenol, infusionvia osmotic minipump (30 µg · g¹ · day¹)for 13 days significantly downregulated both caveolin subtypes in theheart. The expression of caveolin-1 was reduced by 48 ± 6.1% andthat of caveolin-3 by 28 ± 4.0%(P < 0.01, n = 8 for each). The subcellulardistribution of caveolin subtypes in ventricular myocardium was notaltered as determined by sucrose gradient fractionation. In contrast,the expression of both caveolin subtypes in skeletal muscles was notsignificantly changed. Our data suggest that the expression of caveolinsubtypes is regulated by -adrenergic receptor stimulation in theheart.

     

Arg16/Gly beta2-adrenergic receptor polymorphism alters the cardiac output response to isometric exercise.

Normotensive adults homozygous for glycine (Gly) of the Arg16/Gly beta2-adrenergic-receptor polymorphism have 1) greater forearm beta2-receptor mediated vasodilation and 2) a higher heart rate (HR) response to isometric handgrip than arginine (Arg) homozygotes. To test the hypothesis that the higher HR response in Gly16 subjects serves to maintain the pressor response [increased cardiac output (CO)] in the setting of augmented peripheral vasodilation to endogenous catecholamines, we measured continuous HR (ECG), arterial pressure (Finapres), and CO (transthoracic echocardiography) during isometric, 40% submaximal handgrip to fatigue in healthy subjects homozygous for Gly (n = 30; mean age +/- SE: 30 +/- 1.2, 13 women) and Arg (n = 17, age 30 +/- 1.6, 11 women). Resting data were similar between groups. Handgrip produced similar increases in arterial pressure and venous norepinephrine and epinephrine concentrations; however, HR increased more in the Gly group (60.1 +/- 4.3% increase from baseline vs. 45.5 +/- 3.9%, P = 0.03), and this caused CO to be higher (Gly: 7.6 +/- 0.3 l/m vs. Arg: 6.5 +/- 0.3 l/m, P = 0.03), whereas the decrease in systemic vascular resistance in the Gly group did not reach significance (P = 0.09). We conclude that Gly16 homozygotes generate a higher CO to maintain the pressor response to handgrip. The influence of polymorphic variants in the beta2-adrenergic receptor gene on the cardiovascular response to sympathoexcitation may have important implications in the development of hypertension and heart failure.     

Beta-arrestin binding to the beta2-adrenergic receptor requires both receptor phosphorylation and receptor activation

Homologous desensitization of beta2-adrenergic receptors has been shown to be mediated by phosphorylation of the agonist-stimulated receptor by G-protein-coupled receptor kinase 2 (GRK2) followed by binding of beta-arrestins to the phosphorylated receptor. Binding of beta-arrestin to the receptor is a prerequisite for subsequent receptor desensitization, internalization via clathrin-coated pits, and the initiation of alternative signaling pathways. In this study we have investigated the interactions between receptors and beta-arrestin2 in living cells using fluorescence resonance energy transfer. We show that (a) the initial kinetics of beta-arrestin2 binding to the receptor is limited by the kinetics of GRK2-mediated receptor phosphorylation; (b) repeated stimulation leads to the accumulation of GRK2-phosphorylated receptor, which can bind beta-arrestin2 very rapidly; and (c) the interaction of beta-arrestin2 with the receptor depends on the activation of the receptor by agonist because agonist withdrawal leads to swift dissociation of the receptor-beta-arrestin2 complex. This fast agonist-controlled association and dissociation of beta-arrestins from prephosphorylated receptors should permit rapid control of receptor sensitivity in repeatedly stimulated cells such as neurons.     

Mutation of tyrosine-141 inhibits insulin-promoted tyrosine phosphorylation and increased responsiveness of the human beta 2-adrenergic receptor.

The ability of insulin to promote phosphorylation of the human beta 2-adrenergic receptor (beta 2AR) was assessed in Chinese hamster fibroblasts transfected with beta 2AR cDNA. Phosphotyrosine residues were detected in purified beta 2AR using a polyclonal anti-phosphotyrosine antibody and by phosphoamino acid analysis following metabolic labelling with inorganic 32P. Treatment of the cells with insulin induced a 2.4-fold increase in the phosphotyrosine content of the receptor. The insulin-promoted phosphorylation of the beta 2AR was accompanied by an increase in the beta-adrenergic-stimulated adenyl cyclase activity. Substitution of a phenylalanine residue for tyrosine-141 completely prevented both the increased tyrosine phosphorylation and the enhanced responsiveness of the beta 2AR promoted by insulin treatment. Mutation of three other tyrosines located in the cytoplasmic domain of the receptor, tyrosine-366, tyrosine-350 and tyrosine-354, did not abolish the insulin-promoted tyrosine phosphorylation. Taken together, these results suggest that insulin promotes phosphorylation of the beta 2AR on tyrosine-141 and that such phosphorylation leads to a supersensitization of the receptor.     

Plasmon-waveguide resonance studies of ligand binding to the human beta 2-adrenergic receptor

Plasmon-waveguide resonance (PWR) spectroscopy is an optical technique that can be used to probe the molecular interactions occurring within anisotropic proteolipid membranes in real time without requiring molecular labeling. This method directly monitors mass density, conformation, and molecular orientation changes occurring in such systems and allows determination of protein-ligand binding constants and binding kinetics. In the present study, PWR has been used to monitor the incorporation of the human beta(2)-adrenergic receptor into a solid-supported egg phosphatidylcholine lipid bilayer and to follow the binding of full agonists (isoproterenol, epinephrine), a partial agonist (dobutamine), an antagonist (alprenolol), and an inverse agonist (ICI-118,551) to the receptor. The combination of differences in binding kinetics and the PWR spectral changes point to the occurrence of multiple conformations that are characteristic of the type of ligand, reflecting differences in the receptor structural states produced by the binding process. These results provide new evidence for the conformational heterogeneity of the liganded states formed by the beta(2)-adrenergic receptor.     

Live cell imaging of Gs and the beta2-adrenergic receptor demonstrates that both alphas and beta1gamma7 internalize upon stimulation and exhibit similar trafficking patterns that differ from that of the beta2-adrenergic receptor

To visualize and investigate the regulation of the localization patterns of Gs and an associated receptor during cell signaling, we produced functional fluorescent fusion proteins and imaged them in HEK-293 cells. alphas-CFP, with cyan fluorescent protein (CFP) inserted into an internal loop of alphas, localized to the plasma membrane and exhibited similar receptor-mediated activity to that of alphas. Functional fluorescent beta1gamma7 dimers were produced by fusing an amino-terminal yellow fluorescent protein (YFP) fragment to beta1 (YFP-N-beta1) and a carboxyl-terminal YFP fragment to gamma7 (YFP-C-gamma7). When expressed together, YFP-N-beta1 and YFP-C-gamma7 produced fluorescent signals in the plasma membrane that were not seen when the subunits were expressed separately. Isoproterenol stimulation of cells co-expressing alphas-CFP, YFP-N-beta1/YFP-C-gamma7, and the beta2-adrenergic receptor (beta2AR) resulted in internalization of both fluorescent signals from the plasma membrane. Initially, alphas-CFP and YFP-N-beta1/YFP-C-gamma7 stained the cytoplasm diffusely, and subsequently they co-localized on vesicles that exhibited minimal overlap with beta2AR-labeled vesicles. Moreover, internalization of beta2AR-GFP, but not alphas-CFP or YFP-N-beta1/YFP-C-gamma7, was inhibited by a fluorescent dominant negative dynamin 1 mutant, Dyn1(K44A)-mRFP, indicating that the Gs subunits and beta2AR utilize different internalization mechanisms. Subsequent trafficking of the Gs subunits and beta2AR also differed in that vesicles labeled with the Gs subunits exhibited less overlap with RhoB-labeled endosomes and greater overlap with Rab11-labeled endosomes. Because Rab11 regulates traffic through recycling endosomes, co-localization of alphas and beta1gamma7 on these endosomes may indicate a means of recycling specific alphasbetagamma combinations to the plasma membrane.     

Decreased maximal heart rate with aging is related to reduced {beta}-adrenergic responsiveness but is largely explained by a reduction in intrinsic heart rate.

A decrease in maximal exercise heart rate (HR(max)) is a key contributor to reductions in aerobic exercise capacity with aging. However, the mechanisms involved are incompletely understood. We sought to gain insight into the respective roles of intrinsic heart rate (HR(int)) and chronotropic beta-adrenergic responsiveness in the reductions in HR(max) with aging in healthy adults. HR(max) (Balke treadmill protocol to exhaustion), HR(int) (HR during acute ganglionic blockade with intravenous trimethaphan), and chronotropic beta-adrenergic responsiveness (increase in HR with incremental intravenous infusion of isoproterenol during ganglionic blockade) were determined in 15 older (65 +/- 5 yr) and 15 young (25 +/- 4 yr) healthy men. In the older men, HR(max) was lower (162 +/- 9 vs. 191 +/- 11 beats/min, P < 0.0001) and was associated with a lower HR(int) (58 +/- 7 vs. 83 +/- 9 beats/min, P < 0.0001) and chronotropic beta-adrenergic responsiveness (0.094 +/- 0.036 vs. 0.154 +/- 0.045 DeltaHR/[isoproterenol]: P < 0.0001). Both HR(int) (r = 0.87, P < 0.0001) and chronotropic beta-adrenergic responsiveness (r = 0.61, P < 0.0001) were positively related to HR(max). Accounting for the effects of HR(int) and chronotropic beta-adrenergic responsiveness reduced the age-related difference in HR(max) by 83%, rendering it statistically nonsignificant (P = 0.2). Maximal oxygen consumption was lower in the older men (34.9 +/- 8.1 vs. 48.6 +/- 6.7 ml x kg(-1) x min(-1), P < 0.0001) and was positively related to HR(max) (r = 0.62, P < 0.0001), HR(int) (r = 0.51, P = 0.002), and chronotropic beta-adrenergic responsiveness (r = 0.47, P = 0.005). Our findings indicate that, together, reductions in HR(int) and chronotropic responsiveness to beta-adrenergic stimulation largely explain decreases in HR(max) with aging, with the reduction in HR(int) playing by far the greatest role.     

Functions of beta2-adrenergic receptor in human periodontal ligament cells

Adrenergic receptors (ARs) are receptors of noradrenalin and adrenalin, of which there are nine different subtypes. In particular, β2 adrenergic receptor (β2-AR) is known to be related to the restoration and maintenance of homeostasis in bone and cardiac tissues; however, the functional role of signaling through β2-AR in periodontal ligament (PDL) tissue has not been fully examined. In this report, we investigated that β2-AR expression in PDL tissues and their features in PDL cells. β2-AR expressed in rat PDL tissues and human PDL cells (HPDLCs) derived from two different patients (HPDLCs-2G and -3S). Rat PDL tissue with occlusal loading showed high β2-AR expression, while its expression was downregulated in that without loading. In HPDLCs, β2-AR expression was increased exposed to stretch loading. The gene expression of PDL-related molecules was investigated in PDL clone cells (2-23 cells) overexpressing β2-AR. Their gene expression and intracellular cyclic adenosine monophosphate (cAMP) levels were also investigated in HPDLCs treated with a specific β2-AR agonist, fenoterol (FEN). Overexpression of β2-AR significantly promoted the gene expression of PDL-related molecules in 2 to 23 cells. FEN led to an upregulation in the expression of PDL-related molecules and increased intracellular cAMP levels in HPDLCs. In both HPDLCs, inhibition of cAMP signaling by using protein kinase A inhibitor suppressed the FEN-induced gene expression of α-smooth muscle actin. Our findings suggest that the occlusal force is important for β2-AR expression in PDL tissue and β2-AR is involved in fibroblastic differentiation and collagen synthesis of PDL cells. The signaling through β2-AR might be important for restoration and homeostasis of PDL tissue.     

Phorbol-ester-induced phosphorylation of the beta 2-adrenergic receptor decreases its coupling to Gs.

Phorbol-esters have been shown to modulate the beta-adrenergic-stimulated adenylyl cyclase in a number of cell lines. Here, using site directed mutagenesis, we investigate the role of the beta 2-adrenergic receptor phosphorylation by protein kinase C in this regulatory process. Mutation of the serine-261, -262, -344 and -345 of the beta 2-adrenergic receptor prevented the phorbol-ester-induced phosphorylation of the receptor. This mutation also abolished the phorbol-ester-induced decrease in high-affinity agonist binding and potency of the beta 2-adrenergic receptor. We suggest that protein kinase C mediated phosphorylation of the receptor promotes its functional uncoupling.     

Toward the active conformations of rhodopsin and the beta2-adrenergic receptor

Using sets of experimental distance restraints, which characterize active or inactive receptor conformations, and the X-ray crystal structure of the inactive form of bovine rhodopsin as a starting point, we have constructed models of both the active and inactive forms of rhodopsin and the beta2-adrenergic G-protein coupled receptors (GPCRs). The distance restraints were obtained from published data for site-directed crosslinking, engineered zinc binding, site-directed spin-labeling, IR spectroscopy, and cysteine accessibility studies conducted on class A GPCRs. Molecular dynamics simulations in the presence of either "active" or "inactive" restraints were used to generate two distinguishable receptor models. The process for generating the inactive and active models was validated by the hit rates, yields, and enrichment factors determined for the selection of antagonists in the inactive model and for the selection of agonists in the active model from a set of nonadrenergic GPCR drug-like ligands in a virtual screen using ligand docking software. The simulation results provide new insights into the relationships observed between selected biochemical data, the crystal structure of rhodopsin, and the structural rearrangements that occur during activation.