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.     

PKA-mediated phosphorylation of the beta1-adrenergic receptor promotes Gs/Gi switching

Recently, it has been shown that PKA-mediated phosphorylation of the β₂-adrenergic receptor (β₂-AR) by the cyclic AMP-dependent protein kinase (PKA) reduces its affinity for G_s and increases its affinity for G_i. Here we demonstrate that, like the β₂-AR, the β₁-AR is also capable of “switching” its coupling from G_s to G_i in a PKA-dependent manner. The β₁-AR is capable of activating adenylate cyclase via G_s, and can also activate the extracellular-regulated kinases, p44 and p42 (ERK1/2). In transfected CHO cells, the observed β₁-AR-mediated activation of ERK is both sensitive to pertussis toxin (PTX), indicating involvement of G_i/G_o, and to the PKA inhibitor, H-89. β₁-ARs with PKA phosphorylation sites mutated to alanines are unable to activate ERK. Mutating these same residues to aspartic acid, mimicking PKA phosphorylation, leads to a decrease in G_s-stimulated cAMP accumulation and an increase in PTX-sensitive ERK activation. These results strongly support the hypothesis that the β₁-AR, like the β₂-AR, can undergo PKA-dependent “G_s/G_i switching”.     

Beta 1/beta 2-adrenergic receptor heterodimerization regulates beta 2-adrenergic receptor internalization and ERK signaling efficacy

Beta(1)- and beta(2)-adrenergic receptors (beta(1)AR and beta(2)AR) are co-expressed in numerous tissues where they play a central role in the responses of various organs to sympathetic stimulation. Although the two receptor subtypes share some signaling pathways, each has been shown to have specific signaling and regulatory properties. Given the recent recognition that many G protein-coupled receptors can form homo- and heterodimers, the present study was undertaken to determine whether the beta(1)AR and beta(2)AR can form dimers in cells and, if so, to investigate the potential functional consequences of such heterodimerization. Using co-immunoprecipitation and bioluminescence resonance energy transfer, we show that beta(1)AR and beta(2)AR can form heterodimers in HEK 293 cells co-expressing the two receptors. Functionally, beta-adrenergic stimulated adenylyl cyclase activity was found to be identical in cells expressing beta(1)AR, beta(2)AR, or both receptors at similar levels, indicating that heterodimerization did not affect this signaling pathway. When considering ERK1/2 MAPK activity, a significant agonist-promoted activation was detected in beta(2)AR- but not beta(1)AR-expressing cells. Similarly to what was observed in cells expressing the beta(1)AR alone, no beta-adrenergic stimulated ERK1/2 phosphorylation was observed in cells co-expressing the two receptors. A similar inhibition of agonist-promoted internalization of the beta(2)AR was observed upon co-expression of the beta(1)AR, which by itself internalized to a lesser extent. Taken together, our data suggest that heterodimerization between beta(1)AR and beta(2)AR inhibits the agonist-promoted internalization of the beta(2)AR and its ability to activate the ERK1/2 MAPK signaling pathway.     

Insights into signaling from the beta2-adrenergic receptor structure

With more than 800 members, the G protein-coupled receptor family constitutes the largest group of membrane proteins involved in signal transduction. Until the end of last year, high-resolution three-dimensional structures were available for only one of them--the light receptor rhodopsin. Recently the structure of the beta(2)-adrenergic receptor has been obtained, and it revealed interesting differences with the structure of rhodopsin. Analyses of these differences raise important questions about the binding modes of diffusible ligands in the receptor and allow formulation of testable hypotheses about the structural determinants linking drug binding to specific signaling responses. The three-dimensional structure derived from the beta(2)-adrenergic receptor crystal has been used to virtually dock ligands with distinct activities. The different binding modes of these ligands, which correlated with their reported efficacy profiles, suggest that it could be possible to predict the structural determinants of drug signaling efficacies.     

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.     

Differential targeting of beta -adrenergic receptor subtypes and adenylyl cyclase to cardiomyocyte caveolae. A mechanism to functionally regulate the cAMP signaling pathway

Differential modes for beta(1)- and beta(2)-adrenergic receptor (AR) regulation of adenylyl cyclase in cardiomyocytes is most consistent with spatial regulation in microdomains of the plasma membrane. This study examines whether caveolae represent specialized subdomains that concentrate and organize these moieties in cardiomyocytes. Caveolae from quiescent rat ventricular cardiomyocytes are highly enriched in beta(2)-ARs, Galpha(i), protein kinase A RIIalpha subunits, caveolin-3, and flotillins (caveolin functional homologues); beta(1)-ARs, m(2)-muscarinic cholinergic receptors, Galpha(s), and cardiac types V/VI adenylyl cyclase distribute between caveolae and other cell fractions, whereas protein kinase A RIalpha subunits, G protein-coupled receptor kinase-2, and clathrin are largely excluded from caveolae. Cell surface beta(2)-ARs localize to caveolae in cardiomyocytes and cardiac fibroblasts (with markedly different beta(2)-AR expression levels), indicating that the fidelity of beta(2)-AR targeting to caveolae is maintained over a physiologic range of beta(2)-AR expression. In cardiomyocytes, agonist stimulation leads to a marked decline in the abundance of beta(2)-ARs (but not beta(1)-ARs) in caveolae. Other studies show co-immunoprecipitation of cardiomyocytes adenylyl cyclase V/VI and caveolin-3, suggesting their in vivo association. However, caveolin is not required for adenylyl cyclase targeting to low density membranes, since adenylyl cyclase targets to low buoyant density membrane fractions of HEK cells that lack prototypical caveolins. Nevertheless, cholesterol depletion with cyclodextrin augments agonist-stimulated cAMP accumulation, indicating that caveolae function as negative regulators of cAMP accumulation. The inhibitory interaction between caveolae and the cAMP signaling pathway as well as domain-specific differences in the stoichiometry of individual elements in the beta-AR signaling cascade represent important modifiers of cAMP-dependent signaling in the heart.     

Identification of a Gs activator region of the beta 2-adrenergic receptor that is autoregulated via protein kinase A-dependent phosphorylation.

We have localized a G protein activator region of the human beta 2-adrenergic receptor to region beta III-2 (from Arg259 to Lys273). The synthetic beta III-2, corresponding to the C-terminal end of the third cytoplasmic loop, activates Gs at nanomolar concentrations and weakly activates Gi. beta III-2 activates adenylyl cyclase at nanomolar concentrations in wild-type S49 lymphoma membranes, but not in membranes of unc mutant S49 cells, in which Gs is uncoupled from beta-adrenergic stimulation. Phosphorylation of beta III-2 by cAMP-dependent protein kinase A, which is involved in the desensitization of the beta-adrenergic receptor from Gs, drastically reduces the effect of beta III-2 on Gs while potentiating its action on Gi, resulting in a total loss of adenylyl cyclase-stimulating activity. These findings indicate that this receptor sequence is a multipotential G protein activator whose G protein specificity is regulated by protein kinase A.     

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.     

A beta1-adrenergic receptor CaM kinase II-dependent pathway mediates cardiac myocyte fetal gene induction

Beta-adrenergic signaling plays an important role in the natural history of dilated cardiomyopathies. Chronic activation of beta-adrenergic receptors (beta1-AR and beta2-AR) during periods of cardiac stress ultimately harms the failing heart by mechanisms that include alterations in gene expression. Here, we show that stimulation of beta-ARs with isoproterenol in neonate rat ventricular myocytes causes a "fetal" response in the relative activities of the human cardiac fetal and/or adult gene promoters that includes repression of the human and rat alpha-myosin heavy chain (alpha-MyHC) promoters with simultaneous activation of the human atrial natriuretic peptide (ANP) and rat beta-MyHC promoters. We also show that the promoter changes correlate with changes in endogenous gene expression as measured by mRNA expression. Furthermore, we show that these changes are specifically mediated by the beta1-AR, but not the beta2-AR, and are independent of alpha1-AR stimulation. We also demonstrate that the fetal gene response is independent of cAMP and protein kinase A, whereas inhibition of Ca2+/calmodulin-dependent protein kinase (CaMK) pathway blocks isoproterenol-mediated fetal gene program induction. Finally, we show that induction of the fetal program is dependent on activation of the L-type Ca2+ channel. We conclude that in neonatal rat cardiac myocytes, agonist-occupied beta1-AR mobilizes Ca2+ stores to activate fetal gene induction through cAMP independent pathways that involve CaMK.     

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.     

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.     

β2肾上腺素受体遗传多态性研究进展

β2肾上腺素受体（β2-adrenergic receptor,132-AR）对血管和支气管平滑肌的紧张性起着重要的调节作用,能介导心脏的正性变力和变时效应。近年来研究发现,人类β2-AR具有遗传多态性,而使受体表现出不同的生物学特性。本文主要对β2-AR的遗传多态性及遗传药理学的研究进展进行简要概述。     

Gravin-mediated formation of signaling complexes in beta 2-adrenergic receptor desensitization and resensitization

Agonist-induced desensitization and resensitization of G-protein-linked receptors involve the interaction of receptors with protein kinases, phosphatases, beta-arrestin, and clathrin organized by at least one scaffold protein. The dynamic composition of the signaling complexes and the role of the scaffold protein AKAP250 (gravin) in agonist-induced attenuation and recovery of beta-adrenergic receptors were explored by co-immunoprecipitation of target elements, antisense suppression, and confocal microscopy. Gravin associated with unstimulated receptor, and the association was increased significantly after agonist stimulation for up to 60 min. Agonist stimulation also induced a robust association of the receptor-gravin complex with protein kinases A and C, G-protein-linked receptor kinase-2, beta-arrestin, and clathrin. Confocal microscopy of the green fluorescence protein-tagged beta(2)-adrenergic receptor showed that the receptor underwent sequestration after agonist stimulation. Suppression of gravin expression via antisense oligodeoxynucleotides disrupted agonist-induced association of the receptor with G-protein-linked receptor kinase-2, beta-arrestin, and clathrin as well as receptor recovery from desensitization. Gravin deficiency also inhibited agonist-induced sequestration. These data reveal that gravin-mediated formation of signaling complexes with protein kinases/phosphatases, beta-arrestin, and clathrin is essential in agonist-induced internalization and resensitization of G-protein-linked receptors.     

Protein kinase A-mediated phosphorylation of the beta 2-adrenergic receptor regulates its coupling to Gs and Gi. Demonstration in a reconstituted system

While classically viewed as a prototypic G(s) and adenylyl cyclase-coupled G protein-coupled receptor, recent studies have indicated that some aspects of beta(2)-adrenergic receptor (beta(2)-AR) signaling are inhibited by pertussis toxin, indicating that they are mediated by G(i)/G(o) proteins. These signals include activation of ERK MAPKs and Akt activation, as well as hypertrophic and anti-apoptotic pathways in cardiac myocytes. Studies in cultured cells have suggested the hypothesis that protein kinase A (PKA)-mediated phosphorylation of the beta(2)-AR regulates its coupling specificity with respect to G(s) and G(i). Using a Chinese hamster ovary cell system, we show that mutant beta(2)-ARs with Ala substituted for Ser at consensus PKA sites stimulate robust cyclic AMP accumulation (G(s)) but are unable to activate ERK (G(i)). In contrast, Ser --> Asp mutants are dramatically impaired in their ability to activate adenylyl cyclase but are significantly more active than wild type receptor in activating ERK. Activation of adenylyl cyclase by wild type and Ser --> Ala mutant receptors is not altered by pertussis toxin, whereas adenylyl cyclase stimulated through the Ser --> Asp mutant is enhanced. Activation of ERK by wild type and Ser --> Asp receptors is inhibited by pertussis toxin. To further rigorously test the hypothesis, we utilized a completely reconstituted system of purified recombinant wild type and PKA phosphorylation site mutant beta(2)-ARs and heterotrimeric G(s) and G(i). G protein coupling was measured by receptor-mediated stimulation of GTPgammaS binding to the G protein. PKA-mediated phosphorylation of the beta(2)-AR significantly decreased its ability to couple to G(s), while simultaneously dramatically increasing its ability to couple to G(i). These results are reproduced when a purified recombinant Ser --> Asp mutant beta(2)-AR is tested, whereas the Ser --> Ala receptor resembles the unphosphorylated wild type. These results provide strong experimental support for the idea that PKA-mediated phosphorylation of the beta(2)-adrenergic receptor switches its predominant coupling from G(s) to G(i).     

beta 1-adrenergic receptor association with PSD-95. Inhibition of receptor internalization and facilitation of beta 1-adrenergic receptor interaction with N-methyl-D-aspartate receptors

The beta(1)-adrenergic receptor (beta(1)AR) is the most abundant subtype of beta-adrenergic receptor in the mammalian brain and is known to potently regulate synaptic plasticity. To search for potential neuronal beta(1)AR-interacting proteins, we screened a rat brain cDNA library using the beta(1)AR carboxyl terminus (beta(1)AR-CT) as bait in the yeast two-hybrid system. These screens identified PSD-95, a multiple PDZ domain-containing scaffolding protein, as a specific binding partner of the beta(1)AR-CT. This interaction was confirmed by in vitro fusion protein pull-down and blot overlay experiments, which demonstrated that the beta(1)AR-CT binds specifically to the third PDZ domain of PSD-95. Furthermore, the full-length beta(1)AR associates with PSD-95 in cells, as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. The interaction between beta(1)AR and PSD-95 is mediated by the last few amino acids of the beta(1)AR, and mutation of the beta(1)AR carboxyl terminus eliminated the binding and disrupted the co-localization of the beta(1)AR and PSD-95 in cells. Agonist-induced internalization of the beta(1)AR in HEK-293 cells was markedly attenuated by PSD-95 co-expression, whereas co-expression of PSD-95 has no significant effect on either desensitization of the beta(1)AR or beta(1)AR-induced cAMP accumulation. Furthermore, PSD-95 facilitated the formation of a complex between the beta(1)AR and N-methyl-d-aspartate receptors, as assessed by co-immunoprecipitation. These data reveal that PSD-95 is a specific beta(1)AR binding partner that modulates beta(1)AR function and facilitates physical association of the beta(1)AR with synaptic proteins, such as the N-methyl-d-aspartate receptors, which are known to be regulated by beta(1)AR stimulation.     

beta-arrestin-biased agonism at the beta2-adrenergic receptor

Classically, the beta 2-adrenergic receptor (beta 2AR) and other members of the seven-transmembrane receptor (7TMR) superfamily activate G protein-dependent signaling pathways in response to ligand stimulus. It has recently been discovered, however, that a number of 7TMRs, including beta 2AR, can signal via beta-arrestin-dependent pathways independent of G protein activation. It is currently unclear if among beta 2AR agonists there exist ligands that disproportionately signal via G proteins or beta-arrestins and are hence "biased." Using a variety of approaches that include highly sensitive fluorescence resonance energy transfer-based methodologies, including a novel assay for receptor internalization, we show that the majority of known beta 2AR agonists exhibit relative efficacies for beta-arrestin-associated activities (beta-arrestin membrane translocation and beta 2AR internalization) identical to the irrelative efficacies for G protein-dependent signaling (cyclic AMP generation). However, for three betaAR ligands there is a marked bias toward beta-arrestin signaling; these ligands stimulate beta-arrestin-dependent receptor activities to a much greater extent than would be expected given their efficacy for G protein-dependent activity. Structural comparison of these biased ligands reveals that all three are catecholamines containing an ethyl substitution on the alpha-carbon, a motif absent on all of the other, unbiased ligands tested. Thus, these studies demonstrate the potential for developing a novel class of 7TMR ligands with a distinct bias for beta-arrestin-mediated signaling.     

Coupling of a mutated form of the human beta 2-adrenergic receptor to Gi and Gs. Requirement for multiple cytoplasmic domains in the coupling process.

We constructed five genes encoding mutant human beta 2-adrenergic receptor sequence (beta 2AR) which contained 12-22 amino acid substitutions with corresponding sequence from the human alpha 2AAR in order to assess the receptor domains involved in Gs versus Gi recognition and coupling. Mutant beta 2AR with substitutions in the N (S1)- and C-terminal (S2) portions of the third intracellular loop, the proximal cytoplasmic tail (S3), and two combinations thereof (S2,3 and S1,2,3), were stably expressed in Chinese hamster fibrobasts (CHW-1102), as were the human beta 2AR and alpha 2AAR at comparable receptor levels. All mutant receptors with S2 substitutions (i.e. S2, S2,3, S1,2,3) were significantly (approximately 85%) uncoupled from Gs. Upon exposure to pertussis toxin, which uncouples receptors from Gi, S1,2,3 exhibited a 526 +/- 99% increase in agonist-stimulated adenylylcyclase activity compared with a 59 +/- 13% increase with the wild type receptor. This enhanced ability of S1,2,3 to interact with Gs following pertussis toxin treatment indicates that, in the absence of toxin exposure, substantial coupling occurs between the mutant receptor and Gi. Mutant beta 2AR bearing only one or two alpha 2AAR-substituted sequences showed no such enhancement. Forskolin-stimulated enzyme activities were increased by pertussis toxin treatment to similar degrees in all clones examined, indicating that the observed effects are confined to the receptor-mediated pathway. In the absence of GTP, competition binding experiments with S1,2,3, beta 2AR and alpha 2AAR revealed that approximately 40-50% of the receptors formed a high affinity binding state for agonist. Pertussis toxin treatment markedly reduced this to approximately 19% with S1,2,3, while having no effect on beta 2AR and completely eliminating high affinity agonist binding to alpha 2AAR. These results suggest that S1,2,3 interacts with Gi as well as Gs, and that receptor:G protein coupling requires the concerted participation of multiple cytoplasmic receptor domains.