Oxytocin and vasopressin V1a and V2 receptors form constitutive homo- and heterodimers during biosynthesis

G protein-coupled receptor (GPCR) oligomerization is a growing concept that has emerged from several studies suggesting that GPCRs can form both homo- and heterodimers. Using both coimmunoprecipitation and bioluminescence resonance energy transfer (BRET) approaches, we established that the vasopressin V1a, V2, and the oxytocin receptors exist as homo- and hetero-dimers in transfected human embryonic kidney 293T cells. Each receptor protomer had a similar propensity to form homo- and heterodimers, indicating that their relative expression levels may determine the homo-/heterodimer ratio. The finding that immature forms of the receptor can be immunoprecipitated as homo- and heterodimers and the detection by BRET of such oligomer in endoplasmic reticulum-enriched fractions suggest that the oligomerization processes take place early during biosynthesis. Treatment with agonists or antagonists did not modify the BRET among any of the vasopressin and oxytocin receptor pairs studied, indicating that the dimerization state of the receptors is not regulated by ligand binding once they have reached the cell surface. Taken together, these results strongly support the notion that GPCR dimerization is a constitutive process.     

Melanocortin receptors form constitutive homo- and heterodimers

A significant number of G protein-coupled receptors are shown to form homo- or heterodimers/oligomers, and oligomerization of GPCRs may be a quite general phenomenon. We have here explored the possibility that the two closely related human melanocortin receptor 1 (MC(1)R) and melanocortin receptor 3 (MC(3)R) form dimers. Using bioluminescence resonance energy transfer (BRET(2)) we demonstrate that MC(1) and MC(3)Rs form homo- and heterodimers, when expressed in Cos-7 cells. Treatment with agonist, partial agonist or antagonists did not modify the BRET(2) signal for any of the receptor pairs studied, suggesting that the dimerization is not regulated by ligand binding. Rather our results indicate that melanocortin receptors exist as constitutively pre-formed dimers.     

Monitoring of ligand-independent dimerization and ligand-induced conformational changes of melatonin receptors in living cells by bioluminescence resonance energy transfer

Several G protein-coupled receptors have been shown to exist as homo-and hetero-oligomeric complexes in living cells. However, the link between ligand-induced receptor activation and its oligomerization state as well as the proportion of the total receptor population that can engage in oligomeric complexes remain open questions. Here, the closely related human MT1 and MT2 melatonin receptors (MT1R, MT2R) were used to address these issues. Bioluminescence resonance energy transfer (BRET) experiments in living HEK 293 cells revealed that these receptors form homo- and hetero-oligomers. Constitutive energy transfer was observed for all receptor combinations at physiological expression levels and could be detected in single cell BRET experiments. Inhibition of the energy transfer by dilution of the BRET partners identified MT1R and MT2R dimers as the predominant receptor species, and this oligomerization state did not change upon agonist and antagonist binding. Agonists, neutral antagonists, and inverse agonists all promoted increases in BRET values for MT2R but not for MT1R homodimers in living cells and isolated plasma membranes. This indicates that no correlation could be inferred between the receptor activation state and the dimerization state of the receptor. This also suggests that ligand-promoted BRET increases represent specific ligand-induced conformational changes of pre-existing dimers rather then increased dimerization. The observation that ligands favored the energy transfer within the hetero-oligomer from MT1R to MT2R but not in the reverse orientation, from MT2R to MT1R, supports this view.     

Opsin oligomerization in a heterologous cell system

Using bioluminescence resonance energy transfer (BRET) we studied opsin oligomerization in heterologous expression systems and quantitatively assessed its oligomerization state. BRET2 saturation and competition experiments were performed with live COS-7 cells expressing Rluc-and GFP2-tagged receptor constructs. BRET2 saturation curves obtained were hyperbolic, and the calculated oligomerization state (N = 1 for dimers) suggested that opsin (N = 1.34 +/- 0.25) forms higher oligomers. Very high BRET2 values obtained for the opsin homo-dimer pair indicated a large energy transfer efficiency (E) and for cases where E > 0.1 a modified saturation curve was proposed. The existence of homo-dimer complexes was additionally supported by competition assay results and was also observed in HEK-293 cells. Furthermore, evidence was provided for homo-and hetero-dimerization of family A (beta2-adrenergic) and B (gastric inhibitory polypeptide, GIP) receptors. In summary, these experiments demonstrate homo-and hetero-dimerization for opsin, beta 2-adrenergic, and GIP receptors.     

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.     

Dimerization Between Vasopressin V1b and Corticotropin Releasing Hormone Type 1 Receptors

1. Increasing evidence indicates that guanyl protein coupled receptors (GPCRs), including members of the vasopressin (VP) receptor family can act as homo- and heterodimers. Regulated expression and interaction of pituitary VP V1b receptor (V1bR) and corticotropin releasing hormone receptor type 1 (CRHR1) are critical for hypothalamic pituitary adrenal (HPA) axis adaptation, but it is unknown whether this involves physical interaction between these receptors. 2. Bioluminescence resonance energy transfer (BRET) experiments using V1bR and CRHR1 fused to either Renilla luciferase (Rluc) or yellow fluorescent protein (YFP) at the N-terminus, but not the carboxyl-terminus, revealed specific interaction (BRET₅₀ = 0.39 ± 0.08, V1bR) that was inhibited by untagged V1b or CRHR1 receptors, suggesting homo- and heterodimerization. The BRET data were confirmed by coimmunoprecipitation experiments using fully bioactive receptors tagged at the aminoterminus with c-myc and Flag epitopes, demonstrating specific homodimerization of the V1b receptor and heterodimerization of the V1b receptor with CRHR1 receptors. 3. Heterodimerization between V1bR and CRHR1 is not ligand dependent since stimulation with CRH and AVP had no effect on coimmunoprecipitation. In membranes obtained from cells cotransfected with CRHR1 and V1bR, incubation with the heterologous nonpeptide antagonist did not alter the binding affinity or capacity of the receptor. 4. The data demonstrate that V1bR and CRHR1 can form constitutive homo- and heterodimers and suggests that the heterodimerization does not influence the binding properties of these receptors.     

Human orexin/hypocretin receptors form constitutive homo- and heteromeric complexes with each other and with human CB1 cannabinoid receptors

Human OX₁ orexin receptors have been shown to homodimerize and they have also been suggested to heterodimerize with CB₁ cannabinoid receptors. The latter has been suggested to be important for orexin receptor responses and trafficking. In this study, we wanted to assess the ability of the other combinations of receptors to also form similar complexes. Vectors for expression of human OX₁, OX₂ and CB₁ receptors, C-terminally fused with either Renilla luciferase or GFP² green fluorescent protein variant, were generated. The constructs were transiently expressed in Chinese hamster ovary cells, and constitutive dimerization between the receptors was assessed by bioluminescence energy transfer (BRET). Orexin receptor subtypes readily formed homo- and hetero(di)mers, as suggested by significant BRET signals. CB₁ receptors formed homodimers, and they also heterodimerized with both orexin receptors. Interestingly, BRET efficiency was higher for homodimers than for almost all heterodimers. This is likely to be due to the geometry of the interaction; the putatively symmetric dimers may place the C-termini in a more suitable orientation in homomers. Fusion of luciferase to an orexin receptor and GFP² to CB₁ produced more effective BRET than the opposite fusions, also suggesting differences in geometry. Similar was seen for the OX₁–OX₂ interaction. In conclusion, orexin receptors have a significant propensity to make homo- and heterodi-/oligomeric complexes. However, it is unclear whether this affects their signaling. As orexin receptors efficiently signal via endocannabinoid production to CB₁ receptors, dimerization could be an effective way of forming signal complexes with optimal cannabinoid concentrations available for cannabinoid receptors.     

Bioluminescence resonance energy transfer reveals ligand-induced conformational changes in CXCR4 homo- and heterodimers

Homo- and heterodimerization have emerged as prominent features of G-protein-coupled receptors with possible impact on the regulation of their activity. Using a sensitive bioluminescence resonance energy transfer system, we investigated the formation of CXCR4 and CCR2 chemokine receptor dimers. We found that both receptors exist as constitutive homo- and heterodimers and that ligands induce conformational changes within the pre-formed dimers without promoting receptor dimer formation or disassembly. Ligands with different intrinsic efficacies yielded distinct bioluminescence resonance energy transfer modulations, indicating the stabilization of distinct receptor conformations. We also found that peptides derived from the transmembrane domains of CXCR4 inhibited activation of this receptor by blocking the ligand-induced conformational transitions of the dimer. Taken together, our data support a model in which chemokine receptor homo- and heterodimers form spontaneously and respond to ligand binding as units that undergo conformational changes involving both protomers even when only one of the two ligand binding sites is occupied.     

Alpha2A- and alpha2C-adrenergic receptors form homo- and heterodimers: the heterodimeric state impairs agonist-promoted GRK phosphorylation and beta-arrestin recruitment

Dimerization of seven transmembrane-spanning receptors diversifies their pharmacologic and physiologic properties. The alpha(2)-adrenergic receptor (alpha(2)AR) subtypes A and C are both expressed on presynaptic nerves and act to inhibit norepinephrine release via negative feedback. However, in vivo and in vitro studies examining the roles of the two individual alpha(2A)- and alpha(2C)AR subtypes are not readily reconciled. We tested the hypothesis that the receptors form homo- and heterodimers and that the alpha(2A)-alpha(2C) heterodimer has unique properties. SDS-PAGE of epitope-tagged receptors revealed potential oligomers including dimers. BRET of live HEK-293 cells transfected with the subtypes fused to Rluc or YFP revealed that both subtypes form dimers and the heterodimer. A lower BRET(50) for the alpha(2A)-alpha(2C) heterodimer (0.79 +/- 0.20) compared to that of the alpha(2A) or alpha(2C) homodimer (2.331 +/- 0.44 or 3.67 +/- 0.69, respectively) suggests that when both subtypes are expressed, there is a greater likelihood that the two receptors will form the heterodimer than homodimers. Co-immunoprecipitation studies confirmed homo- and heterodimer formation. The presence of the alpha(2C)AR within the heterodimer resulted in a marked reduction in the level of GRK2-mediated alpha(2A)AR phosphorylation, which was accompanied by a qualitative attenuation of beta-arrestin recruitment. Signaling of the alpha(2A)-alpha(2C) heterodimer to the beta-arrestin-dependent activation of Akt was decreased compared to that of the alpha(2A)AR homodimer, while p44/p42 MAP kinase activation was unaffected. Thus, the alpha(2C)AR alters alpha(2A)AR signaling by forming oligomers, and these complexes, which appear to be preferred over the homodimers, should be considered a functional signaling unit in cells in which both subtypes are expressed.     

The G protein beta subunit is a determinant in the coupling of Gs to the beta 1-adrenergic and A2a adenosine receptors

The signaling specificity of five purified G protein betagamma dimers, beta(1)gamma(2), beta(2)gamma(2), beta(3)gamma(2), beta(4)gamma(2), and beta(5)gamma(2), was explored by reconstituting them with G(s) alpha and receptors or effectors in the adenylyl cyclase cascade. The ability of the five betagamma dimers to support receptor-alpha-betagamma interactions was examined using membranes expressing the beta(1)-adrenergic or A2a adenosine receptors. These receptors discriminated among the defined heterotrimers based solely on the beta isoform. The beta(4)gamma(2) dimer demonstrated the highest coupling efficiency to either receptor. The beta(5)gamma(2) dimer coupled poorly to each receptor, with EC(50) values 40-200-fold higher than those observed with beta(4)gamma(2). Strikingly, whereas the EC(50) of the beta(1)gamma(2) dimer at the beta(1)-adrenergic receptor was similar to beta(4)gamma(2), its EC(50) was 20-fold higher at the A2a adenosine receptor. Inhibition of adenylyl cyclase type I (AC1) and stimulation of type II (AC2) by the betagamma dimers were measured. betagamma dimers containing Gbeta(1-4) were able to stimulate AC2 similarly, and beta(5)gamma(2) was much less potent. beta(1)gamma(2), beta(2)gamma(2), and beta(4)gamma(2) inhibited AC1 equally; beta(3)gamma(2) was 10-fold less effective, and beta(5)gamma(2) had no effect. These data argue that the beta isoform in the betagamma dimer can determine the specificity of signaling at both receptors and effectors.     

Biochemical and biophysical demonstration of GPCR oligomerization in mammalian cells

In contrast to other families of cell surface receptors, like tyrosine kinase receptors, for which dimerization is an integral part of the activation process, G-protein-coupled receptors (GPCRs) were thought, until recently, to function as monomeric units. However, a growing body of evidence indicates that GPCRs could exist and be active as oligomeric complexes. Because they are major pharmacological targets, their existence as homo- or hetero- oligomers could have important implications for the development and screening of new drugs. The major evidences supporting the idea of GPCR oligomerization come from indirect biochemical or pharmacological experiments. Here we report, using traditional co-immunoprecipitation methods, the existence of differentially epitope-tagged beta2-adrenergic receptor (beta2AR) oligomers in mammalian HEK-293 cells. Moreover, we validate the existence of receptor oligomers in living cells by a new Bioluminescence Resonance Energy Transfer (BRET) technique. Our results clearly demonstrate the presence of constitutive beta2AR oligomers in living cells that can be modulated by the selective adrenergic agonist isoproterenol, suggesting a pertinent physiological role for GPCR oligomerization.     

Expression of mRNA of beta 1- and beta 2-adrenergic receptors in Xenopus oocytes results from structurally distinct receptor mRNAs

Poly(A)+-selected RNA prepared from cells or tissues that express a homogeneous population of either beta 1- or beta 2-adrenergic receptors was isolated and then microinjected into Xenopus laevis oocytes. Following microinjection, the expression of beta-adrenergic receptors was assessed by equilibrium radioligand binding analysis using the antagonist ligand [3H]dihydroalprenolol. The pharmacology of the newly- expressed beta-adrenergic receptors in oocyte membranes was the same as that of the original tissue used as a source of RNA. Hybridization of nick-translated cDNA of hamster beta 2-adrenergic receptor to poly(A)+-selected RNA from tissues containing beta 2-adrenergic receptors was to a mRNA species of 2.2 kilobases. In contrast, hybridization of the cDNA probe to poly(A)+-selected RNA from tissues containing beta 1-adrenergic receptors was to a mRNA species of 2.0 kilobases. A single-stranded fragment of hamster beta 2-adrenergic receptor cDNA corresponding to nucleotides 730-886 was isolated and uniformly radiolabeled. This region of the gene is predicted to encode for the entire second exofacial loop (L4-5), the entire fifth transmembrane-spanning region, and the first 5 amino acid residues of the third cytoplasmic loop (L5-6) of the beta 2-adrenergic receptor. Hybridization at 48 and 56 degrees C of poly(A)+-selected RNA prepared from sources that express either beta 1 or beta 2-adrenergic receptors to the antisense orientation strand of this region of the beta 2-adrenergic receptor cDNA was followed by S1 endonuclease digestion of nonhybridized sequences. At 48 degrees C, S1-resistant hybrids from both sources of RNA protected the probe from S1 endonuclease digestion. At 56 degrees C, however, only the RNA prepared from the source of beta 2-adrenergic receptors protected the probe from S1 endonuclease digestion. These results demonstrate that the mRNAs encoding for the structurally homologous beta 1- and beta 2-adrenergic receptors are distinct in the pharmacological specificity of their translation products and in their size and structure.     

Constraints on GPCR Heterodimerization Revealed by the Type-4 Induced-Association BRET Assay

G-protein-coupled receptors (GPCRs) comprise the largest and most pharmacologically important family of cell-surface receptors encoded by the human genome. In many instances, the distinct signaling behavior of certain GPCRs has been explained in terms of the formation of heteromers with, for example, distinct signaling properties and allosteric cross-regulation. Confirmation of this has, however, been limited by the paucity of reliable methods for probing heteromeric GPCR interactions in situ. The most widely used assays for GPCR stoichiometry, based on resonance energy transfer, are unsuited to reporting heteromeric interactions. Here, we describe a targeted bioluminescence resonance energy transfer (BRET) assay, called type-4 BRET, which detects both homo- and heteromeric interactions using induced multimerization of protomers within such complexes, at constant expression. Using type-4 BRET assays, we investigate heterodimerization among known GPCR homodimers: the CXC chemokine receptor 4 and sphingosine-1-phosphate receptors. We observe that CXC chemokine receptor 4 and sphingosine-1-phosphate receptors can form heterodimers with GPCRs from their immediate subfamilies but not with more distantly related receptors. We also show that heterodimerization appears to disrupt homodimeric interactions, suggesting the sharing of interfaces. Broadly, these observations indicate that heterodimerization results from the divergence of homodimeric receptors and will therefore likely be restricted to closely related homodimeric GPCRs.     

beta-Adrenergic stimulated lipolysis in pony adipocytes is exclusively via a beta2-subtype and is not affected by lactation

Catecholamines are important lipolytic agents in horses and ponies but the nature of the adrenergic receptor subtype distribution in their adipocytes is uncertain. A first objective was to identify the beta-adrenergic receptor subtype(s) present in adipocytes from horses and ponies. A second objective was to evaluate if the lipolytic responsiveness of isolated adipocytes to beta-adrenergic agonists is altered during lactation, a condition known to affect markedly maternal fat metabolism. Isoproterenol and salbutamol elicited strong lipolytic responses in adipocytes isolated from horse and pony subcutaneous adipose tissue. There were weak lipolytic responses to norepinephrine, dobutamine and BRL37344. The weak lipolytic response to NE compared to isoproterenol or salbutamol suggests an antilipolytic action from alpha2-adrenergic receptors. The relative order of potency for the beta-adrenergic agonists was isoproterenol>/=salbutamol>dobutamine=BRL37344. There was expression of beta2-adrenergic receptor mRNA in pony and horse adipose tissues, as estimated by relative RT-PCR, but no expression of mRNAs for beta1- or beta3-adrenergic receptors. Early lactation did not alter the lipolytic responses to beta-adrenergic agonists, nor the expression of beta2-adrenergic receptor mRNA. Thus, these results indicate a dominant if not exclusive presence of beta2-adrenergic receptors in pony and horse adipocytes that is not affected by lactation.     

Biochemical and biophysical characterization of serotonin 5-HT2C receptor homodimers on the plasma membrane of living cells

While many studies have provided evidence of homodimerization and heterodimerization of G-protein-coupled receptors (GPCRs), few studies have used fluorescence resonance energy transfer (FRET) combined with confocal microscopy to visualize receptor dimerization on the plasma membrane, and there have been no reports demonstrating the expression of serotonin receptor dimers/oligomers on the plasma membrane of living cells. In the study presented here, biochemical and biophysical techniques were used to determine if 5-HT(2C) receptors exist as homodimers on the plasma membrane of living cells. Immunoprecipitation followed by Western blotting revealed the presence of immunoreactive bands the predicted size of 5-HT(2C) receptor monomers and homodimers that were detergent and cross-linker sensitive. Bioluminescence resonance energy transfer (BRET) was assessed in HEK293 cells expressing 5-HT(2C) receptors labeled with Renilla luciferase and yellow fluorescent protein. BRET levels were not altered by pretreatment with serotonin. Confocal microscopy provided direct visualization of FRET on the plasma membrane of live cells expressing 5-HT(2C) receptors labeled with cyan (donor) and yellow (acceptor) fluorescent proteins. FRET, assessed by acceptor photobleaching, was dependent on the donor/acceptor ratio and independent of acceptor expression levels, indicating that FRET resulted from receptor clustering and not from overexpression of randomly distributed receptors, providing evidence for GPCR dimers/oligomers in a clustered distribution on the plasma membrane. The results of this study suggest that 5-HT(2C) receptors exist as constitutive homodimers on the plasma membrane of living cells. In addition, a confocal-based FRET method for monitoring receptor dimerization directly on the plasma membrane of living cells is described.     

Mammalian beta 1- and beta 2-adrenergic receptors. Immunological and structural comparisons

Beta 1- and beta 2-adrenergic receptors, pharmacologically distinct proteins, have been reported to be structurally dissimilar. In the present study three techniques were employed to compare the nature of mammalian beta 1- and beta 2-adrenergic receptors. Antibodies against each of the receptor subtypes were raised separately. Polyclonal antisera against beta 1-receptors of rat fat cells were raised in mice, and antisera against beta 2-receptors of guinea pig lung were raised in rabbits. Receptors purified from rat fat cells (beta 1-), S49 mouse lymphoma cells (beta 2-), and rat liver (beta 2-) were probed with these antisera. Each anti-receptor antisera demonstrated the ability to immunoprecipitate purified receptors of both beta 1- and beta 2- subtypes. The mobility of beta-receptors subjected to polyacrylamide gel electrophoresis was probed using antireceptor antibodies and nitrocellulose blots of the gels. Fat cell beta 1-adrenergic receptors display Mr = 67,000 under reducing conditions and Mr = 54,000 under nonreducing conditions, as previously reported (Moxham, C. P., and Malbon, C. C. (1985) Biochemistry 24, 6072-6077). Both beta 1- and beta 2-receptors displayed this same shift in electrophoretic mobility observed in the presence as compared to the absence of disulfide bridge-reducing agents, as detected both by autoradiography of the radiolabeled receptors and by immunoblotting of native receptors. Finally, isoelectric focusing of purified radioiodinated beta 1- and beta 2-adrenergic receptors revealed identical isoelectric points. These data are the first to provide analyses of immunological, structural, and biochemical features of beta 1- and beta 2-subtypes in tandem and underscore the structural similarities that exist between these pharmacologically distinct receptors.     

Cross-regulation between G-protein-coupled receptors. Activation of beta 2-adrenergic receptors increases alpha 1-adrenergic receptor mRNA levels.

Stimulation of DDT1 MF-2 vas deferens cells with epinephrine resulted in a time- and dose-dependent loss of alpha 1-adrenergic receptor-specific ligand binding. Regulation of alpha 1-adrenergic receptor mRNA was characterized. In monolayer culture, cells displayed 0.7 +/- 0.05 amol of alpha 1-adrenergic receptor mRNA/microgram of total cellular RNA. Epinephrine, which acts at both alpha 1- and beta 2-adrenergic receptors of DDT1 MF-2 cells, induced a short term (2-8 h) increase (50-70%) in the abundance of alpha 1-adrenergic receptor mRNA. Propranolol, a beta 2-adrenergic receptor antagonist, attenuated the epinephrine-mediated increase in alpha 1-adrenergic receptor mRNA but did not affect the decrease in alpha 1-adrenergic receptor-specific ligand binding. Phentolamine, an alpha 1-adrenergic receptor antagonist, did not attenuate the epinephrine-mediated increase in alpha 1-adrenergic receptor mRNA at 4 h but did block the decrease in alpha 1-adrenergic receptor-specific ligand binding. The half-life of the alpha 1-adrenergic receptor mRNA was approximately 7 h in untreated cells as well as in cells challenged with epinephrine. The epinephrine-promoted increase in alpha 1-adrenergic receptor mRNA was found to result from cross-regulation via beta 2-adrenergic receptors. Cholera toxin, forskolin, as well as the cyclic AMP analog CPT cAMP (8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate) increased the alpha 1-adrenergic receptor mRNA at 4 h, as did epinephrine in the presence of alpha 1-antagonists but not in the presence of a beta-adrenergic antagonist. This is the first report of heterologous up-regulation of mRNA levels of adrenergic receptors. Cross-regulation between alpha 1- and beta 2-adrenergic receptor-mediated pathways at 4 h occurs at the level of mRNA whereas later down-regulation of alpha 1-receptor mRNA and binding proceed via agonist activation of alpha 1-adrenergic receptors.     

GIPC interacts with the beta1-adrenergic receptor and regulates beta1-adrenergic receptor-mediated ERK activation

Beta1-adrenergic receptors, expressed at high levels in the human heart, have a carboxyl-terminal ESKV motif that can directly interact with PDZ domain-containing proteins. Using the beta1-adrenergic receptor carboxyl terminus as bait, we identified the novel beta1-adrenergic receptor-binding partner GIPC in a yeast two-hybrid screen of a human heart cDNA library. Here we demonstrate that the PDZ domain-containing protein, GIPC, co-immunoprecipitates with the beta1-adrenergic receptor in COS-7 cells. Essential for this interaction is the Ser residue of the beta1-adrenergic receptor carboxyl-terminal ESKV motif. Our data also demonstrate that beta1-adrenergic receptor stimulation activates the mitogen-activated protein kinase, ERK1/2. beta1-adrenergic receptor-mediated ERK1/2 activation was inhibited by pertussis toxin, implicating Gi, and was substantially decreased by the expression of GIPC. Expression of GIPC had no observable effect on beta1-adrenergic receptor sequestration or receptor-mediated cAMP accumulation. This GIPC effect was specific for the beta1-adrenergic receptor and was dependent on an intact PDZ binding motif. These data suggest that GIPC can regulate beta1-adrenergic receptor-stimulated, Gi-mediated, ERK activation while having no effect on receptor internalization or Gs-mediated cAMP signaling.     

Homodimerization of adenosine A2A receptors: qualitative and quantitative assessment by fluorescence and bioluminescence energy transfer

The results presented in this paper show that adenosine A2A receptor (A2AR) form homodimers and that homodimers but not monomers are the functional species at the cell surface. Fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) techniques have been used to demonstrate in transfected HEK293 cells homodimerization of A2AR, which are heptaspanning membrane receptors with enriched expression in striatum. The existence of homodimers at the cell surface was demonstrated by time-resolved FRET. Although agonist activation of the receptor leads to the formation of receptor clusters, it did not affect the degree of A2AR-A2AR dimerization. Both monomers and dimers were detected by immunoblotting in cell extracts. However, cell surface biotinylation of proteins has made evident that more than 90% of the cell surface receptor is in its dimeric form. Thus, it seems that homodimers are the functional form of the receptor present on the plasma membrane. A deletion mutant version of the A2A receptor, lacking its C-terminal domain, was also able to form both monomeric and dimeric species when cell extracts from transfected cells were analyzed by immunoblotting. This suggests that the C-terminal tail does not participate in the dimerization. This is relevant as the C-terminal tail of A2AR is involved in heteromers formed by A2AR and dopamine D2 receptors. BRET ratios corresponding to A2AR-A2AR homodimers were higher than those encountered for heterodimers formed by A2AR and dopamine D2 receptors. As A2AR and dopamine D2 receptors do indeed interact, these results indicate that A2AR homodimers are the functional species at the cell surface and that they coexist with A2AR/D2 receptor heterodimers.