Terence E. Hébert

A Peptide Derived from a β2-Adrenergic Receptor Transmembrane Domain Inhibits Both Receptor Dimerization and Activation

One of the assumptions of the mobile receptor hypothesis as it relates to G protein-coupled receptors is that the stoichiometry of receptor, G protein, and effector is 1:1:1 (Bourne, H. R., Sanders, D. A., and McCormick, F. (1990) Nature 348, 125–132). Many studies on the cooperativity of agonist binding are incompatible with this notion and have suggested that both G proteins and their associated receptors can be oligomeric. However, a clear physical demonstration that G protein-coupled receptors can indeed interact as dimers and that such interactions may have functional consequences was lacking. Here, using differential epitope tagging we demonstrate that β2-adrenergic receptors do form SDS-resistant homodimers and that transmembrane domain VI of the receptor may represent part of an interface for receptor dimerization. The functional importance of dimerization is supported by the observation that a peptide derived from this domain that inhibits dimerization also inhibits β-adrenergic agonist-promoted stimulation of adenylyl cyclase activity. Moreover, agonist stimulation was found to stabilize the dimeric state of the receptor, while inverse agonists favored the monomeric species, which suggests that interconversion between monomeric and dimeric forms may be important for biological activity.

Real-time monitoring of receptor and G-protein interactions in living cells.

G protein–coupled receptors (GPCRs) represent the largest family of proteins involved in signal transduction. Here we present a bioluminescence resonance energy transfer (BRET) assay that directly monitors in real time the early interactions between human GPCRs and their cognate G-protein subunits in living human cells. In addition to detecting basal precoupling of the receptors to Gα-, Gβ- and Gγ-subunits, BRET measured very rapid ligand-induced increases in the interaction between receptor and Gαβγ-complexes (t1/2 ∼ 300 ms) followed by a slower (several minutes) decrease, reflecting receptor desensitization. The agonist-promoted increase in GPCR-Gβγ interaction was highly dependent on the identity of the Gα-subunit present in the complex. Therefore, this G protein–activity biosensor provides a novel tool to directly probe the dynamics and selectivity of receptor-mediated, G-protein activation-deactivation cycles that could be advantageously used to identify ligands for orphan GPCRs.

IDENTIFICATION OF A GABAB RECEPTOR SUBUNIT, GB2, REQUIRED FOR FUNCTIONAL GABAB RECEPTOR ACTIVITY

G protein-coupled receptors are commonly thought to bind their cognate ligands and elicit functional responses primarily as monomeric receptors. In studying the recombinant γ-aminobutyric acid, type B (GABAB) receptor (gb1a) and a GABAB-like orphan receptor (gb2), we observed that both receptors are functionally inactive when expressed individually in multiple heterologous systems. Characterization of the tissue distribution of each of the receptors by in situhybridization histochemistry in rat brain revealed co-localization of gb1 and gb2 transcripts in many brain regions, suggesting the hypothesis that gb1 and gb2 may interact in vivo. In three established functional systems (inwardly rectifying K+channel currents in Xenopus oocytes, melanophore pigment aggregation, and direct cAMP measurements in HEK-293 cells), GABA mediated a functional response in cells coexpressing gb1a and gb2 but not in cells expressing either receptor individually. This GABA activity could be blocked with the GABAB receptor antagonist CGP71872. In COS-7 cells coexpressing gb1a and gb2 receptors, co-immunoprecipitation of gb1a and gb2 receptors was demonstrated, indicating that gb1a and gb2 act as subunits in the formation of a functional GABAB receptor.

Structural and functional aspects of G protein-coupled receptor oligomerization

G protein-coupled receptors (GPCRs) represent the single largest family of cell surface receptors involved in signal transduction. It is estimated that several hundred distinct members of this receptor family in humans direct responses to a wide variety of chemical transmitters, including biogenic amines, amino acids, peptides, lipids, nucleosides, and large polypeptides. These transmembrane receptors are key controllers of such diverse physiological processes as neurotransmission, cellular metabolism, secretion, cellular differentiation, and growth as well as inflammatory and immune responses. GPCRs therefore represent major targets for the development of new drug candidates with potential application in all clinical fields. Many currently used therapeutics act by either activating (agonists) or blocking (antagonists) GPCRs. Studies over the past two decades have provided a wealth of information on the biochemical events underlying cellular signalling by GPCRs. However, our understanding of the molecular interactions between ligands and the receptor protein and, particularly, of the structural correlates of receptor activation or inhibition by agonists and inverse agonists, respectively, is still rudimentary. Most of the work in this area has focused on mapping regions of the receptor responsible for drug binding affinity. Although binding of ligand molecules to specific receptors represents the first event in the action of drugs, the efficacy with which this binding is translated into a physiological response remains the only determinant of therapeutic utility. In the last few years, increasing evidence suggested that receptor oligomerization and in particular dimerization may play an important role in the molecular events leading to GPCR activation. In this paper, we review the biochemical and functional evidence supporting this notion.

The Role of Gβγ Subunits in the Organization, Assembly, and Function of GPCR Signaling Complexes

The role of Gbetagamma subunits in cellular signaling has become well established in the past 20 years. Not only do they regulate effectors once thought to be the sole targets of Galpha subunits, but it has become clear that they also have a unique set of binding partners and regulate signaling pathways that are not always localized to the plasma membrane. However, this may be only the beginning of the story. Gbetagamma subunits interact with G protein-coupled receptors, Galpha subunits, and several different effector molecules during assembly and trafficking of receptor-based signaling complexes and not simply in response to ligand stimulation at sites of receptor cellular activity. Gbetagamma assembly itself seems to be tightly regulated via the action of molecular chaperones and in turn may serve a similar role in the assembly of specific signaling complexes. We propose that specific Gbetagamma subunits have a broader role in controlling the architecture, assembly, and activity of cellular signaling pathways.

A comparison of currents carried by HERG, with and without coexpression of MiRP1, and the native rapid delayed rectifier current. Is MiRP1 the missing link?

Although it has been suggested that coexpression of minK related peptide (MiRP1) is required for reconstitution of native rapid delayed‐rectifier current (IKr) by human ether‐a‐go‐go related gene (HERG), currents resulting from HERG (IHERG) and HERG plus MiRP1 expression have not been directly compared with native IKr. We compared the pharmacological and selected biophysical properties of IHERG with and without MiRP1 coexpression in Chinese hamster ovary (CHO) cells with those of guinea‐pig IKr under comparable conditions. Comparisons were also made with HERG expressed in Xenopus oocytes. MiRP1 coexpression significantly accelerated IHERG deactivation at potentials negative to the reversal potential, but did not affect more physiologically relevant deactivation of outward IHERG, which remained slower than that of IKr. MiRP1 shifted IHERG activation voltage dependence in the hyperpolarizing direction, whereas IKr activated at voltages more positive than IHERG. There were major discrepancies between the sensitivity to quinidine, E‐4031 and dofetilide of IHERG in Xenopus oocytes compared to IKr, which were not substantially affected by coexpression with MiRP1. On the other hand, the pharmacological sensitivity of IHERG in CHO cells was indistinguishable from that of IKr and was unaffected by MiRP1 coexpression. We conclude that the properties of IHERG in CHO cells are similar in many ways to those of native IKr under the same recording conditions, and that the discrepancies that remain are not reduced by coexpression with MiRP1. These results suggest that the physiological role of MiRP1 may not be to act as an essential consituent of the HERG channel complex carrying native IKr.

Dopamine modulates the plasticity of mechanosensory responses in Caenorhabditis elegans

Dopamine‐modulated behaviors, including information processing and reward, are subject to behavioral plasticity. Disruption of these behaviors is thought to support drug addictions and psychoses. The plasticity of dopamine‐mediated behaviors, for example, habituation and sensitization, are not well understood at the molecular level. We show that in the nematode Caenorhabditis elegans, a D1‐like dopamine receptor gene (dop‐1) modulates the plasticity of mechanosensory behaviors in which dopamine had not been implicated previously. A mutant of dop‐1 displayed faster habituation to nonlocalized mechanical stimulation. This phenotype was rescued by the introduction of a wild‐type copy of the gene. The dop‐1 gene is expressed in mechanosensory neurons, particularly the ALM and PLM neurons. Selective expression of the dop‐1 gene in mechanosensory neurons using the mec‐7 promoter rescues the mechanosensory deficit in dop‐1 mutant animals. The tyrosine hydroxylase‐deficient C. elegans mutant (cat‐2) also displays these specific behavioral deficits. These observations provide genetic evidence that dopamine signaling modulates behavioral plasticity in C. elegans.

Characterization of a hyperpolarization‐activated time‐dependent potassium current in canine cardiomyocytes from pulmonary vein myocardial sleeves and left atrium

Cardiomyocytes from the pulmonary vein sleeves (PVs) are known to play an important role in atrial fibrillation. PVs have been shown to exhibit time‐dependent hyperpolarization‐induced inward currents of uncertain nature. We observed a time‐dependent K+ current upon hyperpolarization of PV and left atrial (LA) cardiomyocytes (IKH) and characterized its biophysical and pharmacological properties. The activation time constant was weakly voltage dependent, ranging from 386 ± 14 to 427 ± 37 ms between −120 and −90 mV, and the half‐activation voltage averaged −93 ± 4 mV. IKH was larger in PV than LA cells (e.g. at −120 mV: −2.8 ± 0.3 versus−1.9 ± 0.2 pA pF−1, respectively, P < 0.01). The reversal potential was ∼−84 mV with 5.4 mm[K+]o and changed by 55.7 ± 2.4 mV per decade [K+]o change. IKH was exquisitely Ba2+ sensitive, with a 50% inhibitory concentration (IC50) of 2.0 ± 0.3 μm (versus 76.0 ± 17.9 μm for instantaneous inward‐rectifier current, P < 0.01), and showed similar Cs+ sensitivity to instantaneous current. IKH was potently blocked by tertiapin‐Q, a selective Kir3‐subunit channel blocker (IC50 10.0 ± 2.1 nm), was unaffected by atropine and was significantly increased by isoproterenol (isoprenaline), carbachol and the non‐hydrolysable guanosine triphosphate analogue GTPγS. IKH activation by carbachol required GTP in the pipette and was prevented by pertussis toxin pretreatment. Tertiapin‐Q delayed repolarization in atropine‐exposed multicellular atrial preparations studied with standard microelectrodes (action potential duration pre‐ versus post‐tertiapin‐Q: 190.4 ± 4.3 versus 234.2 ± 9.9 ms, PV; 202.6 ± 2.6 versus 242.7 ± 6.2 ms, LA; 2 Hz, P < 0.05 each). Seven‐day atrial tachypacing significantly increased IKH (e.g. at −120 mV in PV: from −2.8 ± 0.3 to −4.5 ± 0.5 pA pF−1, P < 0.01). We conclude that IKH is a time‐dependent, hyperpolarization‐activated K+ current that likely involves Kir3 subunits and appears to play a significant role in atrial physiology.

Seven Transmembrane Receptor Core Signaling Complexes Are Assembled Prior to Plasma Membrane Trafficking

Much is known about β2-adrenergic receptor trafficking and internalization following prolonged agonist stimulation. However, less is known about outward trafficking of the β2-adrenergic receptor to the plasma membrane or the role that trafficking might play in the assembly of receptor signaling complexes, important for targeting, specificity, and rapidity of subsequent signaling events. Here, by using a combination of bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and confocal microscopy, we evaluated the steps in the formation of the core receptor-G protein heterotrimer complex. By using dominant negative Rab and Sar GTPase constructs, we demonstrate that receptor dimers and receptor-Gβγ complexes initially associate in the endoplasmic reticulum, whereas Gα subunits are added to the complex during endoplasmic reticulum-Golgi transit. We also observed that G protein heterotrimers adopt different trafficking itineraries when expressed alone or with stoichiometric co-expression with receptor. Furthermore, deliberate mistargeting of specific components of these complexes leads to diversion of other members from their normal subcellular localization, confirming the role of these early interactions in targeting and formation of specific signaling complexes.

Heterotrimeric G proteins form stable complexes with adenylyl cyclase and Kir3.1 channels in living cells.

Bioluminescence resonance energy transfer (BRET) and co-immunoprecipitation experiments revealed that heterotrimeric G proteins and their effectors were found in stable complexes that persisted during signal transduction. Adenylyl cyclase, Kir3.1 channel subunits and several G-protein subunits (Gαs, Gαi, Gβ1 and Gγ2) were tagged with luciferase (RLuc) or GFP, or the complementary fragments of YFP (specifically Gβ1-YFP1-158 and Gγ2-YFP159-238, which heterodimerize to produce fluorescent YFP-Gβ1γ2). BRET was observed between adenylyl-cyclase-RLuc or Kir3.1-RLuc and GFP-Gγ2, GFP-Gβ1 or YFP-Gβ1γ2. Gα subunits were also stably associated with both effectors regardless of whether or not signal transduction was initiated by a receptor agonist. Although BRET between effectors and Gβγ was increased by receptor stimulation, our data indicate that these changes are likely to be conformational in nature. Furthermore, receptor-sensitive G-protein-effector complexes could be detected before being transported to the plasma membrane, providing the first direct evidence for an intracellular site of assembly.