Single-molecule spectroscopy of the β2 adrenergic receptor: Observation of conformational substates in a membrane protein

Single-molecule studies of the conformations of the intact β2 adrenergic receptor were performed in solution. Photon bursts from the fluorescently tagged adrenergic receptor in a micelle were recorded. A photon-burst algorithm and a Poisson time filter were implemented to characterize single molecules diffusing across the probe volume of a confocal microscope. The effects of molecular diffusion and photon number fluctuations were deconvoluted by assuming that Poisson distributions characterize the molecular occupation and photon numbers. Photon-burst size histograms were constructed, from which the source intensity distributions were extracted. Different conformations of the β2 adrenergic receptor cause quenching of the bound fluorophore to different extents and hence produce different photon-burst sizes. An analysis of the photon-burst histograms shows that there are at least two distinct substates for the native adrenergic membrane receptor. This behavior is in contrast to one peak observed for the dye molecule, rhodamine 6G. We test the reliability and robustness of the substate number determination by investigating the application of different binning criteria. Conformational changes associated with agonist binding result in a marked change in the distribution of photon-burst sizes. These studies provide insight into the conformational heterogeneity of G protein-coupled receptors in the presence and absence of a bound agonist.

Evolutionarily conserved Galphabetagamma binding surfaces support a model of the G protein-receptor complex.

The pivotal role of G proteins in sensory, hormonal, inflammatory, and proliferative responses has provoked intense interest in understanding how they interact with their receptors and effectors. Nonetheless, the locations of the receptors and effector binding sites remain poorly characterized, although nearly complete structures of the alphabetagamma heterotrimeric complex are available. Here we apply evolutionary trace (ET) analysis [Lichtarge, O., Bourne, H. R. & Cohen, F. E. (1996) J. Mol. Biol. 257, 342-358] to propose plausible locations for these sites. On each subunit, ET identifies evolutionarily selected surfaces composed of residues that do not vary within functional subgroups and that form spatial clusters. Four clusters correctly identify subunit interfaces, and additional clusters on Galpha point to likely receptor or effector binding sites. Our results implicate the conformationally variable region of Galpha in an effector binding role. Furthermore the range of predicted interactions between the receptor and Galphabetagamma, is sufficiently limited that we can build a low resolution and testable model of the receptor-G protein complex.

A heterotrimeric G protein complex couples the muscarinic m1 receptor to phospholipase C-beta.

We addressed the question as to which subtypes of G protein subunits mediate the activation of phospholipase C-beta by the muscarinic m1 receptor. We used the rat basophilic leukemia cell line RBL-2H3-hm1 stably transfected with the human muscarinic m1 receptor cDNA. We microinjected antisense oligonucleotides into the nuclei of the cells to inhibit selectively the expression of G protein subunits; 48 hr later muscarinic receptors were activated by carbachol, and the increase in free cytosolic calcium concentration ([Ca2+]i) was measured. Antisense oligonucleotides directed against the mRNA coding for alpha(q) and alpha11 subunits both suppressed the carbachol-induced increase in [Ca2+]i. In cells injected with antisense oligonucleotides directed against alpha(o1) and alpha14 subunits, the carbachol effect was unchanged. A corresponding reduction of Galpha(q), and Galpha11 proteins by 70-80% compared to uninjected cells was immunochemically detected 2 days after injection of a mixture of alpha(q) and alpha11 antisense oligonucleotides. Expression of Galpha(q) and Galpha11 completely recovered after 4 days. Cells injected with antisense oligonucleotides directed against the mRNAs encoding for beta1, beta4, and gamma4 subunits showed a suppression of the carbachol-induced increase in [Ca2+]i compared to uninjected cells measured at the same time from the same coverslip, whereas in cells injected with antisense oligonucleotides directed against the beta2, beta3, gamma1, gamma2, gamma3, gamma5, and gamma7 subunits, no suppression of carbachol effect was observed. In summary, the results from RBL-2H3-hm1 cells indicate that the m1 receptor utilizes a G protein complex composed of the subunits alpha(q), alpha11, beta1, beta4, and gamma4 to activate phospholipase C.

Receptor-G protein coupling is established by a potential conformational switch in the beta gamma complex.

Receptor-G protein interaction is characterized by cycles of association and dissociation. We present evidence which indicates that during receptor-G protein interaction, the C-terminal tail of the G protein gamma subunit, which is masked in the beta gamma complex, is exposed and establishes high-affinity contact with the receptor. This potential conformational switch provides a mechanism to regulate receptor-G protein coupling. This switch may also be significant for the role of the beta gamma complex in regulation of effector function.

G protein beta gamma subunits stimulate phosphorylation of Shc adapter protein.

The mechanism of mitogen-activated protein (MAP) kinase activation by pertussis toxin-sensitive Gi-coupled receptors is known to involve the beta gamma subunits of heterotrimeric G proteins (G beta gamma), p21ras activation, and an as-yet-unidentified tyrosine kinase. To investigate the mechanism of G beta gamma-stimulated p21ras activation, G beta gamma-mediated tyrosine phosphorylation was examined by overexpressing G beta gamma or alpha 2-C10 adrenergic receptors (ARs) that couple to Gi in COS-7 cells. Immunoprecipitation of phosphotyrosine-containing proteins revealed a 2- to 3-fold increase in the phosphorylation of two proteins of approximately 50 kDa (designated as p52) in G beta gamma-transfected cells or in alpha 2-C10 AR-transfected cells stimulated with the agonist UK-14304. The latter response was pertussis toxin sensitive. These proteins (p52) were also specifically immunoprecipitated with anti-Shc antibodies and comigrated with two Shc proteins, 46 and 52 kDa. The G beta gamma- or alpha 2-C10 AR-stimulated p52 (Shc) phosphorylation was inhibited by coexpression of the carboxyl terminus of beta-adrenergic receptor kinase (a G beta gamma-binding pleckstrin homology domain peptide) or by the tyrosine kinase inhibitors genistein and herbimycin A, but not by a dominant negative mutant of p21ras. Worthmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K) inhibited phosphorylation of p52 (Shc), implying involvement of PI3K. These results suggest that G beta gamma-stimulated Shc phosphorylation represents an early step in the pathway leading to p21ras activation, similar to the mechanism utilized by growth factor tyrosine kinase receptors.

The β2-adrenergic receptor/βarrestin complex recruits the clathrin adaptor AP-2 during endocytosis

βarrestins mediate the desensitization of the β2-adrenergic receptor (β2AR) and many other G protein-coupled receptors (GPCRs). Additionally, βarrestins initiate the endocytosis of these receptors via clathrin coated-pits and interact directly with clathrin. Consequently, it has been proposed that βarrestins serve as clathrin adaptors for the GPCR family by linking these receptors to clathrin lattices. AP-2, the heterotetrameric clathrin adaptor protein, has been demonstrated to mediate the internalization of many types of plasma membrane proteins other than GPCRs. AP-2 interacts with the clathrin heavy chain and cytoplasmic domains of receptors such as those for epidermal growth factor and transferrin. In the present study we demonstrate the formation of an agonist-induced multimeric complex containing a GPCR, βarrestin 2, and the β2-adaptin subunit of AP-2. β2-Adaptin binds βarrestin 2 in a yeast two-hybrid assay and coimmunoprecipitates with βarrestins and β2AR in an agonist-dependent manner in HEK-293 cells. Moreover, β2-adaptin translocates from the cytosol to the plasma membrane in response to the β2AR agonist isoproterenol and colocalizes with β2AR in clathrin-coated pits. Finally, expression of βarrestin 2 minigene constructs containing the β2-adaptin interacting region inhibits β2AR endocytosis. These findings point to a role for AP-2 in GPCR endocytosis, and they suggest that AP-2 functions as a clathrin adaptor for the endocytosis of diverse classes of membrane receptors.

Design, synthesis, and biological characterization of a peptide-mimetic antagonist for a tethered-ligand receptor

Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G protein-coupled receptors, which are enzymatically cleaved to expose a truncated extracellular N terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease α-thrombin, is expressed in various tissues (e.g., platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. We have discovered a series of potent peptide-mimetic antagonists of PAR-1, exemplified by RWJ-56110. Spatial relationships between important functional groups of the PAR-1 agonist peptide epitope SFLLRN were employed to design and synthesize candidate ligands with appropriate groups attached to a rigid molecular scaffold. Prototype RWJ-53052 was identified and optimized via solid-phase parallel synthesis of chemical libraries. RWJ-56110 emerged as a potent, selective PAR-1 antagonist, devoid of PAR-1 agonist and thrombin inhibitory activity. It binds to PAR-1, interferes with PAR-1 calcium mobilization and cellular function (platelet aggregation; cell proliferation), and has no effect on PAR-2, PAR-3, or PAR-4. By flow cytometry, RWJ-56110 was confirmed as a direct inhibitor of PAR-1 activation and internalization, without affecting N-terminal cleavage. At high concentrations of α-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, albeit not in human platelets; whereas, at high concentrations of SFLLRN-NH2, RWJ-56110 blocked activation responses in both cell types. Thus, thrombin activates human platelets independently of PAR-1, i.e., through PAR-4, which we confirmed by PCR analysis. Selective PAR-1 antagonists, such as RWJ-56110, should serve as useful tools to study PARs and may have therapeutic potential for treating thrombosis and restenosis.

RGS proteins reconstitute the rapid gating kinetics of Gβγ-activated inwardly rectifying K+ channels

G protein-gated inward rectifier K+ (GIRK) channels mediate hyperpolarizing postsynaptic potentials in the nervous system and in the heart during activation of Gα(i/o)-coupled receptors. In neurons and cardiac atrial cells the time course for receptor-mediated GIRK current deactivation is 20–40 times faster than that observed in heterologous systems expressing cloned receptors and GIRK channels, suggesting that an additional component(s) is required to confer the rapid kinetic properties of the native transduction pathway. We report here that heterologous expression of “regulators of G protein signaling” (RGS proteins), along with cloned G protein-coupled receptors and GIRK channels, reconstitutes the temporal properties of the native receptor → GIRK signal transduction pathway. GIRK current waveforms evoked by agonist activation of muscarinic m2 receptors or serotonin 1A receptors were dramatically accelerated by coexpression of either RGS1, RGS3, or RGS4, but not RGS2. For the brain-expressed RGS4 isoform, neither the current amplitude nor the steady-state agonist dose-response relationship was significantly affected by RGS expression, although the agonist-independent “basal” GIRK current was suppressed by ≈40%. Because GIRK activation and deactivation kinetics are the limiting rates for the onset and termination of “slow” postsynaptic inhibitory currents in neurons and atrial cells, RGS proteins may play crucial roles in the timing of information transfer within the brain and to peripheral tissues.

Role of the Y5 neuropeptide Y receptor in limbic seizures

Neuropeptide Y (NPY) is an inhibitory neuromodulator expressed abundantly in the central nervous system that is suspected of being an endogenous antiepileptic agent that can control propagation of limbic seizures. Electrophysiological and pharmacological data suggest that these actions of NPY are mediated by G protein-coupled NPY Y2 and NPY Y5 receptors. To determine whether the NPY Y5 receptor (Y5R) is required for normal control of limbic seizures, we examined hippocampal function and responsiveness to kainic acid-induced seizures in Y5R-deficient (Y5R−/−) mice. We report that Y5R−/− mice do not exhibit spontaneous seizure-like activity; however, they are more sensitive to kainic acid-induced seizures. Electrophysiological examination of hippocampal slices from mutant mice revealed normal function, but the antiepileptic effects of exogenously applied NPY were absent. These data demonstrate that Y5R has an important role in mediating NPY’s inhibitory actions in the mouse hippocampus and suggest a role for Y5R in the control of limbic seizures.

Molecular uncoupling of C-C chemokine receptor 5-induced chemotaxis and signal transduction from HIV-1 coreceptor activity

The C-C chemokine receptor 5 (CCR5) plays a crucial role in facilitating the entry of macrophage-tropic strains of the HIV-1 into cells, but the mechanism of this phenomenon is completely unknown. To explore the role of CCR5-derived signal transduction in viral entry, we introduced mutations into two cytoplasmic domains of CCR5 involved in receptor-mediated function. Truncation of the terminal carboxyl-tail to eight amino acids or mutation of the highly conserved aspartate-arginine-tyrosine, or DRY, sequence in the second cytoplasmic loop of CCR5 effectively blocked chemokine-dependent activation of classic second messengers, intracellular calcium fluxes, and the cellular response of chemotaxis. In contrast, none of the mutations altered the ability of CCR5 to act as an HIV-1 coreceptor. We conclude that the initiation of signal transduction, the prototypic function of G protein coupled receptors, is not required for CCR5 to act as a coreceptor for HIV-1 entry into cells.

A dileucine motif in the C terminus of the β2-adrenergic receptor is involved in receptor internalization

The cytoplasmic C terminus of the β2-adrenergic receptor and many other G protein-coupled receptors contains a dileucine sequence that has been implicated in endosome/lysosome targeting of diverse proteins. In the present study, we provide evidence for an essential role of this motif in the agonist-induced internalization of the β2-adrenergic receptor. Mutation of Leu-339 and/or Leu-340 to Ala caused little changes in surface expression, ligand binding, G protein coupling, and signaling to adenylyl cyclase, when these receptors were transiently or stably expressed in CHO or HEK-293 cells. However, agonist-induced receptor internalization was markedly impaired in the L339,340A double mutant and reduced in the two single mutants. This impairment in receptor internalization was seen by using various approaches to determine internalization: binding of hydrophobic vs. hydrophilic ligands, loss of surface β2-adrenergic receptor immunoreactivity, and immunofluorescence microscopy. The selective effects of these mutations suggest that the C-terminal dileucine motif is involved in agonist-induced internalization of the β2-adrenergic receptor.

Regulation of dopaminergic pathways by retinoids: Activation of the D2 receptor promoter by members of the retinoic acid receptor–retinoid X receptor family

Dopamine is a neuromodulator involved in the control of key physiological functions. Dopamine-dependent signal transduction is activated through the interaction with membrane receptors of the seven-transmembrane domain G protein-coupled family. Among them, dopamine D2 receptor is highly expressed in the striatum and the pituitary gland as well as by mesencephalic dopaminergic neurons. Lack of D2 receptors in mice leads to a locomotor parkinsonian-like phenotype and to pituitary tumors. The D2 receptor promoter has characteristics of a housekeeping gene. However, the restricted expression of this gene to particular neurons and cells points to a strict regulation of its expression by cell-specific transcription factors. We demonstrate here that the D2 receptor promoter contains a functional retinoic acid response element. Furthermore, analysis of retinoic acid receptor-null mice supports our finding and shows that in these animals D2 receptor expression is reduced. This finding assigns to retinoids an important role in the control of gene expression in the central nervous system.

Testosterone Signaling through Internalizable Surface Receptors in Androgen Receptor-free Macrophages

Testosterone acts on cells through intracellular transcription-regulating androgen receptors (ARs). Here, we show that mouse IC-21 macrophages lack the classical AR yet exhibit specific nongenomic responses to testosterone. These manifest themselves as testosterone-induced rapid increase in intracellular free [Ca2+], which is due to release of Ca2+ from intracellular Ca2+ stores. This Ca2+ mobilization is also inducible by plasma membrane-impermeable testosterone-BSA. It is not affected by the AR blockers cyproterone and flutamide, whereas it is completely inhibited by the phospholipase C inhibitor U-73122 and pertussis toxin. Binding sites for testosterone are detectable on the surface of intact IC-21 cells, which become selectively internalized independent on caveolae and clathrin-coated vesicles upon agonist stimulation. Internalization is dependent on temperature, ATP, cytoskeletal elements, phospholipase C, and G-proteins. Collectively, our data provide evidence for the existence of G-protein-coupled, agonist-sequestrable receptors for testosterone in plasma membranes, which initiate a transcription-independent signaling pathway of testosterone.

Selection of Gβ Subunits with Point Mutations That Fail to Activate Specific Signaling Pathways In Vivo: Dissecting Cellular Responses Mediated by a Heterotrimeric G Protein in Dictyostelium discoideum

In Dictyostelium discoideum, a unique Gβ subunit is required for a G protein–coupled receptor system that mediates a variety of cellular responses. Binding of cAMP to cAR1, the receptor linked to the G protein G2, triggers a cascade of responses, including activation of adenylyl cyclase, gene induction, actin polymerization, and chemotaxis. Null mutations of the cAR1, Gα2, and Gβ genes completely impair all these responses. To dissect specificity in Gβγ signaling to downstream effectors in living cells, we screened a randomly mutagenized library of Gβ genes and isolated Gβ alleles that lacked the capacity to activate some effectors but retained the ability to regulate others. These mutant Gβ subunits were able to link cAR1 to G2, to support gene expression, and to mediate cAMP-induced actin polymerization, and some were able to mediate to chemotaxis toward cAMP. None was able to activate adenylyl cyclase, and some did not support chemotaxis. Thus, we separated in vivo functions of Gβγ by making point mutations on Gβ. Using the structure of the heterotrimeric G protein displayed in the computer program CHAIN, we examined the positions and the molecular interactions of the amino acids substituted in each of the mutant Gβs and analyzed the possible effects of each replacement. We identified several residues that are crucial for activation of the adenylyl cyclase. These residues formed an area that overlaps but is not identical to regions where bovine Gtβγ interacts with its regulators, Gα and phosducin.

The role of members of the pertussis toxin-sensitive family of G proteins in coupling receptors to the activation of the G protein-gated inwardly rectifying potassium channel

Inwardly rectifying potassium (K+) channels gated by G proteins (Kir3.x family) are widely distributed in neuronal, atrial, and endocrine tissues and play key roles in generating late inhibitory postsynaptic potentials, slowing the heart rate and modulating hormone release. They are directly activated by Gβγ subunits released from G protein heterotrimers of the Gi/o family upon appropriate receptor stimulation. Here we examine the role of isoforms of pertussis toxin (PTx)-sensitive G protein α subunits (Giα1–3 and GoαA) in mediating coupling between various receptor systems (A1, α2A, D2S, M4, GABAB1a+2, and GABAB1b+2) and the cloned counterpart of the neuronal channel (Kir3.1+3.2A). The expression of mutant PTx-resistant Gi/oα subunits in PTx-treated HEK293 cells stably expressing Kir3.1+3.2A allows us to selectively investigate that coupling. We find that, for those receptors (A1, α2A) known to interact with all isoforms, Giα1–3 and GoαA can all support a significant degree of coupling to Kir3.1+3.2A. The M4 receptor appears to preferentially couple to Giα2 while another group of receptors (D2S, GABAB1a+2, GABAB1b+2) activates the channel predominantly through Gβγ liberated from GoA heterotrimers. Interestingly, we have also found a distinct difference in G protein coupling between the two splice variants of GABAB1. Our data reveal selective pathways of receptor activation through different Gi/oα isoforms for stimulation of the G protein-gated inwardly rectifying K+ channel.