Ali Salahpour

Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET).

Heptahelical receptors that interact with heterotrimeric G proteins represent the largest family of proteins involved in signal transduction across biological membranes. Although these receptors generally were believed to be monomeric entities, a growing body of evidence suggests that they may form functionally relevant dimers. However, a definitive demonstration of the existence of G protein-coupled receptor (GPCR) dimers at the surface of living cells is still lacking. Here, using bioluminescence resonance energy transfer (BRET), as a protein-protein interaction assay in whole cells, we unambiguously demonstrate that the human beta(2)-adrenergic receptor (beta(2)AR) forms constitutive homodimers when expressed in HEK-293 cells. Receptor stimulation with the hydrophilic agonist isoproterenol led to an increase in the transfer of energy between beta(2)AR molecules genetically fused to the BRET donor (Renilla luciferase) and acceptor (green fluorescent protein), respectively, indicating that the agonist interacts with receptor dimers at the cell surface. Inhibition of receptor internalization did not prevent agonist-promoted BRET, demonstrating that it did not result from clustering of receptors within endosomes. The notion that receptor dimers exist at the cell surface was confirmed further by the observation that BS3, a cell-impermeable cross-linking agent, increased BRET between beta(2)AR molecules. The selectivity of the constitutive interaction was documented by demonstrating that no BRET occurred between the beta(2)AR and two other unrelated GPCR. In contrast, the well characterized agonist-dependent interaction between the beta(2)AR and the regulatory protein beta-arrestin could be monitored by BRET. Taken together, the data demonstrate that GPCR exist as functional dimers in vivo and that BRET-based assays can be used to study both constitutive and hormone-promoted selective protein-protein interactions.

Pharmacological chaperones rescue cell-surface expression and function of misfolded V2 vasopressin receptor mutants

Over 150 mutations within the coding sequence of the V2 vasopressin receptor (V2R) gene are known to cause nephrogenic diabetes insipidus (NDI). A large number of these mutant receptors fail to fold properly and therefore are not routed to the cell surface. Here we show that selective, nonpeptidic V2R antagonists dramatically increase cell-surface expression and rescue the function of 8 mutant NDI-V2Rs by promoting their proper folding and maturation. A cell-impermeant V2R antagonist could not mimic these effects and was unable to block the rescue mediated by a permeant agent, indicating that the nonpeptidic antagonists act intracellularly, presumably by binding to and stabilizing partially folded mutants. In addition to opening new therapeutic avenues for NDI patients, these data demonstrate that by binding to newly synthesized mutant receptors, small ligands can act as pharmacological chaperones, promoting the proper folding and maturation of receptors and their targeting to the cell surface.

Antagonism of dopamine D2 receptor/β-arrestin 2 interaction is a common property of clinically effective antipsychotics

Since the unexpected discovery of the antipsychotic activity of chlorpromazine, a variety of therapeutic agents have been developed for the treatment of schizophrenia. Despite differences in their activities at various neurotransmitter systems, all clinically effective antipsychotics share the ability to interact with D2 class dopamine receptors (D2R). D2R mediate their physiological effects via both G protein-dependent and independent (β-arrestin 2-dependent) signaling, but the role of these D2R-mediated signaling events in the actions of antipsychotics remains unclear. We demonstrate here that while different classes of antipsychotics have complex pharmacological profiles at G protein-dependent D2R long isoform (D2LR) signaling, they share the common property of antagonizing dopamine-mediated interaction of D2LR with β-arrestin 2. Using two cellular assays based on a bioluminescence resonance energy transfer (BRET) approach, we demonstrate that a series of antipsychotics including haloperidol, clozapine, aripiprazole, chlorpromazine, quetiapine, olanzapine, risperidone, and ziprasidone all potently antagonize the β-arrestin 2 recruitment to D2LR induced by quinpirole. However, these antipsychotics have various effects on D2LR mediated Gi/o protein activation ranging from inverse to partial agonists and antagonists with highly variable efficacies and potencies at quinpirole-induced cAMP inhibition. These results suggest that the different classes of clinically effective antipsychotics share a common molecular mechanism involving inhibition of D2LR/β-arrestin 2 mediated signaling. Thus, selective targeting of D2LR/β-arrestin 2 interaction and related signaling pathways may provide new opportunities for antipsychotic development.

Regulation of Akt Signaling by D2 and D3 Dopamine Receptors In Vivo

The serine/threonine kinase Akt is a downstream target of dopamine receptor signaling that is inhibited/dephosphorylated in response to direct and indirect dopamine receptor agonists. Although pharmacological studies uncovered the involvement of D2-class dopamine receptors in Akt regulation, they did not identify the role of individual receptor subtypes in this process. Here we used knock-out mice lacking the D1, D2, D2 long, or D3 dopamine receptors as well as a D4 receptor-selective antagonist to address the function of each of these receptors in the regulation of Akt in vivo. Under basal conditions, D2, D2 long, and D3 knock-out mice display enhanced striatal Akt activation, whereas D1 knock-out mice and mice treated with the D4 receptor antagonist L745870 (3-[[4-(4-chlorophenyl)piperazin-1-yl]methyl]-1H-pyrrolo[2,3-b]pyridine trihydrochloride) have phospho-Akt levels comparable with those of normal control animals. Furthermore, both amphetamine and apomorphine lose their ability to inhibit Akt in D2 knock-out mice but retain their normal effect on this signaling molecule in D1 knock-out animals. Finally, D3 knock-out mice show a reduced sensitivity of Akt-mediated signaling to dopaminergic drugs but retain the action of these drugs on Akt at high dose regimens. These results indicate that D2 receptors are essential for the inhibition of Akt by dopamine and that D3 receptors also participate in this signaling potentially by enhancing D2 receptor response. Identification of the functions of individual dopamine receptor subtypes in Akt regulation may help the development of new pharmaceutical approaches for mental disorders related to abnormal dopamine transmission such as bipolar disorder and schizophrenia.

Pharmacologic Chaperones as a Potential Treatment for X-Linked Nephrogenic Diabetes Insipidus

In many mendelian diseases, some mutations result in the synthesis of misfolded proteins that cannot reach a transport-competent conformation. In X-linked nephrogenic diabetes insipidus, most of the mutant vasopressin 2 (V2) receptors are trapped in the endoplasmic reticulum and degraded. They are unable to reach the plasma membrane and promote water reabsorption through the principal cells of the collecting ducts. Herein is reported two types of experiments: In vivo studies to assess clinically a short-term treatment with a nonpeptide V1a receptor antagonist (SR49059) and in vitro studies in cultured cell systems. In patients, SR49059 decreased 24- h urine volume (11.9 +/- 2.3 to 8.2 +/- 2.0 L; P = 0.005) and water intake (10.7 +/- 1.9 to 7.2 +/- 1.6 L; P < 0.05). Maximum increase in urine osmolality was observed on day 3 (98 +/- 22 to 170 +/- 52 mOsm/kg; P = 0.05). Sodium, potassium, and creatinine excretions and plasma sodium were constant throughout the study. In vitro studies indicate that the nonpeptide V1a receptor antagonist SR49059 and the V1a/V2 receptor antagonist YM087 (Conivaptan) rescued cell surface expression and function of mutant V2 receptors. Mutant V2 receptors with nonsense mutations were not affected by the treatment. Misfolded V2 receptor mutants were rescued in vitro and also in vivo by nonpeptide antagonists. This therapeutic approach could be applied to the treatment of several hereditary diseases that result from errors in protein folding and kinesis.

Smoothened Signal Transduction Is Promoted by G Protein-Coupled Receptor Kinase 2

ABSTRACT Deregulation of the Sonic hedgehog pathway has been implicated in an increasing number of human cancers. In this pathway, the seven-transmembrane (7TM) signaling protein Smoothened regulates cellular proliferation and differentiation through activation of the transcription factor Gli. The activity of mammalian Smoothened is controlled by three different hedgehog proteins, Indian, Desert, and Sonic hedgehog, through their interaction with the Smoothened inhibitor Patched. However, the mechanisms of signal transduction from Smoothened are poorly understood. We show that a kinase which regulates signaling by many “conventional” 7TM G-protein-coupled receptors, G protein-coupled receptor kinase 2 (GRK2), participates in Smoothened signaling. Expression of GRK2, but not catalytically inactive GRK2, synergizes with active Smoothened to mediate Gli-dependent transcription. Moreover, knockdown of endogenous GRK2 by short hairpin RNA (shRNA) significantly reduces signaling in response to the Smoothened agonist SAG and also inhibits signaling induced by an oncogenic Smoothened mutant, Smo M2. We find that GRK2 promotes the association between active Smoothened and β-arrestin 2. Indeed, Gli-dependent signaling, mediated by coexpression of Smoothened and GRK2, is diminished by β-arrestin 2 knockdown with shRNA. Together, these data suggest that GRK2 plays a positive role in Smoothened signaling, at least in part, through the promotion of an association between β-arrestin 2 and Smoothened.

Functional interaction between trace amine-associated receptor 1 and dopamine D2 receptor.

The ability of dopamine receptors to interact with other receptor subtypes may provide mechanisms for modulating dopamine-related functions and behaviors. In particular, there is evidence suggesting that the trace amine-associated receptor 1 (TAAR1) affects the dopaminergic system by regulating the firing rate of dopaminergic neurons or by altering dopamine D2 receptor (D2R) responsiveness to ligands. TAAR1 is a Gαs protein-coupled receptor that is activated by biogenic amines, “trace amines,” such as β-phenylethylamine (β-PEA) and tyramine that are normally found at low concentrations in the mammalian brain. In the present study, we investigated the biochemical mechanism of interaction between TAAR1 and D2R and the role this interaction plays in D2R-related signaling and behaviors. Using a bioluminescence resonance energy transfer biosensor for cAMP, we demonstrated that the D2R antagonists haloperidol, raclopride, and amisulpride were able to enhance selectively a TAAR1-mediated β-PEA increase of cAMP. Moreover, TAAR1 and D2R were able to form heterodimers when coexpressed in human embryonic kidney 293 cells, and this direct interaction was disrupted in the presence of haloperidol. In addition, in mice lacking TAAR1, haloperidol-induced striatal c-Fos expression and catalepsy were significantly reduced. Taken together, these data suggest that TAAR1 and D2R have functional and physical interactions that could be critical for the modulation of the dopaminergic system by TAAR1 in vivo.

Increased vesicular monoamine transporter enhances dopamine release and opposes Parkinson disease-related neurodegeneration in vivo

Significance Several therapeutic strategies have been used to enhance monoamine neurotransmitter signaling. However, many of these interventions have deleterious side effects or lose effectiveness due to off-target actions and system feedback. These undesirable consequences likely occur because of temporal dysregulation of neurotransmitter release and uptake. We demonstrate that increasing vesicular packaging enhances dopamine neurotransmission without this signaling disruption. Mice with elevated vesicular monoamine transporter display increased dopamine release, improved outcomes on anxiety and depressive behaviors, enhanced locomotion, and protection from a Parkinson disease-related neurotoxic insult. The malleable nature of the dopamine vesicle suggests that interventions aimed at enhancing vesicle filling may be of therapeutic benefit. Disruption of neurotransmitter vesicle dynamics (transport, capacity, release) has been implicated in a variety of neurodegenerative and neuropsychiatric conditions. Here, we report a novel mouse model of enhanced vesicular function via bacterial artificial chromosome (BAC)-mediated overexpression of the vesicular monoamine transporter 2 (VMAT2; Slc18a2). A twofold increase in vesicular transport enhances the vesicular capacity for dopamine (56%), dopamine vesicle volume (33%), and basal tissue dopamine levels (21%) in the mouse striatum. The elevated vesicular capacity leads to an increase in stimulated dopamine release (84%) and extracellular dopamine levels (44%). VMAT2-overexpressing mice show improved outcomes on anxiety and depressive-like behaviors and increased basal locomotor activity (41%). Finally, these mice exhibit significant protection from neurotoxic insult by the dopaminergic toxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), as measured by reduced dopamine terminal damage and substantia nigra pars compacta cell loss. The increased release of dopamine and neuroprotection from MPTP toxicity in the VMAT2-overexpressing mice suggest that interventions aimed at enhancing vesicular capacity may be of therapeutic benefit in Parkinson disease.

BRET biosensors to study GPCR biology, pharmacology, and signal transduction.

Bioluminescence resonance energy transfer (BRET)-based biosensors have been extensively used over the last decade to study protein–protein interactions and intracellular signal transduction in living cells. In this review, we discuss the various BRET biosensors that have been developed to investigate biology, pharmacology, and signaling of G protein-coupled receptors (GPCRs). GPCRs form two distinct types of multiprotein signal transduction complexes based upon their inclusion of G proteins or β-arrestins that can be differentially affected by drugs that exhibit functional selectivity toward G protein or β-arrestin signaling. BRET has been especially adept at illuminating the dynamics of protein–protein interactions between receptors, G proteins, β-arrestins, and their many binding partners in living cells; as well as measuring the formation and accumulation of second messengers following receptor activation. Specifically, we discuss in detail the application of BRET to study dopamine and trace amine receptors signaling, presenting examples of an exchange protein activated by cAMP biosensor to measure cAMP, β-arrestin biosensors to determine β-arrestin recruitment to the receptor, and dopamine D2 receptor and trace amine-associated receptor 1 biosensors to investigate heterodimerization between them. As the biochemical spectrum of BRET biosensors expands, the number of signaling pathways that can be measured will concomitantly increase. This will be particularly useful for the evaluation of functional selectivity in which the real-time BRET capability to measure distinct signaling modalities will dramatically shorten the time to characterize new generation of biased drugs. These emerging approaches will further expand the growing application of BRET in the screening for novel pharmacologically active compounds.

Biochemical characterization of β2-Adrenergic receptor dimers and oligomers

Abstract G Protein-coupled receptor dimerization/oligomerization has been well established during the last several years. Studies have demonstrated the existence of dimers/digomers both in vitro and in living cells. However, a thorough characterization of the biochemical nature of receptor dimers and oligomers as well as their occurrence at the cell surface has not been properly addressed. In this study, we show that both β2-adrenergic receptor (β2AR) dimers and oligomers exist at the plasma membrane and that the detection of such species, following receptor solubilization and resolution by denaturing polyacrylamide gel electrophoresis (SDS-PAGE), does not result from the formation of spurious disulfide bonds during cell lysis. Moreover, our results indicate that the biochemical nature of β2AR dimers is different from that of the oligomers. Although both complexes are partially resistant to SDS denaturation, disulfide bonding is absolutely required for the stability of β2AR oligomers but not dimers in SDS-PAGE. Indeed, dimeric species can be detected even in the presence of high concentrations of reducing and alkylating agents. Although the different biochemical nature of the dimers and oligomers may be indicative of distinct biological roles in cells, additional studies will be required to further elucidate the biosynthesis and function of these receptor forms.