Val J. Watts

Polydopamine-Based Simple and Versatile Surface Modification of Polymeric Nano Drug Carriers

The surface of a polymeric nanoparticle (NP) is often functionalized with cell-interactive ligands and/or additional polymeric layers to control NP interaction with cells and proteins. However, such modification is not always straightforward when the surface is not chemically reactive. For this reason, most NP functionalization processes employ reactive linkers or coupling agents or involve prefunctionalization of the polymer, which are complicated and inefficient. Moreover, prefunctionalized polymers can lose the ability to encapsulate and retain a drug if the added ligands change the chemical properties of the polymer. To overcome this challenge, we use dopamine polymerization as a way of functionalizing NP surfaces. This method includes brief incubation of the preformed NPs in a weak alkaline solution of dopamine, followed by secondary incubation with desired ligands. Using this method, we have functionalized poly(lactic-co-glycolic acid) (PLGA) NPs with three representative surface modifiers: a small molecule (folate), a peptide (Arg-Gly-Asp), and a polymer [poly(carboxybetaine methacrylate)]. We confirmed that the modified NPs showed the expected cellular interactions with no cytotoxicity or residual bioactivity of dopamine. The dopamine polymerization method is a simple and versatile surface modification method, applicable to a variety of NP drug carriers irrespective of their chemical reactivity and the types of ligands.

Ligand-Dependent Oligomerization of Dopamine D2 and Adenosine A2A Receptors in Living Neuronal Cells

Adenosine A2A and dopamine D2 receptors (A2A and D2) associate in homo- and heteromeric complexes in the striatum, providing a structural basis for their mutual antagonism. At the cellular level, the portion of receptors engaging in homo- and heteromers, as well as the effect of persistent receptor activation or antagonism on the cell oligomer repertoire, are largely unknown. We have used bimolecular fluorescence complementation (BiFC) to visualize A2A and D2 oligomerization in the Cath.a differentiated neuronal cell model. Receptor fusions to BiFC fluorescent protein fragments retained their function when expressed alone or in A2A/A2A, D2/D2, and A2A/D2 BiFC pairs. Robust fluorescence complementation reflecting A2A/D2 heteromers was detected at the cell membrane as well as in endosomes. In contrast, weaker BiFC signals, largely confined to intracellular domains, were detected with A2A/dopamine D1 BiFC pairs. Multicolor BiFC was used to simultaneously visualize A2A and D2 homo- and heteromers in living cells and to examine drug-induced changes in receptor oligomers. Prolonged D2 stimulation with quinpirole lead to the internalization of D2/D2 and A2A/D2 oligomers and resulted in decreased A2A/D2 relative to A2A/A2A oligomer formation. Opposing effects were observed in cells treated with D2 antagonists or with the A2A agonist 5′-N-methylcarboxamidoadenosine (MECA). Subsequent radioreceptor binding analysis indicated that the drug-induced changes in oligomer formation were not readily explained by alterations in receptor density. These observations support the hypothesis that long-term drug exposure differentially alters A2A/D2 receptor oligomerization and provide the first demonstration for the use of BiFC to monitor drug-modulated GPCR oligomerization.

Adenosine A2A receptors assemble into higher‐order oligomers at the plasma membrane

MINT‐6797156, MINT‐6797142: A2AR (uniprotkb:P29274) physically interacts (MI:0218) with A2AR (uniprotkb:P29274) by bimolecular fluorescence complementation (MI:0809) MINT‐6797129: A2AR (uniprotkb:P29274) physically interacts (MI:0218) with A2AR (uniprotkb:P29274) by fluorescent resonance energy transfer (MI:0055)

A “Genome-to-Lead” Approach for Insecticide Discovery: Pharmacological Characterization and Screening of Aedes aegypti D1-like Dopamine Receptors

Background Many neglected tropical infectious diseases affecting humans are transmitted by arthropods such as mosquitoes and ticks. New mode-of-action chemistries are urgently sought to enhance vector management practices in countries where arthropod-borne diseases are endemic, especially where vector populations have acquired widespread resistance to insecticides. Methodology/Principal Findings We describe a “genome-to-lead” approach for insecticide discovery that incorporates the first reported chemical screen of a G protein-coupled receptor (GPCR) mined from a mosquito genome. A combination of molecular and pharmacological studies was used to functionally characterize two dopamine receptors (AaDOP1 and AaDOP2) from the yellow fever mosquito, Aedes aegypti. Sequence analyses indicated that these receptors are orthologous to arthropod D1-like (Gαs-coupled) receptors, but share less than 55% amino acid identity in conserved domains with mammalian dopamine receptors. Heterologous expression of AaDOP1 and AaDOP2 in HEK293 cells revealed dose-dependent responses to dopamine (EC50: AaDOP1 = 3.1±1.1 nM; AaDOP2 = 240±16 nM). Interestingly, only AaDOP1 exhibited sensitivity to epinephrine (EC50 = 5.8±1.5 nM) and norepinephrine (EC50 = 760±180 nM), while neither receptor was activated by other biogenic amines tested. Differential responses were observed between these receptors regarding their sensitivity to dopamine agonists and antagonists, level of maximal stimulation, and constitutive activity. Subsequently, a chemical library screen was implemented to discover lead chemistries active at AaDOP2. Fifty-one compounds were identified as “hits,” and follow-up validation assays confirmed the antagonistic effect of selected compounds at AaDOP2. In vitro comparison studies between AaDOP2 and the human D1 dopamine receptor (hD1) revealed markedly different pharmacological profiles and identified amitriptyline and doxepin as AaDOP2-selective compounds. In subsequent Ae. aegypti larval bioassays, significant mortality was observed for amitriptyline (93%) and doxepin (72%), confirming these chemistries as “leads” for insecticide discovery. Conclusions/Significance This research provides a “proof-of-concept” for a novel approach toward insecticide discovery, in which genome sequence data are utilized for functional characterization and chemical compound screening of GPCRs. We provide a pipeline useful for future prioritization, pharmacological characterization, and expanded chemical screening of additional GPCRs in disease-vector arthropods. The differential molecular and pharmacological properties of the mosquito dopamine receptors highlight the potential for the identification of target-specific chemistries for vector-borne disease management, and we report the first study to identify dopamine receptor antagonists with in vivo toxicity toward mosquitoes.

Ligand-Induced Regulation and Localization of Cannabinoid CB1 and Dopamine D2L Receptor Heterodimers

The cannabinoid CB1 (CB1) and dopamine D2 (D2) receptors are coexpressed in the basal ganglia, an area of the brain involved in such processes as cognition, motor function, and emotional control. Several lines of evidence suggest that CB1 and D2 receptors may oligomerize, providing a unique pharmacology in vitro and in vivo. However, limited information exists on the regulation of CB1 and D2 receptor dimers. We used a novel technique, multicolor bimolecular fluorescence complementation (MBiFC) to examine the subcellular localization of CB1-D2L heterodimers as well as D2L-D2L homodimers in a neuronal cell model, Cath. a differentiated cells. MBiFC was then used to explore the effects of persistent ligand treatment on receptor dimerization at the plasma membrane and intracellularly. Persistent (20-h) agonist treatment resulted in increased formation of CB1-D2L heterodimers relative to the D2L-D2L homodimers. The effects of the D2 agonist quinpirole were restricted to the intracellular compartment and may reflect increased D2L receptor expression. In contrast, treatment with the CB1 receptor agonist (2)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol (CP55, 940) produced increases in both membrane and intracellular CB1-D2L heterodimers independently of alterations in CB1 receptor expression. The effects of CB1 receptor activation were attenuated by the CB1 antagonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide (AM281) and were both time- and dose-dependent. The effects of CB1 activation were examined further by combining MBiFC with a constitutively active CB1 receptor mutant, CB1T210I. These studies demonstrated that the expression of CB1T210I increased intracellular CB1-D2L heterodimer formation. In summary, agonist-induced modulation of CB1-D2L oligomerization may have physiological implications in diseases such as Parkinsons disease and drug abuse.

Fluorescent and Bioluminescent Protein-Fragment Complementation Assays in the Study of G Protein-Coupled Receptor Oligomerization and Signaling

Most cellular functions, including signaling by G protein-coupled receptors (GPCRs), are mediated by protein-protein interactions, making the identification and localization of protein complexes key to the understanding of cellular processes. In complement to traditional biochemical techniques, noninvasive resonance energy transfer (RET) and protein-fragment complementation assays (PCAs) now allow protein interactions to be detected in the context of living cells. In this review, fluorescent and bioluminescent PCAs are discussed and their application illustrated with studies on GPCR signaling. Newly developed techniques combining PCA and RET assays for the detection of ternary and quaternary protein complexes are also presented.

A Point Mutation to Gαi Selectively Blocks GoLoco Motif Binding DIRECT EVIDENCE FOR Gα·GoLoco COMPLEXES IN MITOTIC SPINDLE DYNAMICS

Heterotrimeric G-protein Gα subunits and GoLoco motif proteins are key members of a conserved set of regulatory proteins that influence invertebrate asymmetric cell division and vertebrate neuroepithelium and epithelial progenitor differentiation. GoLoco motif proteins bind selectively to the inhibitory subclass (Gαi) of Gα subunits, and thus it is assumed that a Gαi·GoLoco motif protein complex plays a direct functional role in microtubule dynamics underlying spindle orientation and metaphase chromosomal segregation during cell division. To address this hypothesis directly, we rationally identified a point mutation to Gαi subunits that renders a selective loss-of-function for GoLoco motif binding, namely an asparagine-to-isoleucine substitution in the αD-αE loop of the Gα helical domain. This GoLoco-insensitivity (“GLi”) mutation prevented Gαi1 association with all human GoLoco motif proteins and abrogated interaction between the Caenorhabditis elegans Gα subunit GOA-1 and the GPR-1 GoLoco motif. In contrast, the GLi mutation did not perturb any other biochemical or signaling properties of Gαi subunits, including nucleotide binding, intrinsic and RGS protein-accelerated GTP hydrolysis, and interactions with Gβγ dimers, adenylyl cyclase, and seven transmembrane-domain receptors. GoLoco insensitivity rendered Gαi subunits unable to recruit GoLoco motif proteins such as GPSM2/LGN and GPSM3 to the plasma membrane, and abrogated the exaggerated mitotic spindle rocking normally seen upon ectopic expression of wild type Gαi subunits in kidney epithelial cells. This GLi mutation should prove valuable in establishing the physiological roles of Gαi·GoLoco motif protein complexes in microtubule dynamics and spindle function during cell division as well as to delineate potential roles for GoLoco motifs in receptor-mediated signal transduction.

Sensitization of adenylate cyclase: A general mechanism of neuroadaptation to persistent activation of Gαi/o-coupled receptors?

Acute activation of Galphas-coupled receptors stimulates cyclic AMP accumulation leading to the activation of downstream signaling cascades. These Galphas-mediated events can be countered by acute activation of inhibitory G proteins (Galpha(i/o)), which inhibit the activity of adenylate cyclase, thereby attenuating cyclic AMP accumulation. Furthermore, an additional, less direct mechanism for Galpha(i/o) proteins modulation of cyclic AMP signaling also has been described. Persistent activation of several Galpha(i/o)-coupled receptors has been shown to result in a subsequent paradoxical enhancement of adenylate cyclase activity in response to drug-stimulated cyclic AMP accumulation. This sensitization of adenylate cyclase likely represents a cellular adaptive response following prolonged activation of inhibitory receptors. Recent advances in our knowledge of G protein signaling, adenylate cyclase regulation, and other cellular signaling mechanisms have extensively increased our insight into this phenomenon. It is now thought that sensitization occurs as part of a compensatory mechanism by which the cell adapts to chronic inhibitory input. Such a mechanism may be involved in modulating Galphas-coupled receptor signaling following neurotransmitter elevations that occur in psychiatric disease states or following the administration of many drugs of abuse. This review will focus on recent advances in the understanding of molecular signaling pathways that are involved in sensitization and describe the potential role of sensitization in neuronal cell function.

Dexamethasone-Induced Ras Protein 1 Negatively Regulates Protein Kinase C δ: Implications for Adenylyl Cyclase 2 Signaling

We identified dexamethasone-induced Ras protein 1 (Dexras1) as a negative regulator of protein kinase C (PKC) δ, and the consequences of this regulation have been examined for adenylyl cyclase (EC 4.6.1.1) type 2 (AC2) signaling. Dexras1 expression in human embryonic kidney 293 cells completely abolished dopamine D2 receptor-mediated potentiation of AC2 activity, which is consistent with previous reports of its ability to block receptor-mediated Gβγ signaling pathways. In addition, Dexras1 significantly reduced phorbol 12-myristate 13-acetate (PMA)-stimulated AC2 activity but did not alter Gαs-mediated cAMP accumulation. Dexras1 seemed to inhibit PMA stimulation of AC2 by interfering with PKCδ autophosphorylation. This effect was selective for the δ isoform because Dexras1 did not alter autophosphorylation of PKCα or PKCϵ. Dexras1 disruption of PKCδ autophosphorylation resulted in a significant blockade of PKC kinase activity as measured by [γ-32P]ATP incorporation using a PKC-specific substrate. Moreover, Dexras1 and PKCδ coimmunoprecipitated from whole-cell lysates. Dexras1 did not alter the membrane translocation of PKCδ; however, the ability of Dexras1 to interfere with PKCδ autophosphorylation was isoprenylation-dependent as determined using the farnesyltransferase inhibitor methyl {N-[2-phenyl-4-N [2(R)-amino-3-mecaptopropylamino] benzoyl]}-methionate (FTI-277) and a CAAX box-deficient Dexras1 (C277S) mutant. PMA-stimulated AC2 activity was also not affected by Dexras1 C277S. Taken as a whole, these data suggest that Dexras1 functionally interacts with PKCδ at the cellular membrane through an isoprenylation-dependent mechanism to negatively regulate PKCδ activity. Moreover our study suggests that Dexras1 acts to modulate the activation of AC2 in an indirect fashion by inhibiting both Gβγ- and PKC-stimulated AC2 activity. The current study provides a novel role for Dexras1 in signal transduction.

Hexahydrobenzo[a]phenanthridines: novel dopamine D3 receptor ligands.

We report that certain substituted hexahydrobenzo[a]phenanthridines are novel high affinity ligands selective for the dopamine D3 receptor. These data demonstrate that substitutions on the heterocyclic nitrogen and the pendant phenyl ring of this nucleus cause a marked increase in both affinity and selectivity for dopamine D3 vs. D2 receptors. Thus, these compounds represent important new tools to study the pharmacology of dopamine D3 receptors, and may also provide an opportunity for the synthesis of new radioligands for dopamine D3 receptors.