Dissecting Dopamine D2 Receptor Signaling

Dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) that activates G protein and arrestin signaling molecules. D2R antagonism has been a hallmark of antipsychotic medications for more than half a century. However, this drug-class is associated with substantial side effects that decrease quality of life and medication compliance. The development of novel antipsychotic medications with superior therapeutic and side effect profiles has been hampered in part due to a poor understanding of the specific D2R populations and downstream signaling molecules that must be blocked to confer therapeutic efficacy. It has been proposed that antipsychotic medications confer their effects through the blockade of arrestin but not G protein signaling downstream of D2R, and thus substantial efforts have gone towards the development of ligands that selectively block arrestin signaling. However, this approach suffers from several major limitations, namely that blockade of G protein signaling may also be important in conferring antipsychotic effects. Moreover, currently available pharmacological and genetic tools that have been used to probe G protein and arrestin signaling downstream of D2R in vivo suffer from on- and off-target effects that add substantial confounds to our understanding of these processes. Herein, we describe the development of several tools that can be used to probe G protein and arrestin-mediated processes in vivo with high specificity, as well as mechanisms by which these processes are activated

In vitro antimicrobial screening of methanolic extracts of Cleome chelidonii and Cleome gynandra

Antimicrobial activity of methanolic extracts of plants was screened by disc diffusion assay against four bacteria and four fungal cultures. Streptomycin (10 ?g/disc) and nystatin (10 ?g/disc) are used as standards for bacteria and fungi respectively. Minimum inhibitory concentration (MIC) of the extracts was evaluated through micro broth dilution method. The antimicrobial potency of plant extracts was assessed by their zone of inhibition and activity index values. Total activity of extracts was evaluated to quantitatively compare the activity of two plants. Methanolic extract of Cleome gynandra showed maximum antibacterial activity against Staphylococcus aureus (IZ- 22 ± 0.22 mm, AI-0.917, MIC- 0.039 mg/mL, MBC- 0.039 mg/mL). Maximum antifungal potential was shown by C. chelidonii against Candida albicans (IZ- 25 ± 0.92 mm, AI-1.000, MIC- 0.039 mg/mL, MFC- 0.039 mg/mL). Both the extracts exhibited good antimicrobial activity with low range of MIC

Arrestin recruitment to dopamine D2 receptor mediates locomotion but not incentive motivation

The dopamine (DA) D2 receptor (D2R) is an important target for the treatment of neuropsychiatric disorders such as schizophrenia and Parkinson's disease. However, the development of improved therapeutic strategies has been hampered by our incomplete understanding of this receptor's downstream signaling processes in vivo and how these relate to the desired and undesired effects of drugs. D2R is a G protein-coupled receptor (GPCR) that activates G protein-dependent as well as non-canonical arrestin-dependent signaling pathways. Whether these effector pathways act alone or in concert to facilitate specific D2R-dependent behaviors is unclear. Here, we report on the development of a D2R mutant that recruits arrestin but is devoid of G protein activity. When expressed virally in "indirect pathway" medium spiny neurons (iMSNs) in the ventral striatum of D2R knockout mice, this mutant restored basal locomotor activity and cocaine-induced locomotor activity in a manner indistinguishable from wild-type D2R, indicating that arrestin recruitment can drive locomotion in the absence of D2R-mediated G protein signaling. In contrast, incentive motivation was enhanced only by wild-type D2R, signifying a dissociation in the mechanisms that underlie distinct D2R-dependent behaviors, and opening the door to more targeted therapeutics

Development of a Rapid Insulin Assay by Homogenous Time-Resolved Fluorescence

Direct measurement of insulin is critical for basic and clinical studies of insulin secretion. However, current methods are expensive and time-consuming. We developed an insulin assay based on homogenous time-resolved fluorescence that is significantly more rapid and cost-effective than current commonly used approaches. This assay was applied effectively to an insulin secreting cell line, INS-1E cells, as well as pancreatic islets, allowing us to validate the assay by elucidating mechanisms by which dopamine regulates insulin release. We found that dopamine functioned as a significant negative modulator of glucose-stimulated insulin secretion. Further, we showed that bromocriptine, a known dopamine D2/D3 receptor agonist and newly approved drug used for treatment of type II diabetes mellitus, also decreased glucose-stimulated insulin secretion in islets to levels comparable to those caused by dopamine treatment

Angiotensin type 1A receptor regulates β-arrestin binding of the β2-adrenergic receptor via heterodimerization

Heterodimerization between angiotensin type 1A receptor (AT1R) and β2-adrenergic receptor (β2AR) has been shown to modulate G protein-mediated effects of these receptors. Activation of G protein-coupled receptors (GPCRs) leads to β-arrestin binding, desensitization, internalization and G protein-independent signaling of GPCRs. Our aim was to study the effect of heterodimerization on β-arrestin coupling. We found that β-arrestin binding of β2AR is affected by activation of AT1Rs. Costimulation with angiotensin II and isoproterenol markedly enhanced the interaction between β2AR and β-arrestins, by prolonging the lifespan of β2AR-induced β-arrestin2 clusters at the plasma membrane. While candesartan, a conventional AT1R antagonist, had no effect on the β-arrestin2 binding to β2AR, TRV120023, a β-arrestin biased agonist, enhanced the interaction. These findings reveal a new crosstalk mechanism between AT1R and β2AR, and suggest that enhanced β-arrestin2 binding to β2AR can contribute to the pharmacological effects of biased AT1R agonists. © 201

Dissecting Dopamine D2 Receptor Signaling

Dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) that activates G protein and arrestin signaling molecules. D2R antagonism has been a hallmark of antipsychotic medications for more than half a century. However, this drug-class is associated with substantial side effects that decrease quality of life and medication compliance. The development of novel antipsychotic medications with superior therapeutic and side effect profiles has been hampered in part due to a poor understanding of the specific D2R populations and downstream signaling molecules that must be blocked to confer therapeutic efficacy. It has been proposed that antipsychotic medications confer their effects through the blockade of arrestin but not G protein signaling downstream of D2R, and thus substantial efforts have gone towards the development of ligands that selectively block arrestin signaling. However, this approach suffers from several major limitations, namely that blockade of G protein signaling may also be important in conferring antipsychotic effects. Moreover, currently available pharmacological and genetic tools that have been used to probe G protein and arrestin signaling downstream of D2R in vivo suffer from on- and off-target effects that add substantial confounds to our understanding of these processes. Herein, we describe the development of several tools that can be used to probe G protein and arrestin-mediated processes in vivo with high specificity, as well as mechanisms by which these processes are activated

A criterion for selecting the probability density function of best fit for hydrologic data

Ph.D.James R. Wallac