Guidelines for Opioid Prescribing in Children and Adolescents After Surgery: An Expert Panel Opinion.

Importance: Opioids are frequently prescribed to children and adolescents after surgery. Prescription opioid misuse is associated with high-risk behavior in youth. Evidence-based guidelines for opioid prescribing practices in children are lacking. Objective: To assemble a multidisciplinary team of health care experts and leaders in opioid stewardship, review current literature regarding opioid use and risks unique to pediatric populations, and develop a broad framework for evidence-based opioid prescribing guidelines for children who require surgery. Evidence Review: Reviews of relevant literature were performed including all English-language articles published from January 1, 1988, to February 28, 2019, found via searches of the PubMed (MEDLINE), CINAHL, Embase, and Cochrane databases. Pediatric was defined as children younger than 18 years. Animal and experimental studies, case reports, review articles, and editorials were excluded. Selected articles were graded using tools from the Oxford Centre for Evidence-based Medicine 2011 levels of evidence. The Appraisal of Guidelines for Research & Evaluation (AGREE) II instrument was applied throughout guideline creation. Consensus was determined using a modified Delphi technique. Findings: Overall, 14 574 articles were screened for inclusion, with 217 unique articles included for qualitative synthesis. Twenty guideline statements were generated from a 2-day in-person meeting and subsequently reviewed, edited, and endorsed externally by pediatric surgical specialists, the American Pediatric Surgery Association Board of Governors, the American Academy of Pediatrics Section on Surgery Executive Committee, and the American College of Surgeons Board of Regents. Review of the literature and guideline statements underscored 3 primary themes: (1) health care professionals caring for children who require surgery must recognize the risks of opioid misuse associated with prescription opioids, (2) nonopioid analgesic use should be optimized in the perioperative period, and (3) patient and family education regarding perioperative pain management and safe opioid use practices must occur both before and after surgery. Conclusions and Relevance: These are the first opioid-prescribing guidelines to address the unique needs of children who require surgery. Health care professionals caring for children and adolescents in the perioperative period should optimize pain management and minimize risks associated with opioid use by engaging patients and families in opioid stewardship efforts.

Applying Experiential Learning to Career Development Training for Biomedical Graduate Students and Postdocs: Perspectives on Program Development and Design.

Experiential learning is an effective educational tool across many academic disciplines, including career development. Nine different institutions bridged by the National Institutes of Health Broadening Experiences in Scientific Training Consortium compared their experiments in rethinking and expanding training of predoctoral graduate students and postdoctoral scholars in the biomedical sciences to include experiential learning opportunities. In this article, we provide an overview of the four types of experiential learning approaches our institutions offer and compare the learning objectives and evaluation strategies employed for each type. We also discuss key factors for shaping experiential learning activities on an institutional level. The framework we provide can help organizations determine which form of experiential learning for career training might best suit their institutions and goals and aid in the successful design and delivery of such training.

Price of High-Throughput 25-Hydroxyvitamin D Immunoassays: Frequency of Inaccurate Results.

BACKGROUND: With a 10-year sustained increase in 25-hydroxyvitamin D [25(OH)D] testing, laboratories have swapped their LC-MS/MS methods for high-throughput automated immunoassays. Although it is generally well-known that immunoassays have poor recoveries for 25-hydroxyvitamin D2 [25(OH)D2], the frequency and extent to which this impacts total 25(OH)D have not been previously demonstrated. We evaluated 3 automated immunoassays against the first FDA-cleared CDC/NIST-traceable LC-MS/MS method. METHODS: Method comparison was performed for the Siemens ADVIA Centaur, Roche Elecsys Cobas, and Abbott Architect 25(OH)D immunoassay methods in real patient samples (n = 105). We calculated the mean bias in samples containing >20 ng/mL 25(OH)D2 and estimated the percent 25(OH)D2 cross-reactivities. We determined the prevalence of appreciable concentrations of 25(OH)D2 in our patient population through random sampling (n = 120) and projected the frequency of inaccurate 25(OH)D immunoassay results. RESULTS: Linear regression for 25(OH)D was y = 1.09x - 4.44 (Centaur), y = 0.84 + 0.43 (Cobas), and y = 0.83x - 0.48 (Architect). The mean biases of 25(OH)D concentrations were 5.6 (11.0) ng/mL (Centaur), -17.5 (7.2) ng/mL (Cobas), and -20.3 (9.8) ng/mL (Architect) in samples containing >20 ng/mL 25(OH)D2. The observed percent cross-reactivities for 25(OH)D2 were 115% (Centaur), 52% (Cobas), and 44% (Architect). We estimate that 8% of our population has >20 ng/mL 25(OH)D2, thereby compromising the accuracy of 25(OH)D results in >3000 samples annually. CONCLUSIONS: We demonstrate that immunoassay manufacturer package inserts indicate much better recoveries of 25(OH)D2 than what is observed in unadulterated real patient samples. We estimate the frequency of inaccurate total 25(OH)D determination by these immunoassay methods to be largely dependent on the concentration of 25(OH)D2 in each sample.

The role of personality traits and barriers to mental health treatment seeking among college students.

Many college students experience a mental health problem yet do not seek treatment from a mental health professional. In the present study, we examined how perceived barriers (stigma perceptions, negative attitudes about treatment, and perceptions of practical barriers), as well as the Big Five personality traits, relate to treatment seeking among college students reporting a current mental health problem. The sample consisted of 261 college students, 115 of which reported experiencing a current problem. Results of a series of logistic regressions revealed that perceived stigma from others (OR = .32), self-stigma (OR = .29), negative attitudes about treatment (OR = .27), and practical barriers (OR = .34) were all associated with a lower likelihood of having sought treatment among students experiencing a problem. Of the five-factor model personality traits, only Neuroticism was associated with a higher likelihood of having sought treatment when experiencing a mental health problem (OR = 2.71). When we considered all significant predictors in a final stepwise conditional model, only self-stigma, practical barriers, and Neuroticism remained significant unique predictors. Implications for addressing barriers to treatment and encouraging treatment seeking among college students are discussed. (PsycINFO Database Record

Enhancing the Meaningfulness of Work for Astronauts on Long Duration Space Exploration Missions.

INTRODUCTION: Numerous authors have identified the stressors likely to be encountered on long duration space exploration missions (e.g., to Mars), including the possibility of significant crises, separation from family, boredom/monotony, and interpersonal conflict. Although many authors have noted that meaningful work may be beneficial for astronauts on these missions, none have detailed the sources of meaningful work for astronauts and how these sources may differ between astronauts. The present article identifies how engagement in meaningful work during long duration missions may mitigate the adverse effects of demands and increase the potential for benefits resulting from the missions. METHOD: Semistructured interviews were conducted with nine NASA personnel, including astronauts, flight directors, and flight surgeons. Questions addressed sources of meaning for astronauts, characteristics of tasks that enhance vs. detract from meaning, and recommendations for enhancing meaning. RESULTS: Personnel mentioned contributing to humanity and the next generation, contributing to the mission, and exploration as the most meaningful aspects of their work. Characteristics of tasks that enhanced meaning included using a variety of skills, feeling personal control over their schedule, autonomy in the execution of tasks, and understanding the importance of the experiments conducted on the mission. Top recommendations to sustain meaning were insuring social needs were met through such activities as the strategic use of social media, giving astronauts autonomy as well as structure, and conducting training during transit. DISCUSSION: Implications are addressed for tailoring meaning-based interventions for astronauts participating on long duration missions and assessing the effectiveness of these interventions.Britt TW, Sytine A, Brady A, Wilkes R, Pittman R, Jennings K, Goguen K. Enhancing the meaningfulness of work for astronauts on long duration space exploration missions. Aerosp Med Hum Perform. 2017; 88(8):779-783.

Case Report: Neonate With Stridor and Subcutaneous Emphysema as the Only Signs of Physical Abuse.

A stridulous, dysphonic cry with no external signs of trauma is a unique and unusual presenting sign for physical abuse. We report a previously healthy neonate with unremarkable birth history and medical history who presented with stridor and hypopharyngeal perforation due to physical abuse. This case highlights the need for further evaluation for traumatic injuries in the setting of unexplained new-onset stridor and consideration of physical abuse in the differential diagnosis.

Study of GPCR-protein interactions using gel overlay assays and glutathione-S-transferase-fusion protein pull-downs.

Numerous recent studies have suggested that the predicted cytosolic domains of G protein-coupled receptors represent a surface for association with proteins that may serve multiple roles in receptor localization, turnover, and signaling beyond the well-characterized interactions of these receptors with heterotrimeric G proteins. This Chapter describes two in vitro methods for ascertaining interactions between G protein-coupled receptors and various binding partners: gel overlay strategies and GST-fusion protein pull-downs.

Differential effects of allosteric M(1) muscarinic acetylcholine receptor agonists on receptor activation, arrestin 3 recruitment, and receptor downregulation.

Muscarinic acetylcholine receptors (mAChRs) are drug targets for multiple neurodegenerative and neuropsychiatric disorders, but the full therapeutic potential of mAChR-targeted drugs has not been realized, mainly because of a lack of subtype-selective agonists. Recent advances have allowed the development of highly selective agonists that bind to an allosteric site on the M(1) mAChR that is spatially distinct from the orthosteric acetylcholine binding site, but less is known about the profile of intracellular signals activated by orthosteric versus allosteric M(1) mAChR agonists. We investigated the activation and regulatory mechanisms of two structurally distinct allosteric M(1) mAChR agonists, AC260584 and TBPB. We show that allosteric agonists potently activate multiple signal transduction pathways linked to the M(1) mAChR receptor but, compared to orthosteric agonists, much less efficiently recruit arrestin 3, a protein involved in regulation of G-protein coupled receptor signaling. Consistent with decreased arrestin recruitment, both allosteric agonists showed blunted responses in measurements of receptor desensitization, internalization, and downregulation. These results advance the understanding of mAChR biology and may shed light on unanticipated differences in the pharmacology of orthosteric vs. allosteric agonists that might be capitalized upon for drug development for the treatment of CNS diseases.

A selective allosteric potentiator of the M1 muscarinic acetylcholine receptor increases activity of medial prefrontal cortical neurons and restores impairments in reversal learning.

M(1) muscarinic acetylcholine receptors (mAChRs) may represent a viable target for treatment of disorders involving impaired cognitive function. However, a major limitation to testing this hypothesis has been a lack of highly selective ligands for individual mAChR subtypes. We now report the rigorous molecular characterization of a novel compound, benzylquinolone carboxylic acid (BQCA), which acts as a potent, highly selective positive allosteric modulator (PAM) of the rat M(1) receptor. This compound does not directly activate the receptor, but acts at an allosteric site to increase functional responses to orthosteric agonists. Radioligand binding studies revealed that BQCA increases M(1) receptor affinity for acetylcholine. We found that activation of the M(1) receptor by BQCA induces a robust inward current and increases spontaneous EPSCs in medial prefrontal cortex (mPFC) pyramidal cells, effects which are absent in acute slices from M(1) receptor knock-out mice. Furthermore, to determine the effect of BQCA on intact and functioning brain circuits, multiple single-unit recordings were obtained from the mPFC of rats that showed BQCA increases firing of mPFC pyramidal cells in vivo. BQCA also restored discrimination reversal learning in a transgenic mouse model of Alzheimer's disease and was found to regulate non-amyloidogenic APP processing in vitro, suggesting that M(1) receptor PAMs have the potential to provide both symptomatic and disease modifying effects in Alzheimer's disease patients. Together, these studies provide compelling evidence that M(1) receptor activation induces a dramatic excitation of PFC neurons and suggest that selectively activating the M(1) mAChR subtype may ameliorate impairments in cognitive function.

Discovery and characterization of novel allosteric potentiators of M1 muscarinic receptors reveals multiple modes of activity.

Activators of M(1) muscarinic acetylcholine receptors (mAChRs) may provide novel treatments for schizophrenia and Alzheimer's disease. Unfortunately, the development of M(1)-active compounds has resulted in nonselective activation of the highly related M(2) to M(5) mAChR subtypes, which results in dose-limiting side effects. Using a functional screening approach, we identified several novel ligands that potentiated agonist activation of M(1) with low micromolar potencies and induced 5-fold or greater leftward shifts of the acetylcholine (ACh) concentration-response curve. These ligands did not compete for binding at the ACh binding site, indicating that they modulate receptor activity by binding to allosteric sites. The two most selective compounds, cyclopentyl 1,6-dimethyl-4-(6-nitrobenzo[d][1,3]-dioxol-5-yl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (VU0090157) and (E)-2-(4-ethoxyphenylamino)-N'-((2-hydroxynaphthalen-1-yl)methylene)acetohydrazide (VU0029767), induced progressive shifts in ACh affinity at M(1) that were consistent with their effects in a functional assay, suggesting that the mechanism for enhancement of M(1) activity by these compounds is by increasing agonist affinity. These compounds were strikingly different, however, in their ability to potentiate responses at a mutant M(1) receptor with decreased affinity for ACh and in their ability to affect responses of the allosteric M(1) agonist, 1-[1'-(2-tolyl)-1,4'-bipiperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one. Furthermore, these two compounds were distinct in their abilities to potentiate M(1)-mediated activation of phosphoinositide hydrolysis and phospholipase D. The discovery of multiple structurally distinct positive allosteric modulators of M(1) is an exciting advance in establishing the potential of allosteric modulators for selective activation of this receptor. These data also suggest that structurally diverse M(1) potentiators may act by distinct mechanisms and differentially regulate receptor coupling to downstream signaling pathways.

Novel selective allosteric activator of the M1 muscarinic acetylcholine receptor regulates amyloid processing and produces antipsychotic-like activity in rats.

Recent studies suggest that subtype-selective activators of M(1)/M(4) muscarinic acetylcholine receptors (mAChRs) may offer a novel approach for the treatment of psychotic symptoms associated with schizophrenia and Alzheimer's disease. Previously developed muscarinic agonists have provided clinical data in support of this hypothesis, but failed in clinical development because of a lack of true subtype specificity and adverse effects associated with activation of other mAChR subtypes. We now report characterization of a novel highly selective agonist for the M(1) receptor with no agonist activity at any of the other mAChR subtypes, termed TBPB [1-(1'-2-methylbenzyl)-1,4'-bipiperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one]. Mutagenesis and molecular pharmacology studies revealed that TBPB activates M(1) through an allosteric site rather than the orthosteric acetylcholine binding site, which is likely critical for its unprecedented selectivity. Whole-cell patch-clamp recordings demonstrated that activation of M(1) by TBPB potentiates NMDA receptor currents in hippocampal pyramidal cells but does not alter excitatory or inhibitory synaptic transmission, responses thought to be mediated by M(2) and M(4). TBPB was efficacious in models predictive of antipsychotic-like activity in rats at doses that did not produce catalepsy or peripheral adverse effects of other mAChR agonists. Finally, TBPB had effects on the processing of the amyloid precursor protein toward the non-amyloidogenic pathway and decreased Abeta production in vitro. Together, these data suggest that selective activation of M(1) may provide a novel approach for the treatment of symptoms associated with schizophrenia and Alzheimer's disease.

Synthesis and SAR of analogs of the M1 allosteric agonist TBPB. Part II: Amides, sulfonamides and ureas--the effect of capping the distal basic piperidine nitrogen.

This letter describes the further synthesis and SAR, developed through an iterative analog library approach, of analogs of the highly selective M1 allosteric agonist TBPB by deletion of the distal basic piperidine nitrogen by the formation of amides, sulfonamides and ureas. Despite the large change in basicity and topology, M1 selectivity was maintained.

Synthesis and SAR of analogues of the M1 allosteric agonist TBPB. Part I: Exploration of alternative benzyl and privileged structure moieties.

This Letter describes the first account of the synthesis and SAR, developed through an iterative analogue library approach, of analogues of the highly selective M1 allosteric agonist TBPB. With slight structural changes, mAChR selectivity was maintained, but the degree of partial M1 agonism varied considerably.

Centrally active allosteric potentiators of the M4 muscarinic acetylcholine receptor reverse amphetamine-induced hyperlocomotor activity in rats.

Previous clinical and animal studies suggest that selective activators of M(1) and/or M(4) muscarinic acetylcholine receptors (mAChRs) have potential as novel therapeutic agents for treatment of schizophrenia and Alzheimer's disease. However, highly selective centrally penetrant activators of either M(1) or M(4) have not been available, making it impossible to determine the in vivo effects of selective activation of these receptors. We previously identified VU10010 [3-amino-N-(4-chlorobenzyl)-4, 6-dimethylthieno[2,3-b]pyridine-2-carboxamide] as a potent and selective allosteric potentiator of M(4) mAChRs. However, unfavorable physiochemical properties prevented use of this compound for in vivo studies. We now report that chemical optimization of VU10010 has afforded two centrally penetrant analogs, VU0152099 [3-amino-N-(benzo[d][1,3]dioxol-5-ylmethyl)-4,6-dimethylthieno[2,3-b]pyridine carboxamide] and VU0152100 [3-amino-N-(4-methoxybenzyl)-4,6-dimethylthieno[2,3-b]pyridine carboxamide], that are potent and selective positive allosteric modulators of M(4). VU0152099 and VU0152100 had no agonist activity but potentiated responses of M(4) to acetylcholine. Both compounds were devoid of activity at other mAChR subtypes or at a panel of other GPCRs. The improved physiochemical properties of VU0152099 and VU0152100 allowed in vivo dosing and evaluation of behavioral effects in rats. Interestingly, these selective allosteric potentiators of M(4) reverse amphetamine-induced hyperlocomotion in rats, a model that is sensitive to known antipsychotic agents and to nonselective mAChR agonists. This is consistent with the hypothesis that M(4) plays an important role in regulating midbrain dopaminergic activity and raises the possibility that positive allosteric modulation of M(4) may mimic some of the antipsychotic-like effects of less selective mAChR agonists.

An allosteric potentiator of M4 mAChR modulates hippocampal synaptic transmission.

Muscarinic acetylcholine receptors (mAChRs) provide viable targets for the treatment of multiple central nervous system disorders. We have used cheminformatics and medicinal chemistry to develop new, highly selective M4 allosteric potentiators. VU10010, the lead compound, potentiates the M4 response to acetylcholine 47-fold while having no activity at other mAChR subtypes. This compound binds to an allosteric site on the receptor and increases affinity for acetylcholine and coupling to G proteins. Whole-cell patch clamp recordings revealed that selective potentiation of M4 with VU10010 increases carbachol-induced depression of transmission at excitatory but not inhibitory synapses in the hippocampus. The effect was not mimicked by an inactive analog of VU10010 and was absent in M4 knockout mice. Selective regulation of excitatory transmission by M4 suggests that targeting of individual mAChR subtypes could be used to differentially regulate specific aspects of mAChR modulation of function in this important forebrain structure.

A novel family of potent negative allosteric modulators of group II metabotropic glutamate receptors.

Group II metabotropic glutamate receptors (mGluRs), mGluR2 and mGluR3, play a number of important roles in mammalian brain and represent exciting new targets for certain central nervous system disorders. We now report synthesis and characterization of a novel family of derivatives of dihydrobenzo[1,4]diazepin-2-one that are selective negative allosteric modulators for group II mGluRs. These compounds inhibit both mGluR2 and mGluR3 but have no activity at group I and III mGluRs. The novel mGluR2/3 antagonists also potently block mGluR2/3-mediated inhibition of the field excitatory postsynaptic potentials at the perforant path synapse in hippocampal slices. These compounds induce a rightward shift and decrease the maximal response in the glutamate concentration-response relationship, consistent with a noncompetitive antagonist mechanism of action. Furthermore, radioligand binding studies revealed no effect on binding of the orthosteric antagonist [(3)H]LY341495 [2S-2-amino-2-(1S,2S-2-carboxycyclopropan-1-yl)-3-(xanth-9-yl)propionic acid]. Site-directed mutagenesis revealed that a single point mutation in transmembrane V (N735D), previously shown to be an important residue for potentiation activity of the mGluR2 allosteric potentiator LY487379 [N-(4-(2-methoxyphenoxy)phenyl)-N-(2,2,2-trifluoroethylsulfonyl)pyrid-3-ylmethylamine], is not critical for the inhibitory activity of negative allosteric modulators of group II mGluRs. However, this single mutation in human GluR2 almost completely blocked the enhancing activity of biphenyl-indanone A, a novel allosteric potentiator of mGluR2. Our data suggest that these two positive allosteric modulators of mGluR2 may share a common binding site and that this site may be distinct from the binding site for the new negative allosteric modulators of group II mGluRs.

A novel class of positive allosteric modulators of metabotropic glutamate receptor subtype 1 interact with a site distinct from that of negative allosteric modulators.

We recently reported a novel class of compounds, represented by 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CD-PPB), that act as positive allosteric modulators (potentiators) of metabotropic glutamate receptor (mGluR) subtype 5. Studies of CDPPB analogs revealed that some compounds in this series serve also as positive allosteric modulators of mGluR1. Although CDPPB is selective for mGluR5 relative to other mGluR subtypes, several CDPPB analogs also showed 2.5-fold potentiation of glutamate-induced calcium transients in cells expressing mGluR1 at 10 muM, with 4-nitro-N-(1,4-diphenyl-1H-pyrazol-5-yl)benzamide (VU-71) being selective for mGluR1. In previous studies, we found that two structural classes of mGluR5-selective allosteric potentiators, including CDPPB, share a common binding site with the allosteric mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine. Negative allosteric modulators of mGluR1, regardless of structural class, have been reported to bind to a common allosteric antagonist site on this receptor. However, neither the novel CDPPB analogs nor previously identified allosteric mGluR1 potentiators [e.g., (S)-2-(4-fluorophenyl)-1-(toluene-4-sulfonyl)pyrrolidine (Ro 67-7476), ethyl diphenylacetylcarbamate (Ro 01-6128), and butyl (9H-xanthene-9-carbonyl)carbamate (Ro 67-4853)] displaced the binding of [(3)H]1-(3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-2-phenyl-1-ethanone (R214127), a high-affinity radioligand for the allosteric antagonist site on mGluR1 at concentrations several orders of magnitude higher than those required to induce allosteric potentiation of mGluR1 responses. These data suggest that allosteric potentiators of mGluR1 act at a site that is distinct from that of allosteric antagonists of mGluR1. Site-directed mutagenesis revealed that valine at position 757 in transmembrane V of mGluR1a is crucial for the activity of multiple classes of allosteric mGluR1 potentiators.

Alpha 2-adrenergic agonist enrichment of spinophilin at the cell surface involves beta gamma subunits of Gi proteins and is preferentially induced by the alpha 2A-subtype.

Agonist activation regulates reciprocal interactions of spinophilin and arrestin with the alpha2A- and alpha2B -adrenergic receptor (AR) subtypes via their 3i loop. Because arrestin association with G protein-coupled receptor is preceded by redistribution of arrestin to the cell surface, the present studies explored whether agonist activation of the alpha2A- and alpha2B -AR subtypes also led to spinophilin enrichment at the cell surface. Live cell imaging studies using a green fluorescent protein-tagged spinophilin examined spinophilin localization and its regulation by alpha2 -AR agonist. Agonist activation of alpha2A-AR preferentially, compared with the alpha2B-AR, led to spinophilin enrichment at the cell surface in human embryonic kidney 293 cells and in mouse embryo fibroblasts derived from spinophilin null mice. Activation of the delta LEESSSS alpha2A-AR, which has enriched association with spinophilin compared with the wild-type (WT) alpha2A-AR, does not show an enhanced redistribution of spinophilin to the surface compared with WT alpha2A-AR, demonstrating that the ability or affinity of the receptor in binding spinophilin may be independent of the ability of the receptor to effect spinophilin redistribution to the surface. Agonist-evoked enrichment of spinophilin at the cell surface seems to involve downstream signaling events, manifested both by the pertussis toxin sensitivity of the process and by the marked attenuation of spinophilin redistribution in cells expressing the beta-adrenergic receptor kinase-C tail, which sequesters beta gamma subunits of G proteins. Together, the data suggest that agonist-evoked spinophilin enrichment at the cell surface is caused by receptor-evoked signaling pathways and is independent of the affinity of the receptor for the spinophilin molecule.

Study of G-protein-coupled receptor-protein interactions using gel overlay assays and glutathione-S-transferase-fusion protein pull-downs.

Numerous recent studies have suggested that the predicted cytosolic domains of G-protein-coupled receptors (GPCRs) represent a surface for association with proteins that may serve multiple roles in receptor localization, turnover, and signaling beyond the well-characterized interactions of these receptors with heterotrimeric G-proteins. This chapter describes two in vitro methods for ascertaining interactions between GPCRs and various binding partners: gel overlay strategies and GST-fusion protein pull-downs.