RESUMEN
The biased signaling has been extensively studied in the original mu opioid receptor (MOR-1), particularly through G protein and ß-arrestin2 signaling pathways. The concept that the G protein pathway is often linked to the therapeutic effect of the drug, while the ß-arrestin pathway is associated to the side effects has been proposed to develop biased analgesic compounds with limited side-effects associated with traditional opiates. The mu opioid receptor gene, OPRM1, undergoes extensive alternative pre-mRNA splicing, generating multiple splice variants or isoforms that are conserved from rodent to human. One type of the Oprm1 splice variants are the full-length 7 transmembrane (7TM) C-terminal splice variants, which have identical receptor structures including entire binding pocket, but contain a different intracellular C-terminal tail resulted from 3' alternative splicing. Increasing evidence suggest that these full-length 7TM C-terminal variants play important roles in mu opioid pharmacology, raising questions regarding biased signaling at these multiple C-terminal variants. In the present study, we investigated the effect of different C-terminal variants on mu agonist-induced G protein coupling, ß-arrestin2 recruitment, and ultimately, signaling bias. We found that mu agonists produced marked differences in G protein activation and ß-arrestin2 recruitment among various C-terminal variants, leading to biased signaling at various level. Particularly, MOR-1O, an exon 7-associated variant, showed greater ß-arrestin2 bias for most mu agonists than MOR-1, an exon 4-associated variant. Biased signaling of G protein-coupled receptors has been defined by evidences that different agonists can produce divergent signaling transduction pathways through a single receptor. Our findings that a single mu agonist can induce differential signaling through multiple 7TM splice variants provide a new perspective on biased signaling at least for Oprm1, which perhaps is important for our understanding of the complex mu opioid actions in vivo where all the 7TM splice variants co-exist.
Asunto(s)
Empalme Alternativo/fisiología , Analgésicos Opioides/metabolismo , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Transducción de Señal/fisiología , Empalme Alternativo/genética , Secuencia de Aminoácidos , Analgésicos Opioides/farmacología , Animales , Células CHO , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Células HEK293 , Humanos , Naltrexona/análogos & derivados , Naltrexona/metabolismo , Naltrexona/farmacología , Unión Proteica/fisiología , Receptores Opioides mu/agonistas , Transducción de Señal/efectos de los fármacosRESUMEN
G-protein-coupled receptors (GPCRs) have varying and diverse physiological roles, transmitting signals from a range of stimuli, including light, chemicals, peptides, and mechanical forces. More than 130 GPCRs are orphan receptors (i.e., their endogenous ligands are unknown), representing a large untapped reservoir of potential therapeutic targets for pharmaceutical intervention in a variety of diseases. Current deorphanization approaches are slow, laborious, and usually require some in-depth knowledge about the receptor pharmacology. In this study we describe a cell-based assay to identify small molecule probes of orphan receptors that requires no a priori knowledge of receptor pharmacology. Built upon the concept of pharmacochaperones, where cell-permeable small molecules facilitate the trafficking of mutant receptors to the plasma membrane, the simple and robust technology is readily accessible by most laboratories and is amenable to high-throughput screening. The assay consists of a target harboring a synthetic point mutation that causes retention of the target in the endoplasmic reticulum. Coupled with a beta-galactosidase enzyme-fragment complementation reporter system, the assay identifies compounds that act as pharmacochaperones causing forward trafficking of the mutant GPCR. The assay can identify compounds with varying mechanisms of action including agonists and antagonists. A universal positive control compound circumvents the need for a target-specific ligand. The veracity of the approach is demonstrated using the beta-2-adrenergic receptor. Together with other existing assay technologies to validate the signaling pathways and the specificity of ligands identified, this pharmacochaperone-based approach can accelerate the identification of ligands for these potentially therapeutically useful receptors.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Sondas Moleculares/análisis , Sondas Moleculares/química , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Bibliotecas de Moléculas Pequeñas/análisis , Bibliotecas de Moléculas Pequeñas/química , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Humanos , Ligandos , Sondas Moleculares/farmacología , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Células Tumorales CultivadasRESUMEN
Extensive 3' alternative splicing of the mu opioid receptor gene OPRM1 creates multiple C-terminal splice variants. However, their behavioral relevance remains unknown. The present study generated 3 mutant mouse models with truncated C termini in 2 different mouse strains, C57BL/6J (B6) and 129/SvEv (129). One mouse truncated all C termini downstream of Oprm1 exon 3 (mE3M mice), while the other two selectively truncated C-terminal tails encoded by either exon 4 (mE4M mice) or exon 7 (mE7M mice). Studies of these mice revealed divergent roles for the C termini in morphine-induced behaviors, highlighting the importance of C-terminal variants in complex morphine actions. In mE7M-B6 mice, the exon 7-associated truncation diminished morphine tolerance and reward without altering physical dependence, whereas the exon 4-associated truncation in mE4M-B6 mice facilitated morphine tolerance and reduced morphine dependence without affecting morphine reward. mE7M-B6 mutant mice lost morphine-induced receptor desensitization in the brain stem and hypothalamus, consistent with exon 7 involvement in morphine tolerance. In cell-based studies, exon 7-associated variants shifted the bias of several mu opioids toward ß-arrestin 2 over G protein activation compared with the exon 4-associated variant, suggesting an interaction of exon 7-associated C-terminal tails with ß-arrestin 2 in morphine-induced desensitization and tolerance. Together, the differential effects of C-terminal truncation illustrate the pharmacological importance of OPRM1 3' alternative splicing.
Asunto(s)
Analgésicos Opioides/farmacología , Morfina/farmacología , Receptores Opioides mu/metabolismo , Empalme Alternativo , Animales , Encéfalo/metabolismo , Codón sin Sentido , Relación Dosis-Respuesta a Droga , Tolerancia a Medicamentos , Exones , Tránsito Gastrointestinal/efectos de los fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Locomoción/efectos de los fármacos , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Dependencia de Morfina/genética , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Opioides mu/genéticaRESUMEN
ß-Arrestins (ßarrs) interact with G protein-coupled receptors (GPCRs) to desensitize G protein signaling, to initiate signaling on their own, and to mediate receptor endocytosis. Prior structural studies have revealed two unique conformations of GPCR-ßarr complexes: the "tail" conformation, with ßarr primarily coupled to the phosphorylated GPCR C-terminal tail, and the "core" conformation, where, in addition to the phosphorylated C-terminal tail, ßarr is further engaged with the receptor transmembrane core. However, the relationship of these distinct conformations to the various functions of ßarrs is unknown. Here, we created a mutant form of ßarr lacking the "finger-loop" region, which is unable to form the core conformation but retains the ability to form the tail conformation. We find that the tail conformation preserves the ability to mediate receptor internalization and ßarr signaling but not desensitization of G protein signaling. Thus, the two GPCR-ßarr conformations can carry out distinct functions.
Asunto(s)
Endocitosis/genética , Proteínas Mutantes/química , Receptores Acoplados a Proteínas G/química , beta-Arrestinas/química , Secuencia de Aminoácidos/genética , Reguladores de Proteínas de Unión al GTP/genética , Células HEK293 , Humanos , Conformación Molecular , Complejos Multiproteicos , Proteínas Mutantes/genética , Receptores Acoplados a Proteínas G/genética , beta-Arrestinas/genéticaRESUMEN
The ß2-adrenergic receptor (ß2AR) has been a model system for understanding regulatory mechanisms of G-protein-coupled receptor (GPCR) actions and plays a significant role in cardiovascular and pulmonary diseases. Because all known ß-adrenergic receptor drugs target the orthosteric binding site of the receptor, we set out to isolate allosteric ligands for this receptor by panning DNA-encoded small-molecule libraries comprising 190 million distinct compounds against purified human ß2AR. Here, we report the discovery of a small-molecule negative allosteric modulator (antagonist), compound 15 [([4-((2S)-3-(((S)-3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)amino)-2-(2-cyclohexyl-2-phenylacetamido)-3-oxopropyl)benzamide], exhibiting a unique chemotype and low micromolar affinity for the ß2AR. Binding of 15 to the receptor cooperatively enhances orthosteric inverse agonist binding while negatively modulating binding of orthosteric agonists. Studies with a specific antibody that binds to an intracellular region of the ß2AR suggest that 15 binds in proximity to the G-protein binding site on the cytosolic surface of the ß2AR. In cell-signaling studies, 15 inhibits cAMP production through the ß2AR, but not that mediated by other Gs-coupled receptors. Compound 15 also similarly inhibits ß-arrestin recruitment to the activated ß2AR. This study presents an allosteric small-molecule ligand for the ß2AR and introduces a broadly applicable method for screening DNA-encoded small-molecule libraries against purified GPCR targets. Importantly, such an approach could facilitate the discovery of GPCR drugs with tailored allosteric effects.
Asunto(s)
Antagonistas Adrenérgicos beta/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Receptores Adrenérgicos beta 2/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Antagonistas Adrenérgicos beta/química , Antagonistas Adrenérgicos beta/metabolismo , Animales , Sitios de Unión/genética , Unión Competitiva/efectos de los fármacos , ADN/genética , Humanos , Ligandos , Estructura Molecular , Mutación , Receptores Adrenérgicos beta 2/genética , Células Sf9 , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , SpodopteraRESUMEN
Buprenorphine has long been classified as a mu analgesic, although its high affinity for other opioid receptor classes and the orphanin FQ/nociceptin ORL1 receptor may contribute to its other actions. The current studies confirmed a mu mechanism for buprenorphine analgesia, implicating several subsets of mu receptor splice variants. Buprenorphine analgesia depended on the expression of both exon 1-associated traditional full length 7 transmembrane (7TM) and exon 11-associated truncated 6 transmembrane (6TM) MOR-1 variants. In genetic models, disruption of delta, kappa1 or ORL1 receptors had no impact on buprenorphine analgesia, while loss of the traditional 7TM MOR-1 variants in an exon 1 knockout (KO) mouse markedly lowered buprenorphine analgesia. Loss of the truncated 6TM variants in an exon 11 KO mouse totally eliminated buprenorphine analgesia. In distinction to analgesia, the inhibition of gastrointestinal transit and stimulation of locomotor activity were independent of truncated 6TM variants. Restoring expression of a 6TM variant with a lentivirus rescued buprenorphine analgesia in an exon 11 KO mouse that still expressed the 7TM variants. Despite a potent and robust stimulation of (35) S-GTPγS binding in MOR-1 expressing CHO cells, buprenorphine failed to recruit ß-arrestin-2 binding at doses as high as 10 µM. Buprenorphine was an antagonist in DOR-1 expressing cells and an inverse agonist in KOR-1 cells. Buprenorphine analgesia is complex and requires multiple mu receptor splice variant classes but other actions may involve alternative receptors.
Asunto(s)
Analgésicos Opioides/farmacología , Buprenorfina/farmacología , Nocicepción , Empalme del ARN , Receptores Opioides mu/genética , Animales , Células CHO , Cricetinae , Cricetulus , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Receptores Opioides mu/metabolismo , Arrestina beta 2/metabolismoRESUMEN
Allosteric modulators of G protein-coupled receptors (GPCRs) have a number of potential advantages compared to agonists or antagonists that bind to the orthosteric site of the receptor. These include the potential for receptor selectivity, maintenance of the temporal and spatial fidelity of signaling in vivo, the ceiling effect of the allosteric cooperativity which may prevent overdose issues, and engendering bias by differentially modulating distinct signaling pathways. Here we describe the discovery, synthesis, and molecular pharmacology of δ-opioid receptor-selective positive allosteric modulators (δ PAMs). These δ PAMs increase the affinity and/or efficacy of the orthosteric agonists leu-enkephalin, SNC80 and TAN67, as measured by receptor binding, G protein activation, ß-arrestin recruitment, adenylyl cyclase inhibition, and extracellular signal-regulated kinases (ERK) activation. As such, these compounds are useful pharmacological tools to probe the molecular pharmacology of the δ receptor and to explore the therapeutic potential of δ PAMs in diseases such as chronic pain and depression.
Asunto(s)
Receptores Opioides delta/metabolismo , Relación Estructura-Actividad , Animales , Arrestinas/metabolismo , Benzamidas/farmacología , Unión Competitiva , Células CHO , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Técnicas de Química Sintética , Cricetulus , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos/métodos , Encefalina Leucina/farmacología , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Estructura Molecular , Terapia Molecular Dirigida , Piperazinas/farmacología , Unión Proteica , Quinolinas/farmacología , beta-ArrestinasRESUMEN
Hetero-oligomeric complexes of G protein-coupled receptors (GPCRs) may represent novel therapeutic targets exhibiting different pharmacology and tissue- or cell-specific site of action compared with receptor monomers or homo-oligomers. An ideal tool for validating this concept pharmacologically would be a hetero-oligomer selective ligand. We set out to develop and execute a 1536-well high-throughput screen of over 1 million compounds to detect potential hetero-oligomer selective ligands using a ß-arrestin recruitment assay in U2OS cells coexpressing recombinant µ- and δ-opioid receptors. Hetero-oligomer selective ligands may bind to orthosteric or allosteric sites, and we might anticipate that the formation of hetero-oligomers may provide novel allosteric binding pockets for ligand binding. Therefore, our goal was to execute the screen in such a way as to identify positive allosteric modulators (PAMs) as well as agonists for µ, δ, and hetero-oligomeric receptors. While no hetero-oligomer selective ligands were identified (based on our selection criteria), this single screen did identify numerous µ- and δ-selective agonists and PAMs as well as nonselective agonists and PAMs. To our knowledge, these are the first µ- and δ-opioid receptor PAMs described in the literature.
Asunto(s)
Regulación Alostérica/efectos de los fármacos , Analgésicos Opioides/farmacología , Ensayos Analíticos de Alto Rendimiento , Receptores Opioides delta/agonistas , Receptores Opioides delta/química , Receptores Opioides mu/agonistas , Receptores Opioides mu/química , Animales , Arrestinas/metabolismo , Células CHO , Colforsina/farmacología , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Humanos , Antagonistas de Narcóticos/farmacología , Multimerización de Proteína , beta-ArrestinasRESUMEN
Chemokines display considerable promiscuity with multiple ligands and receptors shared in common, a phenomenon that is thought to underlie their biochemical "redundancy." Their receptors are part of a larger seven-transmembrane receptor superfamily, commonly referred to as G protein-coupled receptors, which have been demonstrated to be able to signal with different efficacies to their multiple downstream signaling pathways, a phenomenon referred to as biased agonism. Biased agonism has been primarily reported as a phenomenon of synthetic ligands, and the biologic prevalence and importance of such signaling are unclear. Here, to assess the presence of biased agonism that may underlie differential signaling by chemokines targeting the same receptor, we performed a detailed pharmacologic analysis of a set of chemokine receptors with multiple endogenous ligands using assays for G protein signaling, ß-arrestin recruitment, and receptor internalization. We found that chemokines targeting the same receptor can display marked differences in their efficacies for G protein- or ß-arrestin-mediated signaling or receptor internalization. This ligand bias correlates with changes in leukocyte migration, consistent with different mechanisms underlying the signaling downstream of these receptors induced by their ligands. These findings demonstrate that biased agonism is a common and likely evolutionarily conserved biological mechanism for generating qualitatively distinct patterns of signaling via the same receptor in response to different endogenous ligands.
Asunto(s)
Receptores de Quimiocina/agonistas , Receptores de Quimiocina/metabolismo , Arrestinas/metabolismo , Quimiocinas/metabolismo , Proteínas de Unión al GTP/metabolismo , Células HEK293 , Humanos , Cinética , Ligandos , Modelos Biológicos , Transducción de Señal , beta-ArrestinasRESUMEN
A variety of G-protein-coupled receptor (GPCR) screening technologies have successfully partnered a number of GPCRs with their cognate ligands. GPCR-mediated ß-arrestin recruitment is now recognized as a distinct intracellular signaling pathway, and ligand-receptor interactions may show a bias toward ß-arrestin over classical GPCR signaling pathways. We hypothesized that the failure to identify native ligands for the remaining orphan GPCRs may be a consequence of biased ß-arrestin signaling. To investigate this, we assembled 10 500 candidate ligands and screened 82 GPCRs using PathHunter ß-arrestin recruitment technology. High-quality screening assays were validated by the inclusion of liganded receptors and the detection and confirmation of these established ligand-receptor pairings. We describe a candidate endogenous orphan GPCR ligand and a number of novel surrogate ligands. However, for the majority of orphan receptors studied, measurement of ß-arrestin recruitment did not lead to the identification of cognate ligands from our screening sets. ß-Arrestin recruitment represents a robust GPCR screening technology, and ligand-biased signaling is emerging as a therapeutically exploitable feature of GPCR biology. The identification of cognate ligands for the orphan GPCRs and the extent to which receptors may exist to preferentially signal through ß-arrestin in response to their native ligand remain to be determined.
Asunto(s)
Arrestinas/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Receptores Acoplados a Proteínas G/agonistas , Animales , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Descubrimiento de Drogas/métodos , Células HEK293 , Humanos , Ligandos , Unión Proteica/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Saccharomyces cerevisiae , Bibliotecas de Moléculas Pequeñas/análisis , beta-ArrestinasRESUMEN
More than two-thirds of all known G-protein coupled receptors are known to modulate the function of adenylate cyclase resulting in altered levels of cAMP. In turn, cAMP fluctuations transform agonist binding events into physiological changes in cell behavior. The advent of nonradioactive, homogeneous methods of measuring intracellular cAMP has enabled the rapid growth of drug discovery and research applications for these GPCR targets. In this chapter, we describe a nonradioactive, chemiluminescent cAMP detection method using enzyme fragment complementation technology to detect a wide range of GPCR modulators which is also suitable for high-throughput screening.
Asunto(s)
Bioensayo/métodos , AMP Cíclico/química , AMP Cíclico/metabolismo , Mediciones Luminiscentes/métodos , Receptores Acoplados a Proteínas G/metabolismo , Regulación Alostérica , Línea Celular , Receptores Acoplados a Proteínas G/agonistasRESUMEN
The recruitment of arrestins to activated 7TMRs results in the activation of alternative signaling pathways, quenching of G-protein activation, and coupling to clathrin-mediated endocytosis. The nearly ubiquitous involvement of arrestin in 7TMR signaling has spurred the development of several methods for monitoring this interaction in mammalian cells. Nonetheless, few maintain the reproducibility and precision necessary for drug discovery applications. Enzyme fragment complementation technology (EFC) is an emerging protein-protein interaction technology based on the forced complementation of a split enzyme that has proven to be highly effective in monitoring the formation of GPCR-arrestin complexes. In these systems, the target proteins are fused to two fragments of an enzyme that show little or no spontaneous complementation. Interaction of the two proteins forces the complementation of the enzyme, resulting in an enzymatic measure of the protein interaction. This chapter discusses the utility and methods involved in using the PathHunter ß-galactosidase complementation system to monitor arrestin recruitment and the advantages of exploiting this pathway in the characterization of 7TMR function.
Asunto(s)
Arrestinas/metabolismo , Descubrimiento de Drogas/métodos , Receptores Acoplados a Proteínas G/metabolismo , Animales , Línea Celular , Humanos , Unión Proteica , beta-Arrestinas , beta-Galactosidasa/metabolismoRESUMEN
The CellCard system enables the analysis of multiple cell types within a single microtiter well. In doing so, the CellCard system not only determines the effect of an experimental condition on a cell type of interest, but also the relative selectivity of that response across nine other cell types. In addition, this approach of cellular multiplexing is a means of miniaturization without the necessity of microfluidic devices. The standard 96-well plate generates ten 96-well plates of data (or, the equivalent of a 960-well plate). Taken together, the CellCard technology enables multiple cell types to be assayed within a single microtiter well allowing for the simultaneous determination of cellular activity and compound selectivity. This chapter will describe a method by which multiple cell types can be simultaneously assayed for biological parameters of interest.
Asunto(s)
Análisis de Matrices Tisulares/métodos , Animales , Células Cultivadas , Humanos , Procesamiento de Imagen Asistido por Computador , Coloración y Etiquetado , Análisis de Matrices Tisulares/instrumentaciónRESUMEN
The authors have developed a cellular analysis platform, based on encoded microcarriers, that enables the multiplexed analysis of a diverse range of cellular assays. At the core of this technology are classes of microcarriers that have unique, identifiable codes that are deciphered using CCD-based imaging and subsequent image analysis. The platform is compatible with a wide variety of cellular imaging-based assays, including calcium flux, reporter gene activation, cytotoxicity, and proliferation. In addition, the platform is compatible with both colorimetric and fluorescent readouts. Notably, this technology has the unique ability to multiplex different cell lines in a single microplate well, enabling scientists to perform assays and data analysis in novel ways.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Biología Molecular/métodos , Animales , Células CHO , Calcio/metabolismo , Carbacol/farmacología , División Celular/efectos de los fármacos , Células Cultivadas , Células Inmovilizadas , Cricetinae , Humanos , Procesamiento de Imagen Asistido por Computador , Calidina/farmacología , Microscopía/instrumentación , Microscopía/métodos , Tamaño de la Partícula , Receptor de Bradiquinina B2/análisis , Receptor de Bradiquinina B2/efectos de los fármacos , Receptor de Bradiquinina B2/genética , Receptores Acoplados a Proteínas G/análisis , Receptores Acoplados a Proteínas G/efectos de los fármacos , Receptores Acoplados a Proteínas G/genética , Receptores Muscarínicos/análisis , Receptores Muscarínicos/efectos de los fármacos , Receptores Muscarínicos/genética , Transducción de Señal , Pruebas de ToxicidadRESUMEN
Quantitative trait loci (QTL) were mapped in the woody perennial Douglas fir (Pseudotsuga menziesii var. menziesii [Mirb.] Franco) for complex traits controlling the timing of growth initiation and growth cessation. QTL were estimated under controlled environmental conditions to identify QTL interactions with photoperiod, moisture stress, winter chilling, and spring temperatures. A three-generation mapping population of 460 cloned progeny was used for genetic mapping and phenotypic evaluations. An all-marker interval mapping method was used for scanning the genome for the presence of QTL and single-factor ANOVA was used for estimating QTL-by-environment interactions. A modest number of QTL were detected per trait, with individual QTL explaining up to 9.5% of the phenotypic variation. Two QTL-by-treatment interactions were found for growth initiation, whereas several QTL-by-treatment interactions were detected among growth cessation traits. This is the first report of QTL interactions with specific environmental signals in forest trees and will assist in the identification of candidate genes controlling these important adaptive traits in perennial plants.
Asunto(s)
Sitios de Carácter Cuantitativo , Árboles/genética , Ligamiento Genético , Genotipo , Fenotipo , TemperaturaRESUMEN
A long-term series of experiments to map QTL influencing wood property traits in loblolly pine has been completed. These experiments were designed to identify and subsequently verify QTL in multiple genetic backgrounds, environments, and growing seasons. Verification of QTL is necessary to substantiate a biological basis for observed marker-trait associations, to provide precise estimates of the magnitude of QTL effects, and to predict QTL expression at a given age or in a particular environment. Verification was based on the repeated detection of QTL among populations, as well as among multiple growing seasons for each population. Temporal stability of QTL was moderate, with approximately half being detected in multiple seasons. Fewer QTL were common to different populations, but the results are nonetheless encouraging for restricted applications of marker-assisted selection. QTL from larger populations accounted for less phenotypic variation than QTL detected in smaller populations, emphasizing the need for experiments employing much larger families. Additionally, 18 candidate genes related to lignin biosynthesis and cell wall structure were mapped genetically. Several candidate genes colocated with wood property QTL; however, these relationships must be verified in future experiments.