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Expression and functional characterization of membrane-integrated mammalian corticotropin releasing factor receptors 1 and 2 in Escherichia coli.

ABSTRACT: Corticotropin-Releasing Factor Receptors (CRFRs) are class B1 G-protein-coupled receptors, which bind peptides of the corticotropin releasing factor family and are key mediators in the stress response. In order to dissect the receptors' binding specificity and enable structural studies, full-length human CRFR1? and mouse CRFR2? as well as fragments lacking the N-terminal extracellular domain, were overproduced in E. coli. The characteristics of different CRFR2?-PhoA gene fusion products expressed in bacteria were found to be in agreement with the predicted ones in the hepta-helical membrane topology model. Recombinant histidine-tagged CRFR1? and CRFR2? expression levels and bacterial subcellular localization were evaluated by cell fractionation and Western blot analysis. Protein expression parameters were assessed, including the influence of E. coli bacterial hosts, culture media and the impact of either PelB or DsbA signal peptide. In general, the large majority of receptor proteins became inserted in the bacterial membrane. Across all experimental conditions significantly more CRFR2? product was obtained in comparison to CRFR1?. Following a detergent screen analysis, bacterial membranes containing CRFR1? and CRFR2? were best solubilized with the zwitterionic detergent FC-14. Binding of different peptide ligands to CRFR1? and CRFR2? membrane fractions were similar, in part, to the complex pharmacology observed in eukaryotic cells. We suggest that our E. coli expression system producing functional CRFRs will be useful for large-scale expression of these receptors for structural studies.

SUBMITTER: Jappelli R

PROVIDER: S-EPMC3894963 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Expression and functional characterization of membrane-integrated mammalian corticotropin releasing factor receptors 1 and 2 in Escherichia coli.

Jappelli Roberto R Perrin Marilyn H MH Lewis Kathy A KA Vaughan Joan M JM Tzitzilonis Christos C Rivier Jean E JE Vale Wylie W WW Riek Roland R

PloS one 20140117 1

Corticotropin-Releasing Factor Receptors (CRFRs) are class B1 G-protein-coupled receptors, which bind peptides of the corticotropin releasing factor family and are key mediators in the stress response. In order to dissect the receptors' binding specificity and enable structural studies, full-length human CRFR1α and mouse CRFR2β as well as fragments lacking the N-terminal extracellular domain, were overproduced in E. coli. The characteristics of different CRFR2β-PhoA gene fusion products expresse ...[more]

PMID: 24465390

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Project description:Stress interacts with addictive processes to increase drug use, drug seeking, and relapse. The hippocampal formation (HF) is an important site at which stress circuits and endogenous opioid systems intersect and likely plays a critical role in the interaction between stress and drug addiction. Our prior studies demonstrate that the stress-related neuropeptide corticotropin-releasing factor (CRF) and the delta-opioid receptor (DOR) colocalize in interneuron populations in the hilus of the dentate gyrus and stratum oriens of CA1 and CA3. While independent ultrastructural studies of DORs and CRF receptors suggest that each receptor is found in CA1 pyramidal cell dendrites and dendritic spines, whether DORs and CRF receptors colocalize in CA1 neuronal profiles has not been investigated. Here, hippocampal sections of adult male and proestrus female Sprague-Dawley rats were processed for dual label pre-embedding immunoelectron microscopy using well-characterized antisera directed against the DOR for immunoperoxidase and against the CRF receptor for immunogold. DOR-immunoreactivity (-ir) was found presynaptically in axons and axon terminals as well as postsynaptically in somata, dendrites and dendritic spines in stratum radiatum of CA1. In contrast, CRF receptor-ir was predominantly found postsynaptically in CA1 somata, dendrites, and dendritic spines. CRF receptor-ir frequently was observed in DOR-labeled dendritic profiles and primarily was found in the cytoplasm rather than at or near the plasma membrane. Quantitative analysis of CRF receptor-ir colocalization with DOR-ir in pyramidal cell dendrites revealed that proestrus females and males show comparable levels of CRF receptor-ir per dendrite and similar cytoplasmic density of CRF receptor-ir. In contrast, proestrus females display an increased number of dual-labeled dendritic profiles and an increased membrane density of CRF receptor-ir in comparison to males. We further examined the functional consequences of CRF receptor-ir colocalization with DOR-ir in the same neuron using the hormone responsive neuronal cell line NG108-15, which endogenously expresses DORs, and assayed intracellular cAMP production in response to CRF receptor and DOR agonists. Results demonstrated that short-term application of DOR agonist SNC80 inhibited CRF-induced cAMP accumulation in NG108-15 cells transfected with the CRF receptor. These studies provide new insights on opioid-stress system interaction in the hippocampus of both males and females and establish potential mechanisms through which DOR activation may influence CRF receptor activity.

Dataset Information

Expression and functional characterization of membrane-integrated mammalian corticotropin releasing factor receptors 1 and 2 in Escherichia coli.

Publications

Expression and functional characterization of membrane-integrated mammalian corticotropin releasing factor receptors 1 and 2 in Escherichia coli.

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