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Abstract

Corticotropin?releasing factor (CRF) and its receptors play a major role in regulating an organism's response to physical, emotional and environmental stress. While the primary role of CRF is the regulation of adrenocorticotropin hormone (ACTH) secretion from the pituitary and modulation of the hypothalamic?pituitary adrenal axis, CRF is also widely distributed in the central nervous system where it produces a broad spectrum of autonomic, electrophysiological and behavioral effects consistent with a neurotransmitter or neuromodulator role in the brain. Methods are provided for characterizing the receptor proteins through which CRF exerts its function. A well?characterized radioligand receptor binding assay is provided that yields quantitative information about the affinity and density of receptors in a variety of tissues, while another procedure utilizes similar kinetic theories but, in contrast to the homogenized or whole cell suspension approach, makes use of slide?mounted tissue sections.

        GO TO THE FULL PROTOCOL: PDF or HTML at Wiley Online Library Table of Contents

• Basic Protocol 1: Association Kinetic Assay to Determine Time Course for Equilibrium Binding
• Alternate Protocol 1: Kinetic Assay to Determine Dissociation Time Course
• Basic Protocol 2: Saturation (Scatchard) Assays to Determine KD and Bmax
• Basic Protocol 3: Competition Assays to Determine Ki Values of Competing Ligands
• Support Protocol 1: Preparation of Corticotropin‐Releasing Factor (CRF) Receptors from Tissues or Cells
• Basic Protocol 4: Receptor Autoradiography to Study Corticotropin‐Releasing Factor (CRF) Receptors
• Reagents and Solutions
• Commentary
• Literature Cited
• Figures
• Tables

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GO TO THE FULL PROTOCOL: PDF or HTML at Wiley Online Library Figures

• Figure 1.13.1 Peptide sequences of human (A ) corticotropin‐releasing factor (CRF) and (B ) urocortin.

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• Figure 1.13.2 Association (A ) and dissociation (B ) of [125 I]sauvagine binding to human CRF2(a) receptors expressed in stable CHO cell lines. (A) For association experiments, cell membrane homogenates were incubated at 22°C with ∼100 to 200 pM [125 I]sauvagine for various times. Nonspecific binding was defined in the presence of 1 µM D ‐PheCRF(12‐41) at each time point. The association rate constant ( k +1 ) was determined (assuming pseudo‐first‐order kinetics) by plotting ln[Be /(Be ‐B)] versus time, where Be is specific binding (fmol/mg protein) at equilibrium and B is specific binding at any given time point. An example of such a plot is found in UNIT . Fig. k +1 was calculated from the equation k ob − k −1 = k +1 × CL, where k ob is the slope of the association plot described above, k −1 is the dissociation rate constant, and CL is ligand concentration. In the experiment described above, k +1 was observed to be 0.415 min−1 nmol−1 . (B) Following equilibrium, dissociation of [125 I]sauvagine was initiated by the addition of 1 µM D ‐PheCRF(12‐41), and the reaction was stopped at various times by centrifugation. Specific binding, B, was calculated for each time point, t , and the dissociation constant was determined from the equation ln(B/B0 ) = k −1 × t , where B0 is specific binding at equilibrium. In the experiment described above, k −1 was observed to be 0.0252 min−1 .

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• Figure 1.13.3 Saturation (A ) and Scatchard (B ) analyses of [125 I]sauvagine binding to human CRF2(a) receptors expressed in stable CHO cell lines. Human CRF2(a) receptor–transfected CHO cell membranes were incubated with 10 to 12 concentrations of [125 I]sauvagine (1 pM to 2 nM) at 22°C as described (see ). Nonspecific binding was defined in the presence of 1 µM D ‐Phe r/hCRF(12‐41) at each concentration. K D and B max values were estimated by nonlinear regression analysis using Prism software (GraphPad).

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• Figure 1.13.4 Competition of CRF‐related peptides for [125 I]sauvagine binding to human CRF2(a) receptors stably expressed in CHO cell lines. CRF2(a) ‐expressing cell membranes were incubated with 200 pM [125 I]sauvagine at 22°C along with varying concentrations of competing peptides, and were analyzed for the ability of competitors to inhibit binding. The rank order of potencies of these peptides defines the receptor subtype. That is, the rank order is identical for the CRF2(a) receptor subtype, regardless of the tissue or species from which it is derived. The rank order of potencies is sauvagine (3 nM), r/hCRF (20 nM), α‐helCRF(9‐41) (150 nM), and oCRF (300 nM). α‐helCRF, α‐helical oCRF(9‐41).

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• Figure 1.13.5 Localization of CRF1 and CRF2 receptor ligand binding sites by autoradiography using [125 I]sauvagine and [125 I]oCRF in rat brain. Horizontal slide‐mounted rat brain sections were incubated with either radiolabeled oCRF or sauvagine and apposed to film as described (see ). [125 I]oCRF (left) labels predominantly the CRF1 receptor subtype in the internal granular layer of the olfactory bulb, and in cortical and cerebellar regions, with virtually no labeling of the CRF2(a) subtype. [125 I]Sauvagine (right), which has equal affinity for both the CRF1 and CRF2(a) receptor subtypes, labels all of the same CRF1 receptor regions as [125 I]oCRF, and also labels regions high in CRF2(a) receptor density, such as the lateral septum, the choroid plexus, and the ependymal layer of the olfactory bulb. This confirms the apparent selectivity profile of the two radioligands, and is consistent with the rank order profile demonstrated in the competition studies.

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