Characterization of Corticotropin‐Releasing Factor (CRF) Receptors

Dimitri E. Grigoriadis1, Marge T. Lorang1, Nicola Duggan1, Errol B. De Souza1

1 Neurocrine Biosciences, San Diego, California
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 1.13
DOI:  10.1002/0471141755.ph0113s02
Online Posting Date:  May, 2001
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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.

     
 
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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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Materials

Basic Protocol 1: Association Kinetic Assay to Determine Time Course for Equilibrium Binding

  Materials
  • Assay buffer (see recipe)
  • 6 µM unlabeled rat/human corticotropin‐releasing factor (r/hCRF; Peninsula Labs) in assay buffer
  • Radioligand in assay buffer (select one):
  •   900 pM [125I]ovine CRF ([125I]oCRF; NEN Life Sciences; for CRF 1 receptor subtype)
  •   900 pM [125I]sauvagine (NEN Life Sciences; for CRF 1, CRF 2(a) or CRF 2(b) receptor subtypes)
  •   900 pM [125I]r/hCRF (2000 to 2200 Ci/mmol; NEN Life Sciences; for CRF 1 receptor subtype)
  •   900 pM [125I]urocortin (2000 to 2200 Ci/mmol; Amersham; nonselective activator of CRF receptors)
  • Membrane suspension (see protocol 5)
  • Wash buffer (see recipe), ice cold
  • 1.5‐ml polypropylene microcentrifuge tubes
  • Tabletop microcentrifuge with 60‐tube capacity and speed >10,000 × g (e.g., Beckman Microcentrifuge 12, Eppendorf model 5403)
  • Vacuum aspirator: aspiration flask connected to tubing with Pasteur pipet/pipet tip (for radioactive waste)
  • Microcentrifuge tube cutter (Fisher) or dog nail clippers (local pet supply)
  • 12 × 75–mm gamma counter tubes

Alternate Protocol 1: Kinetic Assay to Determine Dissociation Time Course

  • 31 µM unlabeled rat/human corticotropin‐releasing factor (r/hCRF; Peninsula Labs) in assay buffer (see recipe)

Basic Protocol 2: Saturation (Scatchard) Assays to Determine KD and Bmax

  Materials
  • Assay buffer (see recipe)
  • 6 µM unlabeled rat/human corticotropin‐releasing factor (r/hCRF; Peninsula Labs) or D‐Phe r/hCRF(12‐41) (Rivier et al., ) in recipeassay buffer
  • 6× radioligand solutions (12, 6, 3, 1.5, 0.75, 0.375, 0.186, and 0.09 nM) in recipeassay buffer (select one):
  •   [125I]ovineCRF ([125I]oCRF; NEN Life Sciences; for CRF 1 receptors)
  •   [125I]sauvagine (NEN Life Sciences; for CRF 1 or CRF 2 receptors)
  •   [125I]r/hCRF (2000 to 2200 Ci/mmol; NEN Life Sciences; for CRF 1 receptors)
  •   [125I]urocortin (2000 to 2200 Ci/mmol; Amersham; nonselective activator of CRF receptors)
  • Membrane suspension (see protocol 5)
  • Wash buffer (see recipe), ice cold
  • 1.5‐ml polypropylene microcentrifuge tubes
  • Tabletop microcentrifuge with 60‐tube capacity and speed >10,000 × g (e.g., Beckman Microcentrifuge 12, Eppendorf model 5403)
  • Vacuum aspirator: aspiration flask connected to tubing with Pasteur pipet/pipet tip (for radioactive waste)
  • Microcentrifuge tube cutter (Fisher) or dog nail clippers (local pet supply)
  • 12 × 75–mm gamma counter tubes

Basic Protocol 3: Competition Assays to Determine Ki Values of Competing Ligands

  Materials
  • Assay buffer (see recipe)
  • 6× competing ligand solutions (see recipe; e.g., 6 µM, 1.9 µM, 600 nM, 190 nM, 60 nM, 19 nM, 6 nM, 1.9 nM, 0.6 nM, and 0.19 nM):
  •   agonists: rat/human corticotropin‐releasing (r/hCRF), ovine CRF (oCRF), sauvagine, urotensin I, urocortin
  •   antagonists:D‐Phe r/h CRF(12‐41), α‐helical oCRF(9‐41), astressin
  • 1.2 nM radioligand in assay buffer: [125I]ovine CRF ([125I]oCRF; NEN Life Sciences; for CRF 1 receptor subtype) or [125I]sauvagine (NEN Life Sciences; for CRF 1 or CRF 2 receptor subtypes)
  • Membrane suspension (see protocol 5)
  • Wash buffer (see recipe), ice cold
  • 1.5‐ml polypropylene microcentrifuge tubes
  • Tabletop microcentrifuge with 60‐tube capacity and speed >10,000 × g (e.g., Beckman microcentrifuge 12, Eppendorf model 5403)
  • Vacuum aspirator: aspiration flask connected to tubing with Pasteur pipet/pipet tip (for radioactive waste)
  • Microcentrifuge tube cutter (Fisher) or dog nail clippers (local pet supply)
  • 12 × 75–mm gamma counter tubes

Support Protocol 1: Preparation of Corticotropin‐Releasing Factor (CRF) Receptors from Tissues or Cells

  Materials
  • Fresh whole rodent brain or cells (e.g., mammalian, insect) transfected transiently or stably, and expressing the desired CRF receptor subtype
  • Tissue buffer (see recipe)
  • 50 mM Tris⋅Cl ( appendix 2A) or Dulbecco's PBS (DPBS; Life Technologies), pH 7.0 at 22°C, containing 5 mM EDTA
  • 5‐ to 15‐ml round‐bottom high‐speed (40,000 × g) centrifuge tubes (e.g., Fisher)
  • Polytron tissue homogenizer (Brinkmann) or Tissue Tearor (Fisher)
  • Additional reagents and solutions for counting cells (Phelan, ) and for BCA protein determination ( appendix 3A)

Basic Protocol 4: Receptor Autoradiography to Study Corticotropin‐Releasing Factor (CRF) Receptors

  Materials
  • Whole rodent brain (dissect fresh, freeze immediately, and store at −80°C)
  • Tissue‐Tek O.C.T. Compound embedding medium (VWR)
  • 0.2 nM radioligand (2000 to 2200 Ci/mmol; NEN Life Sciences) in recipeassay buffer:
  •  [125I]oCRF or r/hCRF (for CRF 1 receptors)
  •  [125I]sauvagine (for CRF 1 and CRF 2 receptors)
  • 100 µM competing ligand solution (see recipe):
  •  agonists: rat/human corticotropin‐releasing factor (r/hCRF), ovine CRF (oCRF), sauvagine, urotensin I, urocortin
  •  antagonists: D‐Phe r/hCRF(12‐41), α‐helical oCRF(9‐41), astressin
  • Assay buffer (see recipe), ice cold
  • Wash buffer (see recipe), ice cold
  • Refrigerated cryostat (e.g., Hacker, Leica)
  • Superfrost/PLUS slides (Fisher)
  • Glass Coplin jars (or equivalent staining dishes) and appropriate slide racks
  • Blow dryer (Fisher)
  • X‐ray cassettes (Sigma)
  • Radioactive standard microscales ([125I]microscales; Amersham)
  • X‐ray film (e.g., Kodak Biomax‐MR)
  • Automated X‐ray film developer or manual X‐ray film developing supplies
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Figures

Videos

Literature Cited

Literature Cited
  (Chadwick, D.J., Marsh, J., and Ackrill, K. (eds.). 1993. Corticotropin‐releasing factor. In CIBA Foundation Symposium, Vol.172. John Wiley & Sons, Chichester, England.
   Chalmers, D.T., Lovenberg, T.W., and De Souza, E.B. 1995. Localization of novel corticotropin‐releasing factor receptor (CRF2) mRNA to specific sub‐cortical nuclei in rat brain: Comparison with CRF1 receptor mRNA expression. J. Neurosci. 15:6340‐6350.
   Chalmers, D.T., Lovenberg, T.W., Grigoriadis, D.E., Behan, D.P., and De Souza, E.B. 1996. Corticotropin‐releasing factor receptors: From molecular biology to drug design. Trends Pharmacol. Sci. 17:166‐172.
   Chang, C.P., Pearse, R.I., O'Connell, S., and Rosenfeld, M.G. 1993. Identification of a seven transmembrane helix receptor for corticotropin‐releasing factor and sauvagine in mammalian brain. Neuron 11:1187‐1195.
   Chen, R., Lewis, K.A., Perrin, M.H., and Vale, W.W. 1993. Expression cloning of a human corticotropin‐releasing‐factor receptor. Proc. Natl. Acad. Sci. U.S.A. 90:8967‐8971.
   De Souza, E.B., and Grigoriadis, D.E. 1994. Corticotropin‐releasing factor: Physiology, pharmacology and role in central nervous system and immune disorders. In Psychopharmacology: The Fourth Generation of Progress (F.E. Bloom and D.J. Kupfer, eds.) pp. 505‐517. Raven Press, New York.
   De Souza, E.B. and Nemeroff, C.B. (eds.) 1990. Corticotropin‐Releasing Factor: Basic and Clinical Studies of a Neuropeptide. CRC Press, Boca Raton, Fla.
   Dunn, A.J. and Berridge, C.W. 1990. Physiological and behavioral responses to corticotropin‐releasing factor administration: Is CRF a mediator of anxiety of stress responses? Br. Res. Rev. 15:71‐100.
   Grigoriadis, D.E., Lovenberg, T.W., Chalmers, D.T., Liaw, C., and De Souza, E.B. 1996. Characterization of corticotropin‐releasing factor receptor subtypes. In Neuropeptides: Basic and Clinical Advances (J.N. Crawley and S. McLean, eds.) pp. 60‐80. The New York Academy of Sciences, New York.
   Gulyas, J., Rivier, C., Perrin, M., Koerber, S.C., Sutton, S., Corrigan, A., Lahrichi, S.L., Craig, A.G., Vale, W., and Rivier, J. 1995. Potent, structurally constrained agonists and competitive antagonists of corticotropin‐releasing factor. Proc. Natl. Acad. Sci. U.S.A. 92:10575‐10579.
   Kishimoto, T., Pearse, R.V.II., Lin, C.R., and Rosenfeld, M.G. 1995. A sauvagine/corticotropin‐releasing factor receptor expressed in heart and skeletal muscle. Proc. Natl. Acad. Sci. U.S.A. 92:1108‐1112.
   Kostich, W., Chen, A., Sperle, K., Horlick, R.A., Patterson, J., and Largent, B.L. 1996. Molecular cloning and expression analysis of human CRF receptor type 2α and β isoforms. Soc. Neurosci. Abstr. 22:1545.
   Liaw, C.W., Lovenberg, T.W., Barry, G., Oltersdorf, T., Grigoriadis, D.E., and De Souza, E.B. 1996. Cloning and characterization of the human CRF2 receptor gene and cDNA. Endocrinology 137:72‐77.
   Lovenberg, T.W., Liaw, C.W., Grigoriadis, D.E., Clevenger, W., Chalmers, D.T., De Souza, E.B., and Oltersdorf, T. 1995. Cloning and characterization of a functionally distinct corticotropin‐releasing factor receptor subtype from rat brain. Proc. Natl. Acad. Sci. U.S.A. 92:836‐840.
   Owens, M.J., and Nemeroff, C.B. 1991. Physiology and pharmacology of corticotropin‐releasing factor. Pharmacol. Rev. 43:425‐473.
   Perrin, M.H., Donaldson, C.J., Chen, R., Lewis, K.A., and Vale, W.W. 1993. Cloning and functional expression of a rat brain corticotropin releasing factor (CRF) receptor. Endocrinology 133:3058‐3061.
   Perrin, M., Donaldson, C., Chen, R., Blount, A., Berggren, T., Bilezikjian, L., Sawchenko, P., and Vale, W. 1995. Identification of a second corticotropin‐releasing factor receptor gene and characterization of a cDNA expressed in heart. Proc. Natl. Acad. Sci. U.S.A. 92:2969‐2973.
   Phelan, M.C. 1998. Techniques for mammalian cell tissue culture. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. A.3F.1‐A.3F.14. John Wiley & Sons, New York.
   Rivier, J., Rivier, C., Galyean, R., Miranda, A., Miller, C., Craig, A.G., Yamamoto, G., Brown, M., and Vale, W. 1993. Single point D‐substituted corticotropin‐releasing factor (CRF) analogs: Effect on potency and physicochemical characteristics. J. Med. Chem. 36:2851‐2859.
   Sperle, K., Chen, A., Kostich, W., and Largent, B.L. 1997. CRH2γ: A novel CRH2 receptor isoform found in human brain. Soc. Neurosci. Abstr. 23:689.14.
   Vita, N., Laurent, P., Lefort, S., Chalon, P., Lelias, J.M., Kaghad, M., Le, F.G., Caput, D., and Ferrara, P. 1993. Primary structure and functional expression of mouse pituitary and human brain corticotrophin releasing factor receptors. FEBS Lett. 335:1‐5.
   Webster, E.L., Lewis, D.B., Torpy, D.J., Zachman, E.K., Rice, K.C., and Chrousos, G.P. 1996. In vivo and in vitro characterization of antalarmin, a nonpeptide corticotropin‐releasing hormone (CRH) receptor antagonist: Suppression of pituitary ACTH release and peripheral inflammation. Endocrinology. 137:5747‐5750.
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