Characterization of CGRP Receptor Binding

Alex C. Conner1, Debbie L. Hay1, David R. Poyner1

1 Aston University, Aston Triangle, Birmingham
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 1.30
DOI:  10.1002/0471141755.ph0130s25
Online Posting Date:  September, 2004
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Abstract

CGRP receptor binding may be measured using homogenates of cell membranes or intact cells. Here a microcentrifugation‐based assay is described that utilizes radioiodinated CGRP in displacement studies to determine the binding parameters for any ligand that interacts with CGRP receptors. A similar assay is described for binding to cultured cells. The protocols may be adapted for other radioligands or for filtration‐based assays. The main problems with CGRP binding assays can usually be traced to degradation of the radioligand or displacing ligands. Also, some cell lines fail to express CGRP receptors after extensive passage. CGRP binding assays provide a rapid and easy approach for distinguishing between receptors for CGRP and related peptides such as adrenomedullin and amylin.

Keywords: CGRP; RAMP1; microcentrifugation; filtration; intact cell binding; membrane binding

     
 
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Table of Contents

  • Basic Protocol 1: Binding of CGRP to Membranes from Homogenized Tissues or Cells
  • Basic Protocol 2: Binding of CGRP to Intact Cells
  • Alternate Protocol 1: Saturation Binding to obtain Kd and Bmax Directly
  • Alternate Protocol 2: Rapid Filtration Assay
  • Alternate Protocol 3: CGRP8‐37 Binding
  • Support Protocol 1: Preparation of Membranes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Binding of CGRP to Membranes from Homogenized Tissues or Cells

  Materials
  • Frozen membrane preparation (see protocol 6)
  • Binding buffer for membranes (see recipe)
  • 0.1 mM displacing ligand (see recipe)
  • Radioligand (see recipe)
  • Unlabeled CGRP (e.g., Bachem)
  • Wash solution (i.e., tap water or 100 mM NaCl in distilled water)
  • Tissue homogenizer (e.g., Polytron, Ultra‐Turrax)
  • 0.5‐ml nonstick microcentrifuge (Anachem) or silanized tubes (see recipe)
  • γ counter with adapters for assay tubes
  • Additional reagents and equipment for determining protein concentration ( appendix 3A)

Basic Protocol 2: Binding of CGRP to Intact Cells

  Materials
  • Cells expressing CGRP receptors
  • Binding buffer for intact cells (see recipe), 4° and 37°C
  • 0.1 mM displacing ligand (see recipe)
  • Radioligand (see recipe)
  • Unlabeled CGRP (e.g., Bachem)
  • 0.1 M NaOH
  • 12‐well flat‐bottom plates
  • γ counter with appropriate adapter tubes
  • 25° or 37°C incubator

Alternate Protocol 1: Saturation Binding to obtain Kd and Bmax Directly

  Materials
  • Tissues or 15‐cm plates of cells expressing CGRP receptors
  • Homogenization buffer, ice‐cold: 20 mM HEPES, pH 7.5 (adjust with 1 M NaOH)/1 mM EDTA
  • Resuspension buffer, ice‐cold: 20 mM HEPES, pH 7.5 (adjust with 1 M NaOH)/1 mM MgCl 2
  • 50‐ml plastic tubes
  • Tissue homogenizer
  • Muslin (optional)
  • Ultracentrifuge and appropriate tubes
  • Additional reagents and equipment for determining protein concentration ( appendix 3A)
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Figures

Videos

Literature Cited

Literature Cited
   Aiyar, N., Disa, J., Siemens, I.R., and Nambi, P. 1997. Differential effects of guanine nucleotides on [125I]‐hCGRP(8‐37) binding to porcine lung and human neuroblastoma cell membranes. Neuropeptides. 31:99‐103.
   Bhogal, R., Smith, D.M., Purkiss, P., and Bloom, S.R. 1993. Molecular identification of binding sites for calcitonin gene‐related peptide (CGRP) and islet amyloid polypeptide (IAPP) in mammalian lung: Species variation and binding of truncated CGRP and IAPP. Endocrinology. 133:2351‐2361.
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   Tilakaratne, N., Christopoulos, G., Zumpe, E.T., Foord, S.M., and Sexton, P.M. 2000. Amylin receptor phenotypes derived from human calcitonin receptor/RAMP coexpression exhibit pharmacological differences dependent on receptor isoform and host cell environment. J. Pharmacol. Exp. Ther. 294:61‐72.
   Van Rossum, D., Dumont, Y., Fournier, A., St‐Pierre, S., and Quirion, R. 1992. Effect of dithiothreitol and Gpp(NH)p on [125I] alpha‐hCGRP binding sites in brain and peripheral tissues. Ann. N.Y. Acad. Sci. 657:429‐431.
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