Characterization of CGRP Receptor Binding

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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 MgCl₂

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

Figure 1.30.1 Displacement of [¹²⁵ I]CGRP by unlabeled CGRP in membranes prepared from Cos 7 cells transfected with CL and RAMP1. Total cpm are shown on the y axis. Nonspecific binding in this example was 440 cpm, with a resulting K _i of 0.6 nM and a Hill coefficient of 0.65. [¹²⁵ I]CGRP was present at 40 pM.

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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.
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	Poyner, D.R., Sexton, P.M., Marshall, I., Smith, D.M., Quirion, R., Born, W., Muff, R., Fischer, J.A., and Foord, S.M. 2002. International Union of Pharmacology XXXII. The mammalian CGRP, adrenomedullin, amylin and calcitonin receptors. Pharmacol. Rev. 54:233‐246.
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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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Characterization of CGRP Receptor Binding

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited