Characterization of Chemokine Receptors

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

This unit describes the procedures for measuring binding of a radiolabeled chemokine to chemokine receptors in cells or cell membranes. The whole?cell binding assay can be used for both purified leukocytes as well as transfected cell lines expressing chemokine receptors. Two basic protocols are described. The first is applicable to all cells expressing chemokine receptors, both primary cultures as well as recombinantly transfected cell lines expressing a single chemokine receptor. The second is used principally for transfected cell lines and measures chemokine binding to cell membranes using scintillation proximity assay (SPA) methodology, but does not require washing steps and thus is applicable to automated high?throughput screening strategies. An alternative procedure is also described which also uses SPA methodology to measure GTP?gamma?S binding to chemokine receptors in cell membranes. The two basic protocols can be used to determine the binding of compounds to chemokine receptors, while the alternate protocol determines the effects of compounds on the chemokine?stimulated activation of the receptor.

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Basic Protocol 1: Binding of Radiolabeled Chemokines to Cells Expressing Chemokine Receptors
Basic Protocol 2: Competition Equilibrium Assay Using Scintillation Proximity Assay (SPA) Technology on Membranes Expressing a Chemokine Receptor
Alternate Protocol 1: SPA‐Based [35S]GTPγS Binding to Cell Membranes Expressing Chemokine Receptors
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Binding of Radiolabeled Chemokines to Cells Expressing Chemokine Receptors

Materials

Unlabeled recombinant chemokine: prepare in‐house or purchase (e.g., PeproTech, R&D Systems, Dictagene, Gryphon Sciences)
HEPES binding buffer (see recipe )
Radiolabeled (i.e., ¹²⁵ I) chemokine ligand (Amersham)
Transfected cells expressing a single chemokine receptor (ATCC: http://www.attc.org) or purified leukocytes (unit 12.4 )
Phosphate buffered saline (PBS; unit 12.4 ) containing recipe1 mM EDTA ( appendix 2A )
0.4% (w/v) trypan blue solution (Life Technologies): store in a dark bottle and filter before use or after prolonged storage
Wash buffer, ice cold: HEPES binding buffer containing 0.5 M NaCl
Optiphase Supermix scintillation liquid (Wallac)

Hemacytometer
96‐well opaque MultiScreen plates fitted with a 0.65‐µm hydrophilic low‐protein‐binding membrane (Millipore)
Multichannel pipettor (optional)
Plate shaker
MultiScreen manifold filtration system (Millipore)
Vacuum pump
Wallac Microbeta Trilux counter
PRISM software (GraphPad Software)

NOTE: The radiolabeled ligand should be prepared in a laboratory equipped for protection from γ‐irradiation.

Basic Protocol 2: Competition Equilibrium Assay Using Scintillation Proximity Assay (SPA) Technology on Membranes Expressing a Chemokine Receptor

Materials

Confluent cells expressing a recombinant chemokine receptor (e.g., CHO, HEK) grown in 75‐ml flasks in appropriate growth medium
PBS (unit 12.4 ) with and without 1 mM EDTA
Breakage buffer (see recipe )
HEPES binding buffer (see recipe )
Wheat‐germ agglutinin (WGA)‐coated SPA beads (Amersham)
¹²⁵ I‐labeled chemokine (Amersham or NEN Life Sciences)
1 to 5 µM unlabeled chemokine (ReproTech)

5‐mm‐diameter Polytron PT1200 homogenizer
Ultracentrifuge and appropriate tubes
96‐deep‐well microtiter plates (i.e., Corning low‐binding plate)
Wallac Microbeta Trilux counter
Graphing software (e.g., GraphPad PRISM)

Additional reagents and equipment for Bradford protein assay ( appendix 3A )

Alternate Protocol 1: SPA‐Based [35S]GTPγS Binding to Cell Membranes Expressing Chemokine Receptors

Cell membranes expressing a given chemokine receptor (see protocol 2 , steps to )
GTPγS assay buffer (see recipe )
GDP (Roche)
Individual or multiple chemokines
Individual or multiple test compounds
1200 Ci/mmol guanosine 5′‐(γ‐[³⁵ S]thio)triphosphate ([³⁵ S]GTPγS; NEN Life Sciences)
1 mM unlabeled guanosine 5′‐O‐3‐thiotriphosphate (GTPγS; Roche)
Shaker
Centrifuge with microplate carrier

CAUTION: Radioactive materials require special handling; all supernatants must be considered radioactive waste and disposed of appropriately.

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Figures

Figure 1.24.1 Equilibrium binding competition assay of TARC binding to mCCR4 expressed in HEK cells using a whole‐cell assay. The competition is homologous in that the radiolabeled and unlabeled ligand are both human TARC, which is species cross‐reactive such that it also binds to the murine CCR4 receptor. The IC₅₀ value obtained in this experiment is 0.9 nM.

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Figure 1.24.2 Heterologous equilibrium competition binding assays of mCCR5 expressed in Chinese hamster ovary (CHO) cells using a whole cell assay. Human [¹²⁵ I]MIP‐1α was used as the tracer, and the competition was determined for mMIP‐1α (solid triangles), RANTES (open squares), and two amino terminally‐modified hRANTES proteins—i.e., Met‐RANTES (open circles) and AOP‐RANTES (solid circles; Proudfoot et al., ; Simmons et al., ). In this assay, mMIP‐1α and hRANTES had IC₅₀ values of 0.04 and 9.7 nM, respectively. AOP‐RANTES had a slightly higher affinity with an IC₅₀ of 3.7 nM, while Met‐RANTES had a significantly lower affinity with an IC₅₀ of 170 nM.

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Figure 1.24.3 Competition binding experiment with RANTES (solid circles) and MIP‐1α (open circles) on membranes prepared from CHO cells stably transfected with hCCR5. The CCR5 cell membranes were incubated for 3 hr at room temperature with 0.2 nM [¹²⁵ I]MIP‐1α and increasing concentrations of RANTES and MIP‐1α.The IC₅₀ values determined in this assay for RANTES and MIP‐1α were 2.7 nM and 0.7 nM, respectively.

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Figure 1.24.4 ANTES (open circles) and MIP‐1α (filled circles) stimulate CCR5‐mediated [³⁵ S]GTPγS binding to CHO cell membranes. CCR5 cell membranes were incubated for 30 min in the presence of increasing concentrations of RANTES or MIP‐1α. In this assay, RANTES had an EC₅₀ value of 12 nM and MIP‐1α had an EC₅₀ value of 760 nM.

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Figure 1.24.5 Stimulation of [³⁵ S]GTPγS binding to CCR5 cell membranes by RANTES (solid bar) and AOP‐RANTES (hashed bar). CCR5 cell membranes were incubated for 30 min in the absence (basal; open bar) or presence of 1 µM RANTES or 0.1 µM AOP‐RANTES; n = 3. This experiment confirms the fact that AOP‐RANTES is a full agonist of CCR5, and as was reported for phosphorylation of the receptor, is even more potent than RANTES in certain assays (Oppermann et al., ).

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Videos

Literature Cited

Literature Cited
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	Simmons, G., Clapham, P.R., Picard, L., Offord, R.E., Rosenkilde, M.M., Schwartz, T.W., Buser, R., Wells, T.N.C. and Proudfoot, A.E. 1997. Potent inhibition of HIV‐1 infectivity in macrophages and lymphocytes by a novel CCR5 antagonist. Science 276:276‐279.
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	Weng, Y.M., Siciliano, S.J., Waldburger, K.E., Sirotinameisher, A., Staruch, M.J.D., Gould, S.L., Daugherty, B.L., Springer, M.S., and DeMartino, J.A. 1998. Binding and functional properties of recombinant and endogenous CXCR3 chemokine receptors. J. Biol. Chem. 273:18288‐18291.

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Characterization of Chemokine Receptors

Abstract

Table of Contents

Materials

Basic Protocol 1: Binding of Radiolabeled Chemokines to Cells Expressing Chemokine Receptors

Basic Protocol 2: Competition Equilibrium Assay Using Scintillation Proximity Assay (SPA) Technology on Membranes Expressing a Chemokine Receptor

Alternate Protocol 1: SPA‐Based [35S]GTPγS Binding to Cell Membranes Expressing Chemokine Receptors

Figures

Videos

Literature Cited