Characterization of chemokine receptors

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.