Fluorescent CXCL12^AF647 as a novel probe for nonradioactive CXCL12/CXCR4 cellular interaction studies

Abstract

BackgroundChemokines drive the migration of leukocytes via interaction with specific G protein–coupled 7‐transmembrane receptors. The chemokine ligand/receptor pair stromal cell–derived factor‐1 (SDF‐1, CXCL12)/CXCR4 is gaining increasing interest because of its involvement in the metastasis of several types of cancer and in certain inflammatory autoimmune disorders such as rheumatoid arthritis. In addition, CXCR4 serves as an important coreceptor for cellular entry of T‐tropic strains of human immunodeficiency virus (HIV). Therefore, potent and specific CXCR4 antagonists may have therapeutic potential as anti‐HIV, anti‐cancer, and anti‐inflammatory drugs.Methods and ResultsChemokine receptor antagonists can be identified by their ability to inhibit ligand binding to the receptor protein. Until now, chemokine binding assays were mostly performed with radiolabeled chemokine ligands such as [125I]CXCL12. To overcome the practical problems associated with such radioactive chemokine binding assays, we have developed a flow cytometric technique using a new, commercially available Alexa Fluor 647 conjugate of CXCL12 (CXCL12AF647). Calcium flux, chemotaxis, and p44/42 mitogen‐activated protein kinase phosphorylation assays showed that the agonistic activity of the fluorescent CXCL12 was unchanged as compared with that of unlabeled CXCL12. Human T‐lymphoid (CXCR4+) SupT1 cells and CXCR4‐transfected, but not CCR5‐ or CXCR3‐transfected, human astroglioma U87.CD4 cells specifically bound CXCL12AF647 in a concentration‐dependent manner. Unlabeled CXCL12 and the well‐known CXCR4 inhibitors, AMD3100 and T22, blocked the binding of CXCL12AF647 to SupT1 cells with 50% inhibitory concentrations of 92, 13, and 8 ng/ml, respectively. We have also used this method to evaluate CXCL12 binding and CXCR4 expression level in different subsets of human peripheral blood mononuclear cells.ConclusionCXCL12AF647 is a valuable, more convenient alternative for [125I]CXCL12 in ligand/receptor interaction studies. © 2004 Wiley‐Liss, Inc.

Probing the pharmacology of G protein-coupled receptors with fluorescent ligands

“…Similarly, using a fluorescent antagonist for the prostanoid EP 3 R, it was shown that the EP 3 R was expressed in murine kidney and in human brain tissue (Tomasch et al, 2012a). In T lymphocytes, a CXCL12 based fluorescent ligand has been used in conjunction with both microscopy and flow cytometry to identify CXCR4 positive Tlymphoid SupT1 cells (Hatse et al, 2004) and expression of the CB2 receptor on CD4þ T cells was demonstrated using a fluorescent ligand for the cannabinoid CB2 receptor (Petrov et al, 2011). For the neuropeptide Y 1 receptor (Y 1 R), the development of a small molecule fluorescent antagonist allow the confirmation of the expression of the receptor in a human breast cancer cell line (MCF-7) and interestingly using flow cytometry they showed that they could measure the affinity of unlabelled antagonists in a erytholeukemia cell line that endogenously expresses the Y 1 R (Schneider et al, 2007).…”

Citrullination of CXCL12 Differentially Reduces CXCR4 and CXCR7 Binding with Loss of Inflammatory and Anti-HIV-1 Activity via CXCR4

“…Competition for CXCL12 AF647 binding was measured on CXCR4-or CXCR7-transfected CHO cells as described previously (36). Briefly, 1.5 ϫ 10 6 cells/ml (300,000 cells in 200 l) were incubated for 1 h at 4°C with 20 ng/ml CXCL12 AF647 and varying concentrations of unlabeled chemokine in RPMI 1640 plus 2% FBS.…”

CXCR7 participates in CXCL12-induced CD34+ cell cycling through β-arrestin–dependent Akt activation

“…16,21 By using Alexafluor-647-coupled CXCL12 (CXCL12 AF647 ), a fully functional and specific CXCL12 chemokine derivative, 24,25 we showed that CXCL12 AF647 could bind to CD34 1 HSPCs (n 5 4, Figure 2A). …”