Assessing the binding and endocytosis activity of cellular receptors using GFP-ligand fusions.
We have developed a simple scheme for characterizing ligand-receptor binding and post-binding activity on living cells. Our approach makes use of green fluorescent protein (GFP) as an auto-fluorescent tag to label protein ligands. We have constructed GFP-tagged ligands that can be expressed in bacteria as soluble fusion proteins. A cell-binding assay using fluorescence-activated cell sorting (FACS) demonstrates that GFP-tagged proteins retain their wild-type receptor-binding specificity. Using this assay, we measure ligand binding on unfixed cells and demonstrate receptor specificity using specific competitors. To determine the ability of receptor targets to internalize, we developed a second FACS-based assay to detect the rate and percentage of internalized ligand in living cells. Noninternalizing control ligands and fluorescence microscopy of treated cells confirm that our assay is reliable for determining receptor internalization activity.
Medina-Kauwe
L K
LK
University of Southern California Keck School of Medicine, Los Angeles, USA.
Leung
V
V
Wu
L
L
Kedes
L
L
D001665:Binding Sites; D001667:Binding, Competitive; D001943:Breast Neoplasms; D019281:Dimerization; D004705:Endocytosis; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics*; D020890:Neuregulin-1 / Q000235:genetics* / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D014407:Tumor Cells, Cultured
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D020890:Neuregulin-1; D010455:Peptides; D015800:Protozoan Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; C117463:heregulin alpha; C042968:knob protein, Plasmodium falciparum