Genome-scale identification of cellular pathways required for cell surface recognition

IGF2R traffics GABBR2 through a clathrin-dependent pathway. (A) Schematic of a flow cytometry–based GABA_B cell surface expression assay. Parental and IGF2R-KO HEK293 cells were cotransfected with plasmids encoding both GABBR1 (R1) and GABBR2 (R2) full-length cDNAs and surface expression of GABA_B receptors quantified by antibody staining and FACS. A plasmid containing a GFP-tagged CD200 cDNA was transfected in parallel and the number of transfected cells quantified by GFP fluorescence. (B) The percentage of GABA_B receptor–positive cells was significantly higher on the surface of IGF2R-KO cells compared with the parental line (P < 0.01 using Welch's two sample t-test, n = 3); control demonstrates both cell lines were transfected at comparable levels. Error bars, ± SD, n = 3 (cells were transfected in triplicate); a representative experiment of two independent experiments is shown. (C) The clathrin-pathway inhibitor sucrose was added to parental and IGF2R-deficient HEK293 cells expressing the GABA_B receptor complex, and the cell surface receptor levels were quantified. The effect of sucrose on the IGF2R–GABA_B receptor complex interaction was determined by calculating the ratio of positively stained cells on the parental cell line relative to the IGF2R-deficient cells and a buffer-only control. Sucrose treatment led to a significant increase (P < 0.05 using Welch's two sample t-test) in cell surface GABA_B receptor complex staining on the parental cell line, almost to the same level as on the IGF2R-deficient cells, demonstrating that that GABA_B receptor trafficking depends on the clathrin pathway. The control represents a transfection control to demonstrate parental and IGF2R-deficient cells were transfected with equal efficiency. Bars, mean ± SD; n = 3 (cells were transfected in triplicate).