Silencing of Gprasp1 or Gprasp2 perturbs CXCR4 localization. (A) GASP-binding motifs in the C terminus of known and putative GPRASP targets. (B) Experimental schematic. LSK cells were treated with Gprasp1, Gprasp2, or control shRNAs, cultured for 72 hours and then treated for 1 hour with stromal cell–derived factor 1 (SDF-1) to induce CXCR4 internalization. Permeabilized and nonpermeabilized cell were then examined for CXCR4 expression and intracellular-to-extracellular ratio calculated. (C) CXCR4 intracellular-to-extracellular ratio. (D) Total CXCR4 40 to 72 hours posttransduction in Gprasp1, Gprasp2, or control-shRNA–treated HSPCs assessed by flow cytometry. (E) Total CXCR4 72 hours posttransduction in Gprasp1, Gprasp2, or control-shRNA–treated HSPCs treated with 5 μg/mL cycloheximide assessed by flow cytometry. (F) LT-HSCs were examined for GPRASP1/2 and CXCR4 colocalization via confocal microscopy. GPRASP1/2-405 and CXCR4-488 stain; scale bar, 5 μm. (G) High Förster resonance energy transfer (FRET) efficiency between GPRASP1/2-405 and CXCR4-488 in LT-HSCs indicates a physical proximity of <10 nm. Data in panels C, D, E, and G from at least 3 independent experiments. Data represented as mean plus or minus SEM. */#, P < .05; **/##, P < .005; ***/###, P < .001 relative to control. In panel E, * refers to Gprasp1 shRNA; # refers to Gprasp2 shRNA. FACS, fluorescence-activated cell sorting; M1, muscarinic receptor 1; M2, muscarinic receptor 2; ORL1/KOR, opioid receptor-like 1/κ opioid receptor.