FIG. 5.

Active PKB/Akt drives αvβ3 integrin recycling. (A and B) NIH 3T3 fibroblasts were transfected with human αvβ3 (hαvβ3) integrin in combination with constitutively active PKB (memb-PKB) or PKB-AAA. Cells were serum starved and surface labeled with 0.2 mg of N-hydroxysuccinimide (NHS)-S-S-biotin/ml for 30 min at 4°C. Internalization was initiated by warming to 37°C for the times shown in the absence (open symbols) and presence (closed symbols) of 0.6 mM primaquine. Biotin was released from proteins remaining at the cell surface, and biotinylated integrin was determined by capture ELISA using microtiter wells coated with an anti-human β3 integrin monoclonal antibody. (C) Cells were transfected with hαvβ3 in combination with the conditionally active fusion memb-PKB and the hormone-binding domain of the ER (memb-PKB-ER). Serum-starved cells were surface labeled with 0.2 mg of NHS-S-S-biotin/ml for 30 min at 4°C, and internalization was allowed to proceed for 15 min at 22°C. Biotin remaining at the cell surface was removed by exposure to β-mercaptoethanesulfonic acid (MESNa) at 4°C, and internalized integrin was chased back to the cell surface at 37°C for 15 min in the absence (open bars; control) and presence (solid bars) of 300 nM 4-HT. Cells were then reexposed to MESNa, and biotinylated integrin was determined by capture ELISA and expressed as for Fig. 3. Values are means ± standard errors of the means (n = 12).