MUTATIONAL ANALYSIS OF THE CARBOXY-TERMINAL PHOSPHORYLATION SITE OF GLUT-4 IN 3T3-L1 ADIPOCYTES

The carboxy terminus of GLUT-4 contains a functional internalization m otif (Leu-489Leu-490) that helps maintain its intracellular distributi on in basal adipocytes. This motif is flanked by the major phosphoryla tion site in this protein (Ser-488), which may play a role in regulati ng GLUT-4 trafficking in adipocytes. In the present study, the targeti ng of GLUT-4 in which Ser-488 has been mutated to alanine (SAG) has be en examined in stably transfected 3T3-L1 adipocytes. The trafficking o f SAG was not significantly different from that of GLUT-I in several r espects. First, in the absence of insulin, the distribution of SAG was similar to GLUT-4 in that it was largely excluded from the cell surfa ce and was enriched in small intracellular vesicles. Second, SAG exhib ited insulin-dependent movement to the plasma membrane (4- to 5-fold) comparable to GLUT-4 (4- to 5-fold). Finally, okadaic acid, which has previously been shown to stimulate both GLUT-4 translocation and its p hosphorylation at Ser-488, also stimulated the movement of SAG to the cell surface similarly to GLUT-4. Using immunoelectron microscopy, we have shown that GLUT-4 is localized to intracellular vesicles containi ng the Golgi-derived gamma-adaptin subunit of AP-1 and that this local ization is enhanced when Ser-488 is mutated to alanine. We conclude th at the carboxy-terminal phosphorylation site in GLUT-4 (Ser-488) may p lay a role in intracellular sorting at the trans-Golgi network but doe s not play a major role in the regulated movement of GLUT-4 to the pla sma membrane in 3T3-L1 adipocytes.