Functional comparison of the role of dynamin 2 splice variants on GLUT-4 endocytosis in 3T3L1 adipocytes

Previously, we reported that expression of a dominant-interfering neuronal- specific dynamin 1 (K44A/dynamin 1) inhibited the plasma membrane internali zation of GLUT-4 in 3T3L1 adipocytes (15). To investigate the role of the u biquitously expressed isoform of dynamin, dynamin 2, on adipocyte GLUT-4 in ternalization, and to determine whether dynamin splice variants have functi onal specificity, we expressed each of the four dynamin 2 isoforms (aa, ab, ba, and bb) as either wild-type proteins or GTPase-defective mutants. When expressed as enhanced green fluorescent protein (EGFP) fusions, these isof orms were found to have overlapping subcellular distributions being localiz ed throughout the cell cytoplasm, on punctate vesicles and in a perinuclear compartment. This distribution was qualitatively similar to that of endoge nous dynamin 2 and overlapped with GLUT-4 in the basal state. Expression of wild-type dynamin 2 isoforms had no effect on the basal or insulin-stimula ted distribution of GLUT-4; however, expression of the dominant-interfering dynamin 2 mutants inhibited GLUT-4 endocytosis. These data demonstrate tha t dynamin 2 is required for GLUT-4 endocytosis in 3T3L1 adipocytes and sugg est that, relative to GLUT-4 trafficking, the dynamin 2 splice variants hav e overlapping functions and are probably not responsible for mediating dist inct GLUT-4 budding events.