ABUNDANCE AND SUBCELLULAR-DISTRIBUTION OF GTP-BINDING PROTEINS IN 3T3-L1 CELLS BEFORE AND AFTER DIFFERENTIATION TO THE INSULIN-SENSITIVE PHENOTYPE

The abundance and the subcellular distribution of GTP-binding proteins was studied in membrane fractions (plasma membranes and low-density microsomes) from 3T3-L1 cells before and after differentiation to the insulin-sensitive phenotype. After differentiation, the abundance of alpha(i) (alpha subunit of GTP-binding protein G(i)), alpha(o) (alpha subunit of GTP-binding protein G(o)), and of a 47-kDa alpha(s) (alpha subunit of GTP-binding protein G(s)) as detected by immunoblotting with specific antisera was reduced by 10-50% when normalized per membrane protein. In contrast, a 43-kDa alpha(s) was increased about threefold after differentiation. Furthermore, cholera-toxin-catalyzed ADP-ribosylation of both 43-kDa and 47-kDa alpha(s) was disproportionately increased ninefold and threefold, respectively, possibly reflecting the increased production of an ADP-ribosylation factor in the differentiated cells. The small GTP-binding protein Ha-ras was reduced by 50%, whereas rab1 and other small GTP-binding proteins tentatively identified as rab-isoforms (ras-homologous gene products from brain) were increased by 100% and 70%, respectively. Since the total protein content of 3T3-LI cells was increased threefold after differentiation, the observed increase of the 43-kDa alpha(s), rab1 and of the other rab isoforms was eightfold, sixfold and fivefold, respectively, when normalized/cell count. With the exception of the rab isoforms, all GTP-binding proteins were predominantly, if not exclusively, located in the plasma membrane; comparable amounts of the rab isoforms were found in plasma membranes and low-density microsomes. Insulin induced the characteristic redistribution of glucose transporters GLUT4 from low-density microsomes to the plasma membranes, but failed to alter the subcellular distribution of any of the GTP-binding proteins investigated. These data suggest that the increase in the abundance of the 43-kDa alpha(s) subunit and of several rab isoforms might be related to specific functions of the adipocyte-like phenotype, but that none of the investigated guanine-nucleotide-binding regulatory (G)-proteins appears to be tightly associated with the GLUT4.