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 Gi), alpha o (alpha subunit of GTP-binding protein G(o)), and of a 47-kDa alpha s (alpha subunit of GTP-binding protein Gs) 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-L1 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.
Huppertz
C
C
Institut für Pharmakologie und Toxikologie der RWTH Aachen, Germany.
Schürmann
A
A
Joost
H G
HG
D016475:3T3 Cells; D000273:Adipose Tissue / Q000166:cytology / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002454:Cell Differentiation; D002462:Cell Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology*; D051379:Mice; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009124:Muscle Proteins; D010641:Phenotype
D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495693:Slc2a4 protein, mouse; D019204:GTP-Binding Proteins