G-PROTEINS IN ADIPOCYTES AND PREADIPOCYTES - CHARACTERIZATION, SUBCELLULAR-DISTRIBUTION, AND POTENTIAL ROLES FOR G(I2) AND OR G(I3) IN THE CONTROL OF CELL-PROLIFERATION/

Guanosine triphosphate (GTP) binding protein subunits were studied by immunoblot analysis in particulate fractions from mature adipocytes, c onfluent preadipocytes, and in vitro-differentiated preadipocytes. Mat ure adipocytes express Gi alpha 1, Gi alpha 2, Gi alpha 3, Go alpha, G q/11 alpha, G13 alpha and the long and shore isoforms of Gsa, but no G z alpha or G12 alpha. Confluent and differentiated preadipocytes diffe r in having a higher content of Gi alpha 3 and G13 alpha and expressin g G12 alpha. In contrast, they lack Gi alpha 1, Go alpha, and the shor t form of Gs alpha. The G-protein alpha subunits Gi alpha 2, Gs alpha (long isoform), and Gq/11 alpha, and G-protein beta subunits were unch anged throughout the differentiation process. By immunoblot and indire ct immunofluorescence studies on confluent preadipocytes, we showed th at Gi alpha 2 is present in the endoplasmic reticulum and marginally i n plasma membranes and nuclei. In contrast, antibodies to Gi alpha 3 s tained the Golgi apparatus. The role of G proteins on preadipocyte pro liferation was studied using Bordetella pertussis toxin. Exposure of g rowing cells to this toxin in the presence of fetal calf serum (FCS) d ecreased [H-3]thymidine incorporation by 40% and induced a 40% increas e in doubling time. This resulted in a 30% decrease in cell number per well after 48 h. These effects of B. pertussis toxin did not appear t o be related to an increase in cyclic adenosine monophosphaee (cAMP) c oncentration, because forskolin had the opposite effect on cell prolif eration. Finally, B. pertussis toxin prevented serum-induced Raf1 asso ciation to the plasma membrane, possibly by disrupting FCS-induced G b eta gamma effects on the Ras/Raf1 pathway. Since Go alpha and Gi alpha 1 subunits were absent in preadipocytes, we conclude that Gi2 and/or Gi3 proteins transduce some mitogenic signals of FCS through release o f G beta gamma subunits. The subcellular distribution of Gi alpha 2 an d Gi alpha 3 suggests that part of their functions result from interac tions with components other than the plasma membrane.