G proteins in adipocytes and preadipocytes: Characterization, subcellular distribution, and potential roles for Gi2 and/or Gi3 in the control of cell proliferation☆

Abstract Guanosine triphosphate (GTP)-binding protein subunits were studied by immunoblot analysis in particulate fractions from mature adipocytes, confluent preadipocytes, and in vitro -differentiated preadipocytes. Mature adipocytes express Gi α 1, Gi α 2, Gi α 3, Go α , Gq/11 α , G13 α and the long and short isoforms of Gs α , but no Gz α or G12 α . Confluent and differentiated preadipocytes differ in having a higher content of Gi α 3 and G13 α and expressing G12 α . In contrast, they lack Gi α 1, Go α , and the short form of Gs α . The G-protein α subunits Gi α2 , Gs α (long isoform), and Gq/11 α , and G-protein β subunits were unchanged throughout the differentiation process. By immunoblot and indirect immunofluorescence studies on confluent preadipocytes, we showed that Gi α 2 is present in the endoplasmic reticulum and marginally in plasma membranes and nuclei. In contrast, antibodies to Gi α 3 stained the Golgi apparatus. The role of G proteins on preadipocyte proliferation was studied using Bordetella pertussis toxin. Exposure of growing cells to this toxin in the presence of fetal calf serum (FCS) decreased [ 3 H]thymidine incorporation by 40% and induced a 40% increase 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 to be related to an increase in cyclic adenosine monophosphate (cAMP) concentration, because forskolin had the opposite effect on cell proliferation. Finally, B. pertussis toxin prevented serum-induced Raf1 association to the plasma membrane, possibly by disrupting FCS-induced G βγ effects on the Ras/Raf1 pathway. Since Go α and Gi α 1 subunits were absent in preadipocytes, we conclude that Gi2 and/or Gi3 proteins transduce some mitogenic signals of FCS through release of G βγ subunits. The subcellular distribution of Gi α 2 and Gi α 3 suggests that part of their functions result from interactions with components other than the plasma membrane.